CN106188055B - The method that complexing abstraction extracts separation catharanthine from catharanthus roseus is aided in using enzyme - Google Patents

Abstract

The invention discloses a method for extracting and separating vinblastine from periwinkle flowers by an enzyme-assisted complex extraction method, which comprises the following steps: (1) enzyme-assisted ultrasonic extraction of vinblastine in periwinkle flowers, and concentrating the extract under reduced pressure (2) using D2EHPA as a complexing agent and petroleum ether as a diluent to form an extraction system, and carrying out complex extraction on the concentrated solution to obtain an extract rich in vinblastine; (3) back-extracting with dilute hydrochloric acid to obtain Changchun Mass alkali crude product; (4) purification. According to the present invention, vinblastine contains amine groups and shows basic structural characteristics, and uses the acidic complexing agent D2EHPA to perform complex extraction on it, so that vinblastine and complexing agent react to form a complex, and transfer to the extraction In the phase, dilute hydrochloric acid is used for back extraction, so that vinblastine can be recovered. The extraction method of the invention effectively improves the recovery rate of vinblastine, and the extractant of complex extraction can be recycled without causing environmental pollution.

Description

Method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction

technical field

The invention relates to a method for efficiently extracting vinblastine from periwinkle flowers, in particular to a method for extracting and separating vinblastine from periwinkle flowers by an enzyme-assisted complexation extraction method.

Background technique

Periwinkle (Catharanthus roseus) is an important medicinal plant of the genus Periwinkle in the family Apocynaceae, native to South Asia, East Africa and tropical America. In my country, it is mainly distributed in Jiangsu, Zhejiang, Fujian, Jiangxi, Sichuan and Henan. The whole plant of periwinkle can be used as medicine. It contains various terpene indole alkaloids such as vinblastine, vindolin, vinblastine, amaline, serpentine, vincristine, etc., and has high medicinal value. Among them, vinblastine not only has antitumor activity, but also has hypoglycemic, blood pressure, analgesic and other effects. At the same time, vinblastine is also an intermediate for the synthesis of vinblastine antitumor drugs such as vinorelbine and vinflunine.

There has been a long history of research on the extraction and separation of vinca alkaloids at home and abroad, but because there are many types of indole alkaloids in vinca flowers, and the content in plants is very low, generally at a few parts per ten thousand, so indole alkaloids in vinca flowers The extraction and separation of alkaloids has become a bottleneck restricting the industrial development of vinblastine drugs. At present, the extraction technology of related enterprises in my country has a low yield rate, a large waste of resources, and the product quality does not meet the international standard (purity > 98%). They can only produce crude extracts or APIs with low purity, which is difficult to meet the international market. demand. Although the product quality of a small number of enterprises can meet the requirements, the yield rate is low, resulting in a large waste of resources and serious environmental pollution. In addition, due to the over-harvesting of wild periwinkle in our country, the resource reserves of periwinkle have dropped sharply and are already on the verge of extinction. The lack of biological raw materials of periwinkle has also seriously restricted the development of the vinca alkaloid drug industry. Therefore, how to improve the utilization rate of periwinkle biological resources and develop methods for efficiently separating and purifying periwinkle alkaloids has become a top priority.

During the extraction of plant active ingredients, due to the thicker cell walls outside the plant cells, the target products inside the cells are not easily released into the solvent, which hinders the extraction rate of active ingredients. The use of biological enzymes can destroy the extracellular structure of plants and increase the dissolution rate of target components.

