Method for extracting isolorydine from dactylicapnos scandens

Abstract

The invention provides a method for extracting isolorydine from dactylicapnos scandens. The extraction method comprises the following steps: drying and crushing roots of dactylicapnos scandens, adding the dactylicapnos scandens into an acidic solvent, heating, decocting and extracting for 2-4 times, concentrating the filtrate, adjusting the pH to be alkaline to obtain a crude dactylicapnos scandens extracting solution, and then adsorbing and desorbing through composite resin, and recrystallizing and purifying to obtain isolorydine. The extraction method is efficient, low in cost, low in equipment requirement and suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and in particular relates to a method for extracting isocollidine from Rhizoma Cyperi. Background Art

[0002] Purple golden dragon is a plant of the genus Purple golden dragon of the Papaveraceae family, also known as pea seven and Sichuan mountain seven. It is widely distributed in Sichuan (Mianning), Yunnan (Deqin in the northwest, west, central to southeast), western Guangxi and southeastern Tibet. It has analgesic, hemostatic, anti-inflammatory and blood pressure lowering effects. It is mainly used to treat various pains, hypertension, hemorrhage, internal bleeding, bruises, etc.

[0003] Pharmacological studies have shown that the alkaloids contained in Zijinlong are the main active ingredients of its analgesic effect. Some plant chemists have isolated more than a dozen alkaloids from Zijinlong, including papaverine, colidine, l-tetrahydropalmatine, isolidine, l-tetrahydroafricanamine, protoopine, sinomenine, palmatine, jatrorrhizine, dihydrosanguinarine, and magnolamine. However, the content of most of them is below 1%, while the content of isolidine is slightly higher and is mainly distributed in its roots.

[0004] Isoceridine belongs to the aporphine alkaloids, a class of alkaloids that are widely distributed in nature and have important pharmacological activities, such as antiplatelet aggregation activity, α-receptor blocking activity, calcium channel blocking activity, antioxidant activity, and central nervous system inhibitory activity, and has therefore been widely studied.

[0005] According to reports on the prior art, the main methods for extracting isocloidine from Zijinlong are: first, extracting the total alkaloids by soaking or reflux extraction with an alcohol solvent, and then obtaining isocloidine by precipitation, solvent extraction, or silica gel column chromatography. Although the operation method is simple, the product yield and quality are low, and the production cycle is long. Therefore, how to efficiently extract and purify isocloidine remains a difficult problem that needs to be solved.

[0006] In view of the problems existing in the prior art, how to provide a method for efficiently extracting isocollidine from Zijinlong is an urgent problem to be solved in the present invention. Summary of the Invention

[0007] The object of the present invention is to provide a method for extracting isocollidine from Zijinlong, so as to solve the problems of low extraction yield and poor product quality in the prior art.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for extracting isocollidine from Zijinlong, and the extraction method comprises the following steps:

[0010] (1) Dry and crush the root of Zijinlong, place it in a flask, add an acidic solvent, heat and boil it for 2-4 times, filter, combine the filtrate and concentrate it, adjust the pH to alkaline, and obtain a crude Zijinlong extract;

[0011] (2) Filling the composite resin into a chromatography column, loading the crude extract of Zijinlong onto the resin chromatography column, applying pressure to allow the crude extract to be adsorbed on the resin, and recirculating the effluent for adsorption until no isocoridine is detected in the effluent of the chromatography column by thin layer chromatography, and then stopping the circulation;

[0012] (3) eluting the resin chromatography column with an eluent to remove impurity liquids, collecting the effluent containing isocloidine, and post-processing to obtain isocloidine;

[0013] The composite resin comprises a macroporous adsorption resin and a modified adsorption resin.

[0014] As a further improvement, the modified adsorption resin is prepared by reacting LSA-21 macroporous resin and tetrachloromethane.

