A method for efficiently extracting colchicine

Abstract

The present invention belongs to the field of medicine, and relates to a method for efficiently extracting colchicine, comprising the following steps: (1) pre-treatment: crushing and sieving Gloriosa seeds; (2) Flash extraction: mixing the pre-treated medicinal material with an extraction solvent, and extracting 1-4 times with a Flash extraction device, 5 minutes / time-20 minutes / time; (3) concentration: filtering the extract, combining the filtrate, and concentrating to dryness for standby use. The weight-to-volume ratio of the medicinal material raw material to the extraction solvent is 1:5-1:12 in g / mL. The colchicine extraction method provided by the present invention is simple to operate, has a high extraction rate, and has a high product purity, is conducive to saving solvents, reducing costs, and is safe and environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a method for efficiently extracting colchicine, and in particular to a method for extracting colchicine from Gloriosa gloriosa seeds by using a Flash extraction method. Background Art

[0002] Colchicine was originally extracted from the lily family plant Colchicum, also known as colchicine. Colchicine is a tropolone alkaloid that can be obtained from Gloriosa seeds, Trillium, lily and other plants. Its molecular formula is C 22 H 25 NO6, molecular weight 399.44, molecular structure is shown in the following formula I:

[0003]

[0004] Colchicine is an off-white to light yellow crystalline powder that is odorless, slightly hygroscopic, and darkens in color when exposed to light. It is easily soluble in ethanol or chloroform, soluble in water (but can form semi-hydrate crystals in aqueous solutions of a certain concentration), and very slightly soluble in ether.

[0005] Colchicine is a highly effective anti-gout drug with a history of more than 200 years of clinical application. Since colchicine can inhibit the growth of cancer cells, it is also used clinically to treat certain cancers, especially breast cancer.

[0006] The existing colchicine extraction method generally uses reflux extraction for extraction. For example, the Chinese application with publication number CN102276493A discloses a method for extracting colchicine, including 1) pre-treating Lijiang Tripterygium wilfordii; 2) drying; 3) reflux extraction; 4) ethanol recovery; 5) water precipitation to take supernatant; 6) adjusting the pH value to 9-13 after eluting the supernatant, and then performing secondary elution; 7) drying to remove methanol; 8) adding sulfuric acid to dissolve and filter; 9) extracting, filtering, and vacuum drying the precipitate; 10) recrystallizing, filtering, and vacuum drying the precipitate, and crushing the crystals to obtain colchicine fine products. This method has high energy consumption cost, long production cycle, and is extracted under high temperature conditions, which easily introduces more impurities or causes partial decomposition of the required active ingredients.

[0007] The Chinese application with publication number CN101602686A discloses a method for preparing colchicine, which comprises taking raw material fine powder, adding it to an alkaline aqueous solution and soaking it overnight, drying it, and placing it in a CO2 supercritical extraction device for supercritical extraction; adding a 2% hydrochloric acid aqueous solution to the extract to dissolve it, filtering it, adding chloroform to the filtrate for extraction, taking the lower layer liquid, adjusting the pH to 13, adding chloroform for extraction, taking the chloroform layer, separating it through a macroporous adsorption resin column, eluting it with an acid aqueous solution as an eluent, collecting the eluate, concentrating it, adding acetone for recrystallization, washing it, and drying it. This method has complicated operation steps, high preparation cost, low extraction rate, and chloroform has large pollution, is unsafe, and is not environmentally friendly.

[0008] Therefore, it is of great significance to provide a method for preparing colchicine with complete extraction, high extraction rate, high product purity, easy operation, low cost, safety and environmental protection.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for efficiently extracting colchicine, in particular a method for extracting colchicine from Gloriosa gloriosa seeds by using the Flash extraction method. The colchicine extraction method provided by the present invention is simple to operate, has a high extraction rate, and has a high product purity, is conducive to saving solvents, reducing costs, and is safe and environmentally friendly.