Extraction separation method has been widely studied for its advantages of high separation efficiency, large production capacity, convenient rapid continuous and safe operation, and low energy consumption. According to the principle of extraction, in the extraction operation, the extraction without chemical reaction between the extractant and the solute is called physical extraction; the extraction with chemical reaction between the extractant and the solute is called chemical extraction. According to the chemical reaction mechanism between the solute and the extractant, chemical extraction can be roughly divided into five categories: complexation reaction, cation exchange reaction, ion association reaction, addition reaction and belt extraction reaction. Complexation extraction is based on the principle of reversible complexation reaction. The solute to be separated in the solution contacts the extractant containing complexing agent, and the complexing agent reacts with the solute to be separated to form a complex, which is transferred to the extraction phase. Then, according to the solvent swing effect, stripping is carried out, the solute is recovered, and the extractant is recycled. Therefore, the complex extraction method has the advantages of high efficiency, high selectivity and low cost, and can be used for the separation and purification of vinblastine alkaloids in Vinca.

Contents of the invention

In order to solve the above problems, the object of the present invention is to provide a simple and efficient method for extracting and separating vinblastine from periwinkle by using an enzyme-assisted complex extraction method.

In order to achieve the above object, the technical solution adopted in the present invention is:

The method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method comprises the following steps:

(1) Enzyme-assisted ultrasonic extraction

Take the above-ground part of periwinkle and dry it in the shade and crush it to obtain dry periwinkle powder, add mixed enzyme solution to incubate, filter, and leave the periwinkle powder, add ethanol to the periwinkle powder, adjust the pH to 5.6-6.8 with hydrochloric acid, and extract by ultrasonic; The extract is concentrated under reduced pressure to obtain a concentrate;

(2) Complex extraction and enrichment

Using di(2-ethylhexyl)phosphoric acid as a complexing agent and petroleum ether as a diluent to form an extraction system, perform complex extraction on the concentrated solution, separate the supernatant, and obtain an extract rich in vinblastine;

(3) Stripping and separation

back-extracting the extract rich in vinblastine with dilute hydrochloric acid, concentrating the dilute hydrochloric acid layer under reduced pressure, and freeze-drying to obtain crude vinblastine;

(4) Purification

The resulting crude vinblastine is recrystallized to obtain.

The mixed enzyme liquid in step (1) includes: cellulase 3-6U/ml, pectinase 4-8U/ml and hemicellulase 6-9U/ml.

The pH of the mixed enzyme solution in step (1) is 4.0-7.0, and the mixed enzyme solution also includes one or more of metal ions Fe ²⁺ , Co ²⁺ , Mn ²⁺ , Ca ²⁺ , and the total amount of metal ions The mass fraction is 1%.

The incubation temperature is 25-50° C. and the incubation time is 4-6 hours.

The added amount of the ethanol is 5 times the weight of the periwinkle powder obtained by filtering, and the volume fraction of the ethanol is 70-80%.

Ultrasonic extraction in step (1) is performed 3-4 times, 4-6 hours each time.

The vacuum degree of vacuum concentration in steps (1) and (3) is -0.9MPa, and the rotating speed is 150-180 rpm.

In step (2), the volume ratio of di(2-ethylhexyl)phosphoric acid and petroleum ether is 1:1-3, the volume ratio of the extraction system and the concentrate is 1-3:1, and the extraction times are 3-5 times.

The mass fraction of dilute hydrochloric acid in step (3) is 1-3%, the volume ratio of dilute hydrochloric acid to extract is 1-3:1, and the number of times of back extraction is 3-5 times.

Step (4) The specific method for recrystallizing the crude vinblastine is: dissolving the crude vinblastine in ultrapure water, adjusting the pH to 8-9 with ammonia water, stirring at 20-25°C for 5-6h, and then Let stand for 12-24h, that is.

In step (4), the crude vinblastine is dissolved in ultrapure water to a final concentration of 4-6 g/L.

Beneficial effects of the present invention:

1. The structure of vinblastine contains amine groups, showing basicity. According to the structural characteristics of vinblastine, the present invention utilizes acidic complexing agent di(2-ethylhexyl) phosphoric acid to carry out complex extraction to make vinblastine and complexing agent react to form a complex, and transfer to in the extraction phase. Then, according to the swing effect, dilute hydrochloric acid is used for back extraction, so that vinblastine can be recovered, and the extractant can be recycled.