[0015] As a further improvement, the synthesis of the modified adsorption resin comprises the following steps:

[0016] (1) Add LSA-21 macroporous resin and anhydrous ethanol to a conical flask, seal and let stand for 20-24 hours, filter to remove the solvent, add the obtained solid to anhydrous ethanol, stir for 30-50 minutes, and then air dry to obtain the pretreated adsorption resin;

[0017] (2) The pretreated adsorption resin and tetrachloromethane were placed in a flask, refluxed and stirred at 50-70°C for 24-36 hours. After the reaction was completed, the flask was transferred to an ultrasonic cleaner, and then zinc chloride, sodium chloride, iron powder and chloromethyl methyl ether were added thereto, and ultrasonicated at 45-60°C for 10-15 hours;

[0018] (3) The mixture obtained in step (2) is filtered and rinsed with methanol and distilled water in sequence until no obvious white precipitate is generated when silver nitrate is added to the filtered distilled water, and then post-treated to obtain a modified adsorption resin.

[0019] As a further improvement, the weight ratio of the macroporous adsorption resin to the modified adsorption resin is 1.5-3:1.

[0020] As a further improvement, the acidic solvent in step (1) is at least one of hydrochloric acid, sulfuric acid, acetic acid, and tartaric acid.

[0021] As a further improvement, the alkaline pH value in step (1) is 8-10.

[0022] As a further improvement, the macroporous adsorption resin model is at least one of D101, DM-130, WLD-3, HPD750, XDA-1, LSD001, ADS-17, DM-130, and YWD01F.

[0023] As a further improvement, the macroporous adsorption resin model is at least one of D101, WLD-3, and DM-130.

[0024] As a further improvement, the loading amount of the crude extract of Zijinlong in step (2) is 0.5-1 times the product of the diameter and height of the composite resin in the chromatography column.

[0025] As a further improvement, the eluent is at least one of water, methanol, ethanol, acetone, and chloroform.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The method for extracting isocollidine from Zijinlong provided by the present invention has higher product yield and product quality, and the extraction method of the present invention is efficient, low-cost, has low requirements on equipment, and is suitable for large-scale production. DETAILED DESCRIPTION

[0028] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following examples are illustrative of the present invention and are intended only to illustrate the present invention and are not intended to limit the present invention. Other combinations and various modifications within the scope of the present invention may be made without departing from the spirit or scope of the present invention.

[0029] In the following examples, except for the modified adsorption resin, the other compound monomers and related reagents used can be purchased from the market. Among them, Zijinlongyuan is cultivated in the wild by our company's planting base, LSA-21 macroporous resin is purchased from Beijing Ruida Henghui Technology Development Co., Ltd. with the item number HA-0946, and D101, WLD-3, and DM-130 macroporous adsorption resins are purchased from Zhengzhou Qinshi Technology Co., Ltd.

[0030] The synthesis of the modified adsorption resin comprises the following steps:

[0031] (1) Add 20 g of LSA-21 macroporous resin and 100 mL of anhydrous ethanol to a conical flask, seal it and let it stand for 20 h, filter it to remove the solvent, and add the resulting solid to 100 mL of anhydrous ethanol and stir for 50 min at a stirring speed of 300 rpm. After stirring, air dry it naturally to obtain the pretreated adsorption resin;

[0032] (2) 10 g of pretreated adsorption resin and 40 mL of tetrachloromethane were placed in a flask, refluxed and stirred at 65 °C for 24 h. After the reaction was completed, the flask was transferred to an ultrasonic cleaner, and then 4.1 g of zinc chloride, 12 g of sodium chloride, 0.2 g of iron powder, and 10 mL of chloromethyl methyl ether were added thereto, and ultrasonicated at 50 °C for 12 h.

[0033] (3) The mixture obtained in step (2) is filtered, and rinsed with methanol and distilled water in sequence until no obvious white precipitate is generated when silver nitrate is added to the filtered distilled water, and then dried at 120° C. to obtain a modified adsorption resin.