[0011] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0012] The first object of the present invention is to provide a method for extracting colchicine, comprising the steps of:

[0013] (1) Pretreatment: crush and sieve Gloriosa seeds;

[0014] (2) Flash extraction: The pre-treated medicinal material is mixed with the extraction solvent and extracted 1-4 times using a Flash extraction device, with a duration of 5 minutes to 20 minutes per time;

[0015] (3) Concentration: Filter the extract, combine the filtrate, and concentrate to dryness for later use.

[0016] The present invention uses the Flash extraction method to extract colchicine from Gloriosa glabra seeds, and can achieve an extraction transfer rate equivalent to that of ethanol reflux extraction in a relatively short time. In addition, the present invention explores and optimizes conditions such as the number of extractions, solvent concentration and dosage, temperature, and extraction time, and obtains applicable conditions, preferred conditions, and optimal conditions for extracting colchicine from Gloriosa glabra seeds using a Flash extraction device. The extracted product has high purity, high extraction rate, and short extraction time, and the quality is guaranteed while the efficiency is improved.

[0017] Colchicine can be extracted and purified from different raw materials, such as arrowhead and autumn daffodil. Due to the different impurities in different raw materials, the extraction methods are also completely different. The extraction method of the present invention is particularly suitable for extracting and preparing colchicine from the seeds of Gloriosa superba L, a plant of the Liliaceae family.

[0018] In the present invention, the seeds of Gloriosa can be wild or cultivated. The collected Gloriosa seeds can be pre-treated by cleaning, cutting and / or crushing. The cleaning is to remove impurities such as ash, mud and sand entrained in the process of collecting Gloriosa seeds. The collected Gloriosa seeds are prepared into a dry product by air drying, sun drying or machine drying, and the obtained dry product is crushed and sieved to facilitate extraction.

[0019] In the extraction method of the present invention, a Flash extraction device is used to extract 1-4 times, 5 minutes / time to 20 minutes / time, so as to obtain a higher colchicine transfer rate, and the required time is short and the loss of colchicine is low.

[0020] In a further embodiment, in step (2), the extraction is performed 2-3 times using a Flash extraction apparatus, with a duration of 5 minutes to 15 minutes per extraction;

[0021] Preferably, extraction is performed twice using a Flash extraction apparatus, each time for 15 minutes.

[0022] The present invention finds that the number of extractions has a great influence on the transfer rate of colchicine. When the extraction is performed 2-3 times with a Flash extraction device at 5 minutes / time to 15 minutes / time, the transfer rate of colchicine is higher. When the extraction is performed 2 times with a time of 15 minutes / time, the effect is the best.

[0023] In a further embodiment, in step (2), the weight-to-volume ratio of the medicinal material to the extraction solvent is 1:5-1:12 in g / mL;

[0024] Preferably, the weight-to-volume ratio of the medicinal material raw material to the extraction solvent is 1:6-1:10 in g / mL.

[0025] When the weight-to-volume ratio of the medicinal material raw material to the extraction solvent is within the above ratio range, the transfer rate of colchicine extraction is high, and it can save costs and help improve the purity of the product. When the amount of extraction solvent used is too much, it is easy to produce residues, which also causes waste and increases costs; when the amount of extraction solvent used is too little, the extraction is incomplete, affecting the transfer rate of colchicine.

[0026] In a further embodiment, in step (2), the extraction solvent comprises a mixture of an aliphatic alcohol containing 1 to 4 carbon atoms and water.

[0027] The mixture is C 1-4A mixture of aliphatic alcohol and water in any proportion. This aliphatic alcohol has good safety, low price and good extraction effect.

[0028] In a further embodiment, in step (2), the extraction solvent comprises 60%-90% ethanol aqueous solution.

[0029] In a further embodiment, in step (2), the extraction solvent is 70% ethanol aqueous solution.

[0030] As a preferred solution, the present invention adopts 60%-90% ethanol aqueous solution. Within this concentration range, the effect of extracting colchicine is better, and 70% ethanol aqueous solution has the best effect.

[0031] In a further embodiment, in step (2), the temperature of the extraction solvent is 20°C-40°C;

[0032] In a further embodiment, in step (2), the temperature of the extraction solvent is 20°C-30°C, preferably 20°C-25°C.

