A method for recrystallization purification of gibberellin GA7

Abstract

The present invention discloses a method for recrystallization purification of gibberellin GA7, which relates to the field of production and extraction of plant growth regulators. In this method, the crude gibberellin GA7 is dissolved in ethanol, and after filtration, a gibberellin GA7 solution is obtained. The GA7 solution is decolorized with activated carbon and then filtered to obtain a decolorized gibberellin GA7 solution. After adjusting the pH of the decolorized gibberellin GA7 solution with hydrochloric acid, deionized water is slowly added until the fine powder of gibberellin GA7 no longer precipitates. After standing, filtration and drying are carried out. The mother liquor contains almost no gibberellin GA7. At the same time, after the mother liquor recovers ethanol, it is basically all water and does not require environmental protection treatment. This method is simple to operate, safe and environmentally friendly, has a short process flow, is easy to implement, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of production and extraction of plant growth regulators, and specifically to a method for recrystallization and purification of gibberellin GA7. Background Art

[0002] Gibberellin is one of the six major hormones of plants, which mainly has various physiological functions on plant growth and can regulate processes such as plant rooting, germination, growth, flowering, and fruiting. However, there are many types of gibberellin, and the most widely used one in the market is gibberellin GA3. In recent years, due to limitations such as the too strong physiological activity and single effect of gibberellin GA3. The mixture GA4+7 of gibberellin GA4 and GA7 also has relatively high physiological activity and has obvious effects on the preservation of fruits and vegetables and flowers. While the physiological activity of gibberellin GA7 is slightly lower, it has good effects on thinning flowers and fruits of stone fruit plants and improving fruit quality. Therefore, gibberellin GA7 has become a research hotspot in the agricultural field.

[0003] Currently, almost all commercially available gibberellin on the market is obtained by microbial fermentation. A small amount of gibberellin GA4 will be produced during the fermentation of gibberellin GA7. At the same time, there are also isomers of gibberellin GA7, as well as a large amount of proteins, sugars, and other organic impurities, which are difficult to remove during the extraction process. Currently, the main extraction method of gibberellin GA7 is the Japanese Patent No. 59106478 in 1982. The purity of the extracted gibberellin GA7 by this method is between 70-82%, and it also contains a large amount of impurities, resulting in the extracted gibberellin GA7 being dark in color and slightly yellowish. Moreover, the finally obtained crystalline powder still contains isomers of gibberellin GA7, which are difficult to remove by traditional purification methods, and this increasingly does not meet the higher requirements for the content of gibberellin GA7 in the market. Summary of the Invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and provide a method for recrystallization and purification of gibberellin GA7.

[0005] The technical solution of the present invention is as follows:

[0006] A method for recrystallization and purification of gibberellin GA7, which comprises the following steps:

[0007] (1) Dissolution: Dissolution of the crude gibberellin GA7: The crude gibberellin GA7 is dissolved in an organic solvent to obtain a GA7 solution.

[0008] (2) Decolorization: Perform decolorization treatment on the GA7 solution.

[0009] (3) Filtration: Filter the decolorized GA7 solution and collect the filtrate.

[0010] (4) pH adjustment: Hydrochloric acid is used to adjust the pH of the decolorized GA7 solution.

[0011] (5) Crystallization: A certain amount of deionized water is added to the GA7 filtrate after decolorization, and the mixture is left to stand until the filtrate is completely crystallized.

[0012] (6) Filtration and drying: After the material liquid is completely crystallized, a filtration operation is carried out to obtain fine GA7 powder, which is then dried, weighed, its content is measured, and the yield of recrystallization is calculated.

[0013] (7) Solvent recovery: The mother liquor after filtration is subjected to solvent recovery. After recovering to a certain volume, a sample is taken to measure the titer of the mother liquor. At this time, the mother liquor is all deionized water, and at the same time, there is no gibberellin GA7, so it can be directly discharged for environmental protection treatment.