[0034] The activation treatment of D101, WLD-3, and DM-130 macroporous adsorption resins includes the following steps:

[0035] Weigh 100 g of the macroporous adsorption resin with a dry weight respectively, put it into a tea bag made of a 200-mesh filter, soak it in 300 mL of ethanol for 24 hours, discard the soaking liquid, and add an appropriate amount of ethanol for washing until no white turbidity appears when 300 mL of distilled water is added. Then, wash the resin repeatedly with an appropriate amount of distilled water until no ethanol is present, thereby obtaining the activated macroporous adsorption resin.

[0036] The preparation method of Example 1 comprises the following steps:

[0037] (1) Dry and crush the root of Zijinlong, take 20 g and place it in a flask, add 50 mL of 1% by mass sulfuric acid aqueous solution, heat and boil at 100°C for 1 hour, extract three times, filter, combine the filtrate and concentrate to 50 mL, adjust the pH to 9 with 1 mol / L sodium hydroxide solution to obtain a crude Zijinlong extract;

[0038] (2) 65 g of activated WLD-3 macroporous adsorption resin and 35 g of modified adsorption resin were mixed to obtain a composite resin, which was filled into a chromatography column (diameter-to-height ratio of 5 cm:30 cm) with a filling height of 10 cm. The chromatography column was filtered and rinsed with distilled water, and then 50 mL of the crude extract of Zijinlong was loaded onto the resin chromatography column. The column was pressurized to allow the extract to adsorb on the resin, and the effluent was recycled for sample adsorption until no isocoridine was detected in the effluent of the chromatography column in thin layer chromatography (compared with an isocoridine standard sample, the developing solvent was chloroform:methanol:ammonia water = 50:1:0.5), and the circulation was stopped;

[0039] (3) First, 200 mL of water was used as the eluent to elute the chromatography column, and the effluent was discarded; then 200 mL of a mixed solution of ethanol and water (volume ratio of 60:40) was used as the eluent for washing for the second time, and the effluent was discarded; finally, 100 mL of a mixed solution of ethanol and water (volume ratio of 70:30) and the pH was adjusted to 5 with hydrochloric acid was used as the eluent for washing for the third time, the effluent was collected, the pH was adjusted to 7 with ammonia water, and the solvent was removed by rotary evaporation to obtain 0.265 g of crude isocloidine, which was detected by HPLC to have a purity of 90.5%. It was then recrystallized from ethanol to obtain isocloidine with a purity of 96%.

[0040] The preparation method of Example 2 comprises the following steps:

[0041] (1) Dry and crush the root of Zijinlong, take 20 g and place it in a flask, add 50 mL of 1% by mass sulfuric acid aqueous solution, heat and boil at 100°C for 1 hour, extract three times, filter, combine the filtrate and concentrate to 50 mL, adjust the pH to 9 with 1 mol / L sodium hydroxide to obtain a crude Zijinlong extract;

[0042] (2) 70 g of activated D101 macroporous adsorption resin and 30 g of modified adsorption resin were mixed to obtain a composite resin, which was filled into a chromatography column (diameter-to-height ratio of 5 cm:30 cm) with a filling height of 10 cm. The chromatography column was filtered and rinsed with distilled water, and then 50 mL of the crude extract of Zijinlong was loaded onto the resin chromatography column. The column was pressurized to allow the extract to adsorb on the resin, and the effluent was recycled for adsorption until no isocoridine was detected in the effluent of the chromatography column in thin layer chromatography (compared with an isocoridine standard sample, the developing solvent was chloroform:methanol:ammonia water = 50:1:0.5), and the circulation was stopped;

[0043] (3) First, 200 mL of water was used as the eluent to elute the chromatography column, and the effluent was discarded; then 200 mL of a mixed solution of ethanol and water (volume ratio of 60:40) was used as the eluent for washing for the second time, and the effluent was discarded; finally, 100 mL of a mixed solution of ethanol and water (volume ratio of 70:30) and the pH was adjusted to 5 with hydrochloric acid was used as the eluent for washing for the third time, the effluent was collected, the pH was adjusted to 7 with ammonia water, and the solvent was removed by rotary evaporation to obtain 0.272 g of crude isocloidine, which was detected by HPLC to have a purity of 93.3%. It was then recrystallized from ethanol to obtain isocloidine with a purity of 98%.