2. Due to the thicker cell wall outside the plant cells, the target product inside the cells is not easily released into the solvent, which hinders the extraction of active ingredients. Therefore, the present invention utilizes cellulase, pectinase, hemicellulase, etc. to destroy the extracellular structure of plants and improve the dissolution rate of target components. In addition, the present invention also adds metal ions to the mixed enzyme liquid, which can increase the activity of the enzyme in the mixed enzyme liquid and further increase the dissolution rate of the target components.

3. The present invention can efficiently separate vinblastine from periwinkle, effectively improves the resource utilization rate of periwinkle, and the extractant of complex extraction can be recycled without causing environmental pollution.

4. The purity of the vinblastine separated and purified by the method of the present invention can reach more than 99%, and the recovery rate can reach more than 92%, which is a great innovation in the separation and purification of vinblastine extracts and has high research and promotion value.

5. The method of the present invention is simple, convenient to operate, low in production cost, easy to industrialize and popularize, and has good social and economic benefits.

Description of drawings

Fig. 1 is the ultraviolet spectrogram of the vinblastine purification product of the present invention.

Fig. 2 is the ultraviolet spectrogram of vinblastine standard substance.

Fig. 3 is the mass spectrogram of the vinblastine purification product of the present invention.

Fig. 4 is the HPLC chromatogram of the extract of the present invention at 230nm.

Fig. 5 is the HPLC chromatogram of the vinblastine purified product of the present invention at 230 nm.

detailed description

The specific implementation of the present invention will be described in further detail below in conjunction with the examples.

The equipment used for concentration under reduced pressure of the present invention is a rotary evaporator.

Example 1

The method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method comprises the following steps:

(1) Enzyme-assisted ultrasonic extraction

Take 100g of periwinkle aboveground part and dry it in the shade and crush it into powder to get periwinkle dry powder, add 400ml of mixed enzyme solution to hatch, the temperature of incubation is 25°C, and the time is 6h; after the hatching, filter, keep periwinkle powder, add periwinkle powder 5 times the weight of 70% ethanol, adjust the pH to 5.6 with hydrochloric acid, ultrasonically extract 3 times, each time for 6 hours; filter, keep the extract, and concentrate the extract under reduced pressure to obtain a concentrate;

The mixed enzyme solution includes: cellulase 3U/ml, pectinase 4U/ml, hemicellulase 6U/ml; the pH of the mixed enzyme solution is 4.0, and the mixed enzyme solution also includes metal ion Ca ²⁺ , The mass fraction of Ca ²⁺ is 1%;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(2) Complex extraction and enrichment

Using di(2-ethylhexyl)phosphoric acid (D2EHPA) as a complexing agent and petroleum ether as a diluent to form an extraction system, the concentrated solution is subjected to complex extraction, the volume ratio of D2EHPA and petroleum ether is 1:1, and the upper layer is separated liquid to obtain an extract rich in vinblastine; the volume ratio of the extraction system and the concentrated solution is 1:1, and the number of extractions is 3 times;

(3) Stripping and separation

The extract rich in vinblastine is back-extracted with 3% dilute hydrochloric acid with a mass fraction of 3%, the volume ratio of dilute hydrochloric acid to the extract is 1:1, the number of back-extraction is 4 times, the dilute hydrochloric acid layer is concentrated under reduced pressure, and freeze-dried , to obtain the crude product of vinblastine;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(4) Purification

The obtained crude vinblastine is recrystallized, the specific method is: dissolve the crude vinblastine with ultrapure water to a final concentration of 4 g/l, adjust the pH to 9 with ammonia water, stir at 25°C for 6 hours, and then Let it stand for 24 hours, and you get it.