[0014] Preferably, in step (1), the organic solvent used is ethanol, and the volume-to-mass ratio thereof to the crude product is 5 - 8:1.

[0015] Preferably, in step (2), the decolorization treatment uses activated carbon, and the dosage is 0.25 - 0.5% of the volume of the GA7 solution.

[0016] Preferably, in step (4), the pH adjustment range is between 1.4 and 1.7, and a 1:4 hydrochloric acid solution (volume ratio) is used.

[0017] Preferably, in step (5), the amount of deionized water added is in a ratio of 2 - 4:1 to the volume of the filtrate.

[0018] Preferably, in step (6), the drying equipment is a vacuum drying oven or a double-cone dryer, the equipment for content detection is a high-performance liquid chromatograph, and at the same time, the formula for calculating the yield is yield = (mass of crystalline powder × content) / (mass of crude product × content).

[0019] Preferably, in step (7), the solvent recovery temperature is controlled at 55 - 65°C, the vacuum degree is controlled at -0.08 to -0.10 MPa. The concentration equipment is a rotary evaporator or a vacuum concentration evaporator.

[0020] Advantages of the present invention:

[0021] The present invention is easy to operate and implement. It uses deionized water for crystallization without the need to use other organic solvents, which is both safe and environmentally friendly. At the same time, it has a large processing capacity and is suitable for industrial production. After ethanol recovery, it can be reused. There is no waste generation. The mother liquor after ethanol recovery has no product residue and is basically all water, which can be directly discharged for environmental protection treatment. Using the present invention for the purification of gibberellin GA7, the process flow is simple, safe and environmentally friendly, with a large processing capacity, very suitable for industrial production, and the content of the obtained gibberellin GA7 crystal powder can reach more than 90%, and the yield of recrystallization purification can also reach more than 85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the process flow chart of the present invention;

[0023] Figure 2 The chromatogram of the product recrystallized and purified using the acetone - water system;

[0024] Figure 3 is the chromatogram of the product before purification in the industrial example;

[0025] Figure 4 is the chromatogram of the product after purification in the industrial example. DETAILED DESCRIPTION OF THE INVENTION

[0026] The technical solution of the present invention will be further described below with specific examples and drawings.

[0027] Before the examples, the applicant screened the solvent combinations for recrystallization purification. The specific experimental steps are as follows: Solubility tests were carried out on the crude GA7 at room temperature (25°C), and the commonly used solvents in production were screened. The solvents with high solubility and those with low solubility were classified and combined to screen out the best recrystallization solvent combination. The specific data are shown in the following table:

[0028] Table 1. Solubility of GA7 crystal powder in solvents at room temperature

[0029]

[0030]

[0031] It can be seen from the table that at room temperature, the solubility of ethanol is the largest, followed by acetone, while petroleum ether and water are almost insoluble at room temperature. Therefore, the ethanol - water system, ethanol - petroleum ether, methyl tert - butyl ether - petroleum ether, ethyl acetate - petroleum ether, and acetone - water system combinations with better and poorer solubility were used to recrystallize the crystal powder, and the experimental results are as follows:

[0032] Table 2. Yield of GA7 recrystallization under various solvent combinations

[0033]

[0034] Note: The total billion is the mass of the active substance, and the billion is the unit of the total billion.

[0035] As can be seen from the table, when using the ethanol-water system for recrystallization, the yield can reach 83.22%. For other solvent combinations, only the acetone-water system has a slightly higher yield, reaching 69.22%. Therefore, this application selects the ethanol-water system as the best solvent combination for recrystallization. Flow chart reference Figure 1 。

[0036] Example 1:

[0037] 1) Dissolution of crude gibberellin GA7

[0038] Take 10.60 g of crude gibberellin GA7 with a content of 82.6% and dissolve it in 53.6 ml of ethanol at a ratio of 1:5 (mass of gibberellin GA7 to volume of ethanol). Stir for 10 min. After the crude product is completely dissolved, proceed to the next step.