[0044] The preparation method of Example 3 comprises the following steps:

[0045] (1) Dry and crush the root of Zijinlong, take 20 g and place it in a flask, add 50 mL of 1% by mass sulfuric acid aqueous solution, heat and boil at 100°C for 1 hour, extract three times, filter, combine the filtrate and concentrate to 50 mL, adjust the pH to 9 with 1 mol / L sodium hydroxide to obtain a crude Zijinlong extract;

[0046] (2) 74 g of activated DM-130 macroporous adsorption resin and 26 g of modified adsorption resin were mixed to obtain a composite resin, which was filled into a chromatography column (diameter-to-height ratio of 5 cm:30 cm) with a filling height of 10 cm. The chromatography column was filtered and rinsed with distilled water, and then 50 mL of the crude extract of Zijinlong was loaded onto the resin chromatography column. The column was pressurized to allow the extract to adsorb on the resin, and the effluent was recycled for sample adsorption until no isocoridine was detected in the effluent of the chromatography column in thin layer chromatography (compared with an isocoridine standard sample, the developing solvent was chloroform:methanol:ammonia water = 50:1:0.5), and the circulation was stopped;

[0047] (3) First, 200 mL of water was used as the eluent to elute the chromatography column, and the effluent was discarded; then 200 mL of a mixed solution of ethanol and water (volume ratio of 60:40) was used as the eluent for washing for the second time, and the effluent was discarded; finally, 100 mL of a mixed solution of ethanol and water (volume ratio of 70:30) and the pH was adjusted to 5 with hydrochloric acid was used as the eluent for washing for the third time, the effluent was collected, the pH was adjusted to 7 with ammonia water, and the solvent was removed by rotary evaporation to obtain 0.260 g of crude isocloidine, which was detected by HPLC to have a purity of 92.0%. It was then recrystallized from ethanol to obtain isocloidine with a purity of 96%.

[0048] The preparation method of Comparative Example 1 comprises the following steps:

[0049] (1) Dry and crush the root of Zijinlong, take 20 g and place it in a flask, add 50 mL of 1% by mass sulfuric acid aqueous solution, heat and boil at 100°C for 1 hour, extract three times, filter, combine the filtrate and concentrate to 50 mL, adjust the pH to 9 with 1 mol / L sodium hydroxide to obtain a crude Zijinlong extract;

[0050] (2) Fill the activated D101 macroporous adsorption resin into a chromatography column (diameter-to-height ratio of 5 cm:30 cm) with a filling height of 10 cm. Filter and rinse the chromatography column with distilled water, then load 50 mL of the crude extract of Zijinlong onto the resin chromatography column and pressurize it to allow the extract to adsorb on the resin. The effluent is recycled and adsorbed until no isocoridine is detected in the effluent of the chromatography column by thin layer chromatography (compared with an isocoridine standard sample, the developing solvent is chloroform:methanol:aqueous ammonia = 50:1:0.5), and then stop the circulation;

[0051] (3) First, 200 mL of water was used as the eluent to elute the chromatography column, and the effluent was discarded; then 200 mL of a mixed solution of ethanol and water (volume ratio of 60:40) was used as the eluent for washing for the second time, and the effluent was discarded; finally, 100 mL of a mixed solution of ethanol and water (volume ratio of 70:30) and the pH was adjusted to 5 with hydrochloric acid was used as the eluent for washing for the third time, the effluent was collected, the pH was adjusted to 7 with ammonia water, and the solvent was removed by rotary evaporation to obtain 0.192 g of crude isocloidine, which was detected by HPLC to have a purity of 85.2%. It was then recrystallized from ethanol to obtain isocloidine with a purity of 93%.