Example 2

The method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method comprises the following steps:

(1) Enzyme-assisted ultrasonic extraction

Take 100g of periwinkle aboveground part and dry it in the shade and crush it into powder to get periwinkle dry powder, add 400ml of mixed enzyme solution to hatch, the temperature of incubation is 30°C, and the time is 6h; after the hatching, filter, keep periwinkle powder, add periwinkle powder 5 times the weight of 70% ethanol by volume, and adjusting the pH to 5.8 with hydrochloric acid, ultrasonically extracting 4 times, 5 hours each time; filtering, leaving the extract, concentrating the extract under reduced pressure to obtain a concentrate;

The mixed enzyme solution includes: cellulase 4U/ml, pectinase 5U/ml, hemicellulase 7U/ml; the pH of the mixed enzyme solution is 5.0;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 180 rpm;

(2) Complex extraction and enrichment

Using D2EHPA as a complexing agent and petroleum ether as a diluent to form an extraction system, the concentrated solution is subjected to complex extraction. The volume ratio of D2EHPA and petroleum ether is 1:2, and the supernatant is separated to obtain an extract rich in vinblastine ; The volume ratio of the extraction system and the concentrate is 2:1, and the number of extractions is 4 times;

(3) Stripping and separation

The extract rich in vinblastine is back-extracted with 3% dilute hydrochloric acid with a mass fraction of 3%, the volume ratio of dilute hydrochloric acid to the extract is 2:1, the number of back-extractions is 3 times, the dilute hydrochloric acid layer is concentrated under reduced pressure, and freeze-dried , to obtain the crude product of vinblastine;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 180 rpm;

(4) Purification

The obtained crude vinblastine is recrystallized, the specific method is: dissolve the crude vinblastine with ultrapure water to a final concentration of 5 g/l, adjust the pH to 8 with ammonia water, stir at 20° C. for 6 hours, and then Stand still for 20h, that is.

Example 3

The method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method comprises the following steps:

(1) Enzyme-assisted ultrasonic extraction

Take 100g of periwinkle aboveground part and dry it in the shade and crush it into powder to get periwinkle dry powder, add 400ml of mixed enzyme solution to hatch, the temperature of incubation is 40°C, and the incubation time is 5h; after hatching, filter, keep periwinkle powder, add periwinkle powder 5 times the weight of 80% ethanol, adjust the pH to 6.2 with hydrochloric acid, ultrasonically extract 3 times, each time for 5 hours; filter, keep the extract, and concentrate the extract under reduced pressure to obtain a concentrate;

The mixed enzyme solution includes: cellulase 5U/ml, pectinase 8U/ml, hemicellulase 9U/ml; the pH of the mixed enzyme solution is 6.0, and the mixed enzyme solution also includes metal ions Mn ²⁺ , The mass fraction of Mn ²⁺ is 1%;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 160 rpm;

(2) Complex extraction and enrichment

Using D2EHPA as a complexing agent and petroleum ether as a diluent to form an extraction system, the concentrated solution is subjected to complex extraction. The volume ratio of D2EHPA and petroleum ether is 1:2, and the supernatant is separated to obtain an extract rich in vinblastine ; The volume ratio of the extraction system and the concentrate is 3:1, and the number of extractions is 4 times;

(3) Stripping and separation

The extract rich in vinblastine is back-extracted with dilute hydrochloric acid with a mass fraction of 1%, the volume ratio of dilute hydrochloric acid to the extract is 2:1, the number of back-extractions is 5 times, the dilute hydrochloric acid layer is concentrated under reduced pressure, and freeze-dried , to obtain the crude product of vinblastine;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 160 rpm;

(4) Purification

The obtained vinblastine crude product is recrystallized, and the specific method is: dissolve the vinblastine crude product with ultrapure water to a final concentration of 6 g/l, adjust the pH to 9 with ammonia water, stir at 25° C. for 5 h, and then Stand still for 12h, that is.