[0033] In the present invention, the temperature of the extraction solvent is low, which can greatly reduce the loss of colchicine during the extraction process. When the temperature of the extraction solvent is 20°C-30°C, preferably 20°C-25°C, the purity of the extracted colchicine is high.

[0034] In a further solution, in step (1), after the Gloriosa galanthus seeds are crushed, they are passed through an 80-mesh sieve, and the particle size of the medicinal material raw material is 180 μm±7.6 μm.

[0035] The present invention found that when the particle size of the medicinal material raw material is within the above range, the transfer rate of colchicine extraction is high. If the particle size is too large, the transfer rate decreases and the extraction is incomplete; if the particle size is too small, it is easy to increase the impurity content, which is not conducive to purification.

[0036] As a specific preferred embodiment, the method for extracting colchicine comprises the following steps:

[0037] (1) Pretreatment: crush and sieve Gloriosa seeds;

[0038] (2) Flash extraction: After pretreatment, add 6 volumes of 70% ethanol at 25°C, calculated on a g / mL basis, and extract twice using a Flash extraction apparatus, 15 minutes per time;

[0039] (3) Filter the extract, combine the filtrate, and concentrate to dryness for later use.

[0040] The parameters for extracting colchicine using the Flash extraction device include: voltage 120V, power 1800W, speed 1000r / min, and interval time 5 minutes.

[0041] In the present invention, colchicine is extracted under the above parameter conditions, and the obtained colchicine has high transfer rate and high purity.

[0042] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0043] 1. The present invention provides a method for efficiently extracting colchicine. The method of the present invention is used to extract colchicine, which is easy to operate, has a high extraction rate, a short extraction time, saves energy consumption required for heating and cooling, has high product purity, is conducive to saving solvents, reduces production costs, is suitable for large-scale industrial production of colchicine, and has extremely high application value and development prospects.

[0044] 2. The method of the present invention is particularly suitable for extracting colchicine from Gloriosa glabra seeds, and the conditions such as the number of extractions, solvent concentration and dosage, and extraction time are optimized through a large number of experiments, and the applicable conditions, preferred conditions, and optimal conditions for extraction using a Flash extraction device are obtained. The extracted product has high purity, high extraction rate, and short extraction time, thereby ensuring quality while improving efficiency. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0046] The instruments, equipment and materials used in this invention are:

[0047] Instruments and equipment: Flash extractor (Henan Zhixing Biotechnology, model: JHBE-50T), 1000g swing high-speed pulverizer (Wenling Linda Machinery Co., Ltd., model: DFY-1000D), electric heating mantle (Henan Aibo Technology, model: ZNHW20000ML), rotary evaporator (Switzerland BUCHI, model: R-3), high performance liquid chromatograph (Agilent, USA, model: Agilent1100).

[0048] Materials: Gloriosa dahurica seeds (purchased from India, batch number 20200927), ethanol, water.

[0049] The detection method used in the present invention: high performance liquid chromatography (General Rule 0512 of Appendix 4 of the 2020 edition of the "Chinese Pharmacopoeia") is used to determine the content of colchicine.

[0050] In the following examples, the parameters for extracting colchicine using a Flash extraction device include: voltage 120V, power 1800W, speed 1000r / min, and interval time 5 minutes.

[0051] Example 1

[0052] (1) Remove impurities from Gloriosa dahurica seeds (purchased from India, batch number 20200927) and crush and sieve into fine powder.

[0053] (2) Flash extraction: Take 100 g of Gloriosa gloriosa seed powder and add 6 times volume (in g / mL) of 70% ethanol to extract three times, 5 minutes each time;

[0054] (3) Filter the extract, combine the filtrate and concentrate to dryness for later use.

[0055] Example 2

[0056] (1) Remove impurities from Gloriosa dahurica seeds (purchased from India, batch number 20200927) and crush and sieve into fine powder.

[0057] (2) Flash extraction: Take 100 g of Gloriosa gloriosa seed powder, add 10 times the volume (in g / mL) of 60% ethanol and extract once, 20 minutes / time;

[0058] (3) Filter the extract, combine the filtrate and concentrate to dryness for later use.