[0052] The preparation method of Comparative Example 2 comprises the following steps:

[0053] (1) Dry and crush the root of Zijinlong, take 20 g and place it in a flask, add 50 mL of 1% by mass sulfuric acid aqueous solution, heat and boil at 100°C for 1 hour, extract three times, filter, combine the filtrate and concentrate to 50 mL, adjust the pH to 9 with 1 mol / L sodium hydroxide to obtain a crude Zijinlong extract;

[0054] (2) 50 g of activated D101 macroporous adsorption resin and 50 g of modified adsorption resin were mixed to obtain a composite resin, which was filled into a chromatography column (diameter-to-height ratio of 5 cm:30 cm) with a filling height of 10 cm. The chromatography column was filtered and rinsed with distilled water, and then 50 mL of the crude extract of Zijinlong was loaded onto the resin chromatography column. The column was pressurized to allow the extract to adsorb on the resin, and the effluent was recycled for sample adsorption until no isocoridine was detected in the effluent of the chromatography column in thin layer chromatography (compared with an isocoridine standard sample, the developing solvent was chloroform:methanol:ammonia water = 50:1:0.5), and the circulation was stopped;

[0055] (3) First, 200 mL of water was used as the eluent to elute the chromatography column, and the effluent was discarded; then 200 mL of a mixed solution of ethanol and water (volume ratio of 60:40) was used as the eluent for washing for the second time, and the effluent was discarded; finally, 100 mL of a mixed solution of ethanol and water (volume ratio of 70:30) and the pH was adjusted to 5 with hydrochloric acid was used as the eluent for washing for the third time, the effluent was collected, the pH was adjusted to 7 with ammonia water, and the solvent was removed by rotary evaporation to obtain 0.205 g of crude isocloidine, which was detected by HPLC to have a purity of 87.7%. It was then recrystallized from ethanol to obtain isocloidine with a purity of 93%.

[0056] The preparation method of Comparative Example 3 comprises the following steps:

[0057] (1) Dry and crush the root of Zijinlong, take 20 g and place it in a flask, add 150 mL of ethanol, reflux at 65°C for 1 hour, extract twice, filter, combine the filtrates and concentrate to dryness, add 50 mL of distilled water to dissolve, and adjust the pH to 9 with 1 mol / L sodium hydroxide to obtain a crude Zijinlong extract;

[0058] (2) 70 g of activated D101 macroporous adsorption resin and 30 g of modified adsorption resin were mixed to obtain a composite resin, which was filled into a chromatography column (diameter-to-height ratio of 5 cm:30 cm) with a filling height of 10 cm. The chromatography column was filtered and rinsed with distilled water, and then 50 mL of the crude extract of Zijinlong was loaded onto the resin chromatography column. The column was pressurized to allow the extract to adsorb on the resin, and the effluent was recycled for adsorption until no isocoridine was detected in the effluent of the chromatography column in thin layer chromatography (compared with an isocoridine standard sample, the developing solvent was chloroform:methanol:ammonia water = 50:1:0.5), and the circulation was stopped;

[0059] (3) First, 200 mL of water was used as the eluent to elute the chromatography column, and the effluent was discarded; then 200 mL of a mixed solution of ethanol and water (volume ratio of 60:40) was used as the eluent for washing for the second time, and the effluent was discarded; finally, 100 mL of a mixed solution of ethanol and water (volume ratio of 70:30) and the pH was adjusted to 5 with hydrochloric acid was used as the eluent for washing for the third time, the effluent was collected, the pH was adjusted to 7 with ammonia water, and the solvent was removed by rotary evaporation to obtain 0.175 g of crude isocloidine, which was detected by HPLC to have a purity of 80.0%. It was then recrystallized from ethanol to obtain isocloidine with a purity of 90%.