[0039] 2) Decolorization

[0040] Add 0.25% activated carbon, i.e., 0.134 g, to the dissolved solution, stir for 5 min, and then let it stand for 10 min.

[0041] 3) Filtration

[0042] After the decolorization process is completed, vacuum filter the dissolved solution to obtain 49.7 ml of clear and transparent filtrate. The titer is 172564 ug / ml. The solid residue is treated environmentally, and the dissolved solution enters the next treatment.

[0043] 4) pH adjustment

[0044] Slowly add a 1:4 hydrochloric acid solution to the dissolved solution, measuring the pH of the dissolved solution while adding to make the pH of the dissolved solution reach 1.54.

[0045] 5) Crystallization

[0046] Slowly add ethanol with a volume ratio of 2:1 to the filtrate from the previous step, i.e., 99.4 ml, while stirring. After the ethanol is added, continue to stir slowly for 10 min, and then let it stand for 60 min until crystallization is complete.

[0047] 6) Filtration and drying

[0048] After crystallization is complete, perform a filtration operation using vacuum filtration. After filtration, take out the crystalline powder and place it in a vacuum drying oven for drying. After drying is completed, weigh it to obtain a crystalline powder mass of 8.43 g and a content of 91.5%. Therefore, the yield = (8.43×91.5%) / (10.60×82.6%) = 88.1%.

[0049] 7) Solvent recovery

[0050] 134.2 ml of the mother liquor was concentrated under vacuum to recover the solvent. The recovery temperature was controlled at about 55 °C, and the vacuum degree was controlled at -0.085 MPa. When the liquid droplets slowly dripped into the recovery bottle or recovery tank, the concentration was stopped. The volume of the recovered ethanol was recorded as 81.7 ml. The recovered ethanol could be reused. The titer of the mother liquor was 12 μg / ml, and the mother liquor was discharged for environmental protection treatment.

[0051] Example 2:

[0052] 1) Dissolution of crude gibberellin GA7

[0053] 20.7 g of crude gibberellin GA7 with a content of 76.89% was taken and dissolved in 124.5 ml of ethanol at a ratio of 1:6 (mass of gibberellin GA7 to volume of ethanol). After stirring for 10 min and complete dissolution of the crude product, filtration was carried out.

[0054] 2) Decolorization

[0055] 0.436 g of activated carbon, which was 0.35% of the dissolved solution, was added to the dissolved solution, stirred for 5 min, and left standing for 10 min.

[0056] 3) Filtration

[0057] After the decolorization process was completed, the dissolved solution was vacuum filtered to obtain 119.7 ml of clear and transparent filtrate with a titer of 131267 μg / ml. The solid residue was treated for environmental protection, and the dissolved solution entered the next step of treatment.

[0058] 4) pH adjustment

[0059] A 1:4 hydrochloric acid solution was slowly added to the dissolved solution while measuring the pH of the dissolved solution to make the pH of the dissolved solution reach 1.63.

[0060] 5) Crystallization

[0061] 360.0 ml of ethanol with a volume ratio of 3:1 to the filtrate volume was slowly added to the filtrate in the previous step while stirring. After the addition of ethanol was completed, it was slowly stirred for 10 min and then left standing for 60 min until crystallization was complete.

[0062] 6) Filtration and drying

[0063] After crystallization was complete, a filtration operation was carried out. Vacuum filtration was used for filtration. After filtration, the crystalline powder was taken out and placed in a vacuum drying oven for drying. After drying was completed, the mass of the crystalline powder was weighed and found to be 15.22 g with a content of 90.7%. Therefore, the recovery rate = (15.22×90.7%) / (20.7×76.89%) = 86.7%.