Example 4

The method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method comprises the following steps:

(1) Enzyme-assisted ultrasonic extraction

Take 100g of periwinkle aboveground part and dry it in the shade and crush it into powder to get periwinkle dry powder, add 400ml of mixed enzyme solution to hatch, the temperature of incubation is 50°C, and the time is 4h; after hatching, filter, keep periwinkle powder, add periwinkle powder 5 times the weight of 80% ethanol, adjust the pH to 6.8 with hydrochloric acid, ultrasonically extract 4 times, each time for 4 hours; filter, keep the extract, concentrate the extract under reduced pressure to obtain a concentrate;

The mixed enzyme solution includes: cellulase 6U/ml, pectinase 7U/ml, hemicellulase 8U/ml; the pH of the mixed enzyme solution is 7.0, and the mixed enzyme solution also includes metal ions Fe ²⁺ , The mass fraction of Fe ²⁺ is 1%;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(2) Complex extraction and enrichment

Using D2EHPA as a complexing agent and petroleum ether as a diluent to form an extraction system, the concentrated solution is subjected to complex extraction, the volume ratio of D2EHPA and petroleum ether is 1:3, and the supernatant is separated to obtain an extract rich in vinblastine ; The volume ratio of the extraction system and the concentrate is 3:1, and the number of extractions is 5 times;

(3) Stripping and separation

Back-extract the vinblastine-rich extract with 2% dilute hydrochloric acid, the volume ratio of dilute hydrochloric acid to the extract is 3:1, and the number of back-extractions is 3 times. After the dilute hydrochloric acid layer is concentrated under reduced pressure, freeze-dry , to obtain the crude product of vinblastine;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(4) Purification

The obtained crude vinblastine is recrystallized, the specific method is: dissolve the crude vinblastine with ultrapure water to a final concentration of 4 g/l, adjust the pH to 9 with ammonia water, stir at 25°C for 6 hours, and then Let it stand for 24 hours, and you get it.

Comparative example 1

The method for extracting and separating vinblastine from periwinkle comprises the following steps:

(1) Enzyme-free ultrasonic extraction

Take 100g of periwinkle aboveground part and dry it in the shade and pulverize it into powder to obtain periwinkle dry powder, add 80% ethanol with a volume fraction of 5 times the weight of periwinkle dry powder, and adjust the pH to 6.8 with hydrochloric acid, ultrasonically extract 4 times, each time for 6h; filter, leave The extract is concentrated under reduced pressure to obtain a concentrate;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(2) Complex extraction and enrichment

Using D2EHPA as a complexing agent and petroleum ether as a diluent to form an extraction system, the concentrated solution is subjected to complex extraction. The volume ratio of D2EHPA and petroleum ether is 1:1, and the supernatant is separated to obtain an extract rich in vinblastine ; The volume ratio of the extraction system and the concentrate is 2:1, and the number of extractions is 4 times;

(3) Stripping and separation

The extract rich in vinblastine is back-extracted with 3% dilute hydrochloric acid with a mass fraction of 3%, the volume ratio of dilute hydrochloric acid to the extract is 2:1, the number of back-extractions is 4 times, the dilute hydrochloric acid layer is concentrated under reduced pressure, and freeze-dried , to obtain the crude product of vinblastine;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(4) Purification

The obtained crude vinblastine is recrystallized, the specific method is: dissolve the crude vinblastine with ultrapure water to a final concentration of 4 g/l, adjust the pH to 9 with ammonia water, stir at 25°C for 6 hours, and then Let it stand for 24 hours, and you get it.

Comparative example 2

The method for extracting and separating vinblastine from periwinkle comprises the following steps:

(1) Enzyme-assisted ultrasonic extraction

Take 100g of periwinkle aboveground part and dry it in the shade and crush it into powder to get periwinkle dry powder, add 400ml of mixed enzyme solution to hatch, the temperature of incubation is 50°C, and the time is 4h; after hatching, filter, keep periwinkle powder, add periwinkle powder 5 times the weight of 80% ethanol, adjust the pH to 6.8 with hydrochloric acid, ultrasonically extract 4 times, each time for 4 hours; filter, keep the extract, concentrate the extract under reduced pressure to obtain a concentrate;