[0059] Example 3

[0060] (1) Remove impurities from Gloriosa dahurica seeds (purchased from India, batch number 20200927) and crush and sieve into fine powder.

[0061] (2) Flash extraction: Take 100 g of Gloriosa gloriosa seed powder, add 12 times volume (in g / mL) of 80% ethanol and extract 4 times, 5 minutes each time;

[0062] (3) Filter the extract, combine the filtrate and concentrate to dryness for later use.

[0063] Example 4

[0064] (1) Remove impurities from Gloriosa dahurica seeds (purchased from India, batch number 20200927) and crush and sieve into fine powder.

[0065] (2) Flash extraction: Take 100 g of Gloriosa gloriosa seed powder and add 8 times volume (in g / mL) of 90% ethanol to extract twice, 15 minutes each time;

[0066] (3) Filter the extract, combine the filtrate and concentrate to dryness for later use.

[0067] Comparative Example 1

[0068] (1) Remove impurities from Gloriosa dahurica seeds (purchased from India, batch number 20200927) and crush and sieve into fine powder.

[0069] (2) Ethanol reflux extraction method: Take 100 g of Gloriosa jasminoides seed powder, add 6 times volume (in g / mL) of 70% ethanol and reflux extract 3 times, 1 hour each time;

[0070] (3) After filtration, the filtrates were combined and concentrated to dryness.

[0071] Test Example 1: Detection of the extraction rate of colchicine

[0072] Taking colchicine content as an indicator, the colchicine residue was determined by high performance liquid chromatography (General Rule 0512 of Appendix 4 of the 2020 edition of the Chinese Pharmacopoeia), and the colchicine residue after extraction in Example 1 and Comparative Example 1 was determined, and the extraction rate was calculated. The results are shown in Table 1.

[0073] Chromatographic conditions and system suitability test: octylsilane bonded silica gel as filler; methanol-water (32:68) as mobile phase; detection wavelength 245nm; flow rate 1.0mL per minute; column temperature 30℃.

[0074] Preparation of reference solution: Accurately weigh an appropriate amount of colchicine reference substance and place it in a brown volumetric flask. Dissolve and dilute it with methanol-water (1:1) to prepare a solution containing 30 μg per 1 mL as the reference solution.

[0075] Preparation of test solution: Take about 0.2 g of the powder of the product (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 50% methanol, ultrasonically treat for 30 minutes (power 100 w, frequency 40 kHz), cool, weigh it again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain the solution.

[0076] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0077] Table 1 Colchicine extraction rate determination results

[0078] sample Colchicine content% Transfer rate % Gloriosa seeds medicinal herbs 0.6188 / Comparative Example 1 (ethanol reflux extraction) 0.0252 95.92 Example 1 (Flash extraction) 0.0235 96.20

[0079] From the results in Table 1, it can be seen that the extraction of colchicine by the Flash extraction method can achieve an extraction transfer rate equivalent to that of ethanol reflux extraction in a shorter time, and the extraction efficiency is greatly improved.

[0080] Experimental Example 2 Orthogonal test of extraction process for colchicine

[0081] Ingredients: Gloriosa seeds (purchased from India, batch number 20200927) were crushed and sieved into fine powder after removing impurities.

[0082] 100 g of fine powder of Gloriosa jasminoides seeds was taken respectively, and Flash extraction was performed according to the conditions in Table 2. After filtration, the residues were taken respectively, dried, and the content was determined and the transfer rate was calculated. The results are shown in Table 2.

[0083] ANOVA was performed based on the transfer rate of each condition, and the results are shown in Table 3.

[0084] Table 2 Results of orthogonal test for colchicine extraction

[0085]

[0086]

[0087] Table 3 Analysis of variance in orthogonal extraction experiments

[0088] factor Sum of Squared Deviations Degrees of Freedom F Ratio F critical value Number of extractions 448.003 2 3.116 4.460 Extraction time (min) 26.814 2 0.186 4.460 Solvent multiple (mL / g) 14.858 2 0.103 4.460 Ethanol concentration (%) 85.477 2 0.594 4.460 error 575.15 8

[0089] It can be seen from Tables 2 and 3 above that under the four extraction factors, the factors that have a greater impact on the transfer rate of colchicine are mainly the number of extractions and the ethanol concentration. Considering energy consumption and cost, after comprehensive analysis, it is preferred to extract twice with 6 times 70% ethanol, 15 minutes per time, which has the best effect.