[0064] 7) Solvent recovery

[0065] 448.2 ml of the mother liquor was concentrated under vacuum to recover the solvent. The recovery temperature was controlled at about 60 °C, and the vacuum degree was controlled at -0.090 MPa. When the liquid droplets slowly dripped into the recovery bottle or recovery tank, the concentration was stopped. The volume of the recovered ethanol was recorded as 92.4 ml. The recovered ethanol could be reused. The titer of the mother liquor was 10 μg / ml, and the mother liquor was discharged for environmental protection treatment.

[0066] Example 3:

[0067] 1) Dissolution of crude gibberellin GA7

[0068] 40.3 g of crude gibberellin GA7 with a content of 81.89% was dissolved in ethanol at a ratio of 1:8 (mass of gibberellin GA7 to volume of ethanol), that is, dissolved in 322.4 ml of ethanol. Stir for 10 min. After the crude product was completely dissolved, filtration was carried out.

[0069] 2) Decolorization

[0070] 0.5% of activated carbon, that is, 1.61 g, was added to the dissolved solution and stirred for 5 min, then left standing for 10 min.

[0071] 3) Filtration

[0072] After the decolorization process was completed, the dissolved solution was vacuum filtered to obtain 312.5 ml of clear and transparent filtrate with a titer of 103457 μg / ml. The solid residue was treated for environmental protection, and the dissolved solution entered the next step of treatment.

[0073] 4) pH adjustment

[0074] A 1:4 hydrochloric acid solution was slowly added to the dissolved solution while measuring the pH of the dissolved solution to make the pH of the dissolved solution reach 1.49.

[0075] 5) Crystallization

[0076] Deionized water with a volume ratio of 4:1 to the filtrate, that is, 1250.0 ml, was slowly added to the filtrate in the previous step while stirring. After the deionized water was added, it was slowly stirred for 10 min and then left standing for 60 min until crystallization was complete.

[0077] 6) Filtration and drying

[0078] After the crystallization is complete, perform a filtration operation. Vacuum filtration is used for filtration. After the filtration is completed, take out the crystalline powder and place it in a vacuum drying oven for drying. After drying is completed, weigh it and obtain that the mass of the crystalline powder is 30.4 g, and the content is 93.2%. Therefore, the yield = (30.4×93.2%) / (40.3×81.89%) = 85.9%.

[0079] 7) Solvent recovery

[0080] Perform vacuum concentration on 1123.8 ml of mother liquor to recover the solvent. Control the recovery temperature at about 65 °C and the vacuum degree at -0.095 MPa. Stop concentration when the liquid droplets slowly drip into the recovery bottle or recovery tank. Record that the volume of the recovered ethanol is 256.2 ml. The recovered ethanol can be reused. The titer of the mother liquor is 8 ug / ml, and the mother liquor is discharged for environmental protection treatment.

[0081] Example 4:

[0082] 1) Dissolution of crude gibberellin GA7

[0083] Take 11.26 g of crude gibberellin GA7 with a content of 81.7% and dissolve it in 1:8 (mass ratio of gibberellin GA7 to volume of acetone), that is, 90.0 ml of acetone. Stir for 10 min. After the crude product is completely dissolved, proceed to the next step.

[0084] 2) Decolorization

[0085] Add 0.25% activated carbon, that is, 0.225 g, to the dissolved solution. Stir for 5 min and then let it stand for 10 min.

[0086] 3) Filtration

[0087] After the decolorization process is completed, perform vacuum filtration on the dissolved solution to obtain 88.4 ml of clear and transparent filtrate with a titer of 104036 ug / ml. The solid residue is subjected to environmental protection treatment, and the dissolved solution enters the next treatment.

[0088] 4) pH adjustment

[0089] Slowly add a 1:4 hydrochloric acid solution to the dissolved solution, and measure the pH of the dissolved solution while adding to make the pH of the dissolved solution reach 1.52.