The mixed enzyme solution includes: cellulase 6U/ml, pectinase 7U/ml, hemicellulase 8U/ml; the pH of the mixed enzyme solution is 7.0, and the mixed enzyme solution also includes metal ions Fe ²⁺ , The mass fraction of Fe ²⁺ is 1%;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(2) Ordinary extraction and enrichment

Adjust the pH of the concentrated solution to 7.8, use chloroform as the extractant to extract the concentrated solution, the volume ratio of the extractant and the concentrated solution is 2:1, the number of extractions is 4 times, and the supernatant is separated to obtain an extraction rich in vinblastine solution, concentrated under reduced pressure, and freeze-dried to obtain crude vinblastine;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(3) Purification

The obtained crude vinblastine is recrystallized, the specific method is: dissolve the crude vinblastine with ultrapure water to a final concentration of 4 g/l, adjust the pH to 9 with ammonia water, stir at 25°C for 6 hours, and then Let it stand for 24 hours, and you get it.

Comparative example 3

The method for extracting and separating vinblastine from periwinkle comprises the following steps:

(1) Enzyme-free ultrasonic extraction

Take 100g of periwinkle aboveground part and dry it in the shade and pulverize it into powder to obtain periwinkle dry powder, add 80% ethanol with a volume fraction of 5 times the weight of periwinkle dry powder, and adjust the pH to 6.8 with hydrochloric acid, ultrasonically extract 4 times, each time for 6h; filter, leave The extract is concentrated under reduced pressure to obtain a concentrate;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(2) Ordinary extraction and enrichment

Adjust the pH of the concentrated solution to 7.8, use chloroform as the extractant to extract the concentrated solution, the volume ratio of the extractant and the concentrated solution is 2:1, the number of extractions is 4 times, and the supernatant is separated to obtain an extraction rich in vinblastine solution, concentrated under reduced pressure, and freeze-dried to obtain crude vinblastine;

The vacuum degree of decompression concentration is -0.9MPa, and the rotation speed is 150 rpm;

(3) Purification

The obtained vinblastine crude product was recrystallized, and the specific method was: dissolve the vinblastine crude product with ultrapure water to a final concentration of 4 g/L, adjust the pH to 9 with ammonia water, stir at 25° C. for 6 h, and then Let it stand for 24 hours, and you get it.

Experimental example

1. Product analysis

1.1. Ultraviolet spectral analysis

The purified product of Example 1 of the present invention and the vinblastine standard were detected simultaneously by an ultraviolet spectrometer, and the results are shown in FIGS. 1 and 2 . The results show that the purified product of the present invention is consistent with the ultraviolet spectrum results of the vinblastine standard substance.

1.2. Mass spectrometry analysis

The crystalline product of Example 1 of the present invention was detected by a mass spectrometer, and the results are shown in FIG. 3 . The mass spectrum data of the purified product of the present invention is compared with the known vinblastine standard mass spectrum data, and the results show that the purified product of the present invention is consistent with the known vinblastine mass spectrum data.

1.3. Detection by HPLC method

HPLC detection of product: using Waters Symmetry 300 C ₁₈ liquid chromatography column, with acetonitrile: water (acetonitrile and water both contain 1% formic acid) as mobile phase, flow rate 1ml/min, gradient elution (30-90%, 30min) , the detection wavelength is 230nm, and better chromatographic analysis results can be obtained.

Detection objects: extracts and purified products rich in vinblastine.

Result analysis: The results of HPLC analysis are shown in Figures 4 and 5. Figure 4 shows that there is a higher content of the target substance in the extract, and the extraction has higher selectivity; Figure 5 shows that the purity of the purified product reaches more than 99% through recrystallization.

1.4. Recovery rate

With the extract of embodiment 1-4 and comparative example 1-3 as sample, research different extraction and purification methods to the extraction and separation effect of vinblastine, adopt high performance liquid chromatography to detect each sample concentration, calculate the yield of each method ( Table 1).