[0060] From the comparison between Example 2 and Comparative Examples 1-2, it can be seen that the yield and purity of isocolidine extracted from Zijinlong by the composite resin obtained by mixing the macroporous adsorption resin and the modified adsorption resin in proportion are better than the yield and purity when the macroporous adsorption resin is used alone or the weight ratio of the macroporous adsorption resin to the modified adsorption resin is not within the appropriate range.

[0061] From the comparison between Example 2 and Comparative Example 3, it can be seen that the yield and purity of isocolidine obtained by the method of decocting the crude extract of Zijinlong by acidic solvent and then extracting isocolidine from the crude extract of Zijinlong are better than those obtained by conventional methods, such as the alcohol solvent reflux method.

[0062] It can be seen from the test results of Examples 1-3 that the method for extracting isocollidine from Zijinlong provided by the present invention can obtain higher product yield and product purity, and the operation method is simple, the process requirements are low, and it is suitable for large-scale production.

[0063] The above embodiments are only for illustrating the technical concept and features of the present invention. Its purpose is to enable people familiar with this technology to understand the content of the present invention and implement it. It cannot be used to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting isocollidine from Zijinlong, characterized in that: The extraction method comprises the following steps: (1) Dry and crush the root of Zijinlong, place it in a flask, add an acidic solvent, heat and boil it for 2-4 times, filter, combine the filtrate and concentrate, adjust the pH to alkaline, and obtain a crude Zijinlong extract; (2) Filling the composite resin into a chromatography column, loading the crude extract of Zijinlong onto the resin chromatography column, applying pressure to allow the crude extract to be adsorbed on the resin, and recirculating the effluent for adsorption until no isocoridine is detected in the effluent of the chromatography column by thin layer chromatography, and then stopping the circulation; (3) eluting the resin chromatography column with an eluent to remove impurity liquids, collecting the effluent containing isocloidine, and post-processing to obtain isocloidine; The composite resin comprises a macroporous adsorption resin and a modified adsorption resin.

2. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The modified adsorption resin is prepared by reacting LSA-21 macroporous resin and tetrachloromethane.

3. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The synthesis of the modified adsorption resin comprises the following steps: (1) Add LSA-21 macroporous resin and anhydrous ethanol to a conical flask, seal and let stand for 20-24 hours, filter to remove the solvent, add the obtained solid to anhydrous ethanol, stir for 30-50 minutes, and then air dry to obtain the pretreated adsorption resin; (2) The pretreated adsorption resin and tetrachloromethane were placed in a flask, refluxed and stirred at 50-70°C for 24-36 hours. After the reaction was completed, the flask was transferred to an ultrasonic cleaner, and then zinc chloride, sodium chloride, iron powder and chloromethyl methyl ether were added thereto, and ultrasonicated at 45-60°C for 10-15 hours; (3) The mixture obtained in step (2) is filtered and rinsed with methanol and distilled water in sequence until no obvious white precipitate is generated when silver nitrate is added to the filtered distilled water, and then post-treated to obtain a modified adsorption resin.

4. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The weight ratio of the macroporous adsorption resin to the modified adsorption resin is 1.5-3:

1.

5. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The acidic solvent in step (1) is at least one of hydrochloric acid, sulfuric acid, acetic acid, and tartaric acid.

6. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The alkaline pH value in the step (1) is 8-10.

7. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The macroporous adsorption resin model is at least one of D101, DM-130, WLD-3, HPD750, XDA-1, LSD001, ADS-17, DM-130, and YWD01F.

8. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The macroporous adsorption resin model is at least one of D101, WLD-3, and DM-130.

9. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The ratio of the loading amount of the crude extract of Zijinlong in step (2) to the product of the diameter and height of the composite resin in the chromatography column is 0.5-1 mL / cm 2 .

10. The method for extracting isocollidine from Zijinlong according to claim 1, characterized in that: The eluent is at least one of water, methanol, ethanol, acetone and chloroform.