[0090] Experimental Example 3 Effect of different extraction solvents on the purity of colchicine extraction

[0091] In order to compare the changes in colchicine purity under the influence of different extraction solvents, after determining the Flash extraction conditions, the extraction solvent temperatures were set at 20°C, 25°C, 30°C, 35°C, and 40°C, respectively, and colchicine was used as an indicator to determine the purity of the extract.

[0092] Ingredients: Gloriosa seeds (purchased from India, batch number 20200927) were crushed and sieved into fine powder after removing impurities.

[0093] Extraction method (Flash extraction method): Take 100g of Gloriosa glabra seed powder, add 6 times volume (in g / mL) of 70% ethanol at different temperatures for extraction twice, each time for 15 minutes, filter, combine the filtrates and concentrate to dryness for later use.

[0094] Take appropriate amount of extracts respectively and determine the content of colchicine according to the method in Test Example 1, and calculate the purity. The results are shown in Table 4.

[0095] Table 4 Purity of colchicine extracted from Gloriosa glabra seeds with different extraction solvent temperatures.

[0096]

[0097]

[0098] It can be seen from Table 4 above that, under the same extraction conditions, the purity of the crude colchicine obtained when the extraction solvent temperature is 20-25° C. is higher. Therefore, it is determined that the extraction solvent temperature is 20-25° C. as a better solution.

[0099] In summary, as a preferred implementation scheme, specifically: after the Gloriosa seeds are crushed and sieved, 6 volumes (in g / mL) of 70% ethanol at 25°C are added, and the seeds are extracted twice with a Flash extractor, 15 minutes each time, and the filtrates are combined and concentrated to dryness after filtration.

[0100] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for extracting colchicine, characterized in that, The steps include: (1) Pretreatment: crush and sieve Gloriosa seeds; (2) Flash extraction: the pre-treated medicinal material is mixed with an extraction solvent, and extracted 2-3 times using a Flash extraction device, 5 minutes / time to 15 minutes / time; the weight volume ratio of the medicinal material to the extraction solvent is 1:5-1:12 in g / mL; the extraction solvent includes 60%-90% ethanol aqueous solution; the temperature of the extraction solvent is 20°C-40°C; (3) Concentration: Filter the extract, combine the filtrate, and concentrate to dryness for later use.

2. A method for extracting colchicine according to claim 1, characterized in that, In step (2), extraction is performed twice using a Flash extraction apparatus, with each extraction lasting 15 minutes.

3. A method for extracting colchicine according to claim 1, characterized in that, In step (2), the weight-to-volume ratio of the medicinal material raw material to the extraction solvent is 1:6-1:10 in g / mL.

4. A method for extracting colchicine according to claim 1, characterized in that, In step (2), the extraction solvent is 70% ethanol aqueous solution.

5. A method for extracting colchicine according to claim 1, characterized in that, In step (2), the temperature of the extraction solvent is 20°C-30°C.

6. A method for extracting colchicine according to claim 1, characterized in that, In step (2), the temperature of the extraction solvent is 20°C-25°C.

7. A method for extracting colchicine according to any one of claims 1 to 6, characterized in that: In step (1), after the Gloriosa Gloriosa seeds are crushed and sieved, the particle size of the medicinal material raw material is 180 μm±7.6 μm.

8. A method for extracting colchicine according to any one of claims 1 to 6, characterized in that: The steps include: (1) Pretreatment: crush and sieve Gloriosa seeds; (2) Flash extraction: After pretreatment, add 6 volumes of 70% ethanol at 25°C, calculated on a g / mL basis, and extract twice using a Flash extraction apparatus, 15 minutes per time; (3) Filter the extract, combine the filtrate, and concentrate to dryness for later use.

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