Table 1 The result analysis of target product yield

Example 1 Example 2 Example 3 Example 4 Comparative example 1 Comparative example 2 Comparative example 3 Yield (%) 93.2 92.3 92.4 93.1 82.1 80.1 70.1

As can be seen from Table 1, the present invention adopts the method that enzyme-assisted ultrasonic extraction and complex extraction combine, extracts and separates vinblastine from periwinkle, and the yield of its vinblastine is obviously higher than adopting enzyme-assisted ultrasonic extraction alone Or the extraction method of complex extraction is far higher than the extraction method that neither adopts enzyme-assisted ultrasonic extraction nor adopts complex extraction, so it can be explained that the enzyme-assisted complex extraction method of the present invention can significantly improve the extraction rate from Changchun. The yield of extracting and separating vinblastine from the flower effectively improves the resource utilization rate of the periwinkle flower.

Claims (6)

1. Utilize enzyme-assisted complex extraction method to extract the method for separating vinblastine from periwinkle, it is characterized in that, comprises the following steps: (1) Enzyme-assisted ultrasonic extraction Take the above-ground part of periwinkle and dry it in the shade and crush it to obtain dry periwinkle powder, add mixed enzyme solution to incubate, filter, and leave the periwinkle powder, add ethanol to the periwinkle powder, adjust the pH to 5.6-6.8 with hydrochloric acid, and extract by ultrasonic; The extract is concentrated under reduced pressure to obtain a concentrate; (2) Complex extraction and enrichment Using di(2-ethylhexyl)phosphoric acid as a complexing agent and petroleum ether as a diluent to form an extraction system, perform complex extraction on the concentrated solution, separate the supernatant, and obtain an extract rich in vinblastine; (3) Stripping and separation back-extracting the extract rich in vinblastine with dilute hydrochloric acid, concentrating the dilute hydrochloric acid layer under reduced pressure, and freeze-drying to obtain crude vinblastine; (4) Purification The obtained crude vinblastine is recrystallized to obtain; The mixed enzyme liquid in step (1) includes: cellulase 3-6U/mL, pectinase 4-8U/mL and hemicellulase 6-9U/mL; The pH of the mixed enzyme solution in step (1) is 4.0-7.0, and the mixed enzyme solution also includes one or more of metal ions Fe ²⁺ , Co ²⁺ , Mn ²⁺ , Ca ²⁺ , and the total amount of metal ions The mass fraction is 1%; Step (4) The specific method for recrystallizing the crude vinblastine is: dissolving the crude vinblastine in ultrapure water, adjusting the pH to 8-9 with ammonia water, stirring at 20-25°C for 5-6h, and then Let it stand for 12-24 hours, and you will get it; In step (4), the crude vinblastine is dissolved in ultrapure water to a final concentration of 4-6 g/L. 2. The method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method according to claim 1, characterized in that, the temperature of the incubation is 25-50°C, and the time is 4-6h. 3. the method utilizing enzyme-assisted complexation extraction method according to claim 1 to extract and separate vinblastine from periwinkle, it is characterized in that, the addition of described ethanol is the weight of the periwinkle powder that 5 times of filtration obtains, The volume fraction of ethanol is 70-80%. 4. The method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method according to claim 1, characterized in that, in step (1), ultrasonic extraction is performed 3-4 times, each 4-6h. 5. the method utilizing enzyme-assisted complex extraction method according to claim 1 to extract and separate vinblastine from periwinkle, it is characterized in that, in step (2), the ratio of two (2-ethylhexyl) phosphoric acid and sherwood oil The volume ratio is 1:1-3, the volume ratio of the extraction system and the concentrate is 1-3:1, and the extraction times are 3-5 times. 6. the method for extracting and separating vinblastine from periwinkle by enzyme-assisted complex extraction method according to claim 1, it is characterized in that, the massfraction of dilute hydrochloric acid is 1-3% in step (3), dilute hydrochloric acid The volume ratio to the extract is 1-3:1, and the number of back extractions is 3-5 times.