[0090] 5) Crystallization

[0091] Slowly add acetone with a volume ratio of 3:1 to the filtrate from the previous step, that is, 176.8 ml, while stirring. After the addition of acetone is complete, stir slowly for 10 min and then let it stand for 60 min until crystallization is complete.

[0092] 6) Filtration and drying

[0093] After the crystallization is complete, perform a filtration operation. Vacuum filtration is used for filtration. After the filtration is completed, take out the crystalline powder and place it in a vacuum drying oven for drying. After drying is completed, weigh it and obtain the mass of the crystalline powder as 6.32 g, and the content is 86.5%. Therefore, the yield = (6.32×86.5%) / (11.26×83.7%) = 59.4%.

[0094] 7) Solvent recovery

[0095] Vacuum concentrate 243.7 ml of the mother liquor to recover the solvent. Control the recovery temperature at about 55 °C and the vacuum degree at -0.085 MPa. When the liquid droplets slowly drip into the recovery bottle or recovery tank, stop the concentration. Record the volume of the recovered ethanol as 76.5 ml. The recovered acetone can be reused. The titer of the mother liquor is 102 μg / ml, and the mother liquor is discharged for environmental protection treatment. The liquid chromatogram of the product in this example (as shown in Figure 2 ) shows that the peak area ratio of the impurity peaks is still 4.152%, and the content of GA7 is only 86.5%. The yield of this recrystallization is only 59.4%, which further shows that acetone-water is not the best solvent combination for purification.

[0096] Example 5: Industrial example

[0097] 1) Dissolution of crude gibberellin GA7

[0098] Take 121.82 kg of crude GA7 (the chromatogram is as shown in Figure 3 ) with a content of 83.81%. Dissolve it in ethanol with a ratio of 1:6 (the mass ratio of gibberellin GA7 to the volume of ethanol), that is, dissolve it in 731 L of ethanol and start stirring. Stir for 10 min. After the crude product is completely dissolved, perform a decolorization treatment.

[0099] 2) Decolorization

[0100] Add 0.25% activated carbon, that is, 1827 g, to the dissolved solution, stir for 5 min, and let it stand for 20 min.

[0101] 3) Filtration

[0102] After the decolorization process is completed, use nitrogen to press the gibberellin GA4+7 solution into the crystallization tank. During this process, the gibberellin GA7 solution will flow through a filter with a filter element for filtration. Finally, obtain 726 L of clear and transparent filtrate with a titer of 140630 μg / ml. The solid residue is treated for environmental protection, and the solution enters the next step of treatment.

[0103] 4) pH adjustment

[0104] Slowly add a 1:4 hydrochloric acid solution to the solution in the crystallization tank, and measure the pH of the solution while adding to make the pH of the solution reach 1.52.

[0105] 5) Crystallization

[0106] Open the valve of the high-level tank of the deionized water storage tank, and slowly add 2178 L of deionized water, whose volume ratio to the filtrate in the previous step is 3:1, to the filtrate. Start stirring, stir while adding, and after the petroleum ether is added completely, continue to stir slowly for 10 min, and then let it stand for 60 min until crystallization is complete.

[0107] 6) Filtration and drying

[0108] After crystallization is complete, perform a filtration operation. Centrifugal filtration is used for filtration. After filtration, take out the crystalline powder and place it in a double-cone dryer for drying. After drying is completed, weigh it, and the mass of the crystalline powder obtained is 100.5 kg, and the content is 92.6%. The specific liquid chromatogram is as Figure 4 shown. Therefore, the yield = (100.5×92.6%) / (121.82×83.81%) = 91.1%.

[0109] 7) Solvent recovery:

[0110] Pump 2756 L of mother liquor into the solvent recovery tank for solvent recovery. The recovery temperature is controlled at about 60 °C, and the vacuum degree is controlled at -0.090 MPa. Stop the recovery when the liquid drops slowly drip into the recovery tank. Record the volume of the recovered ethanol as 627 L. The recovered ethanol can be reused, and the titer of the mother liquor after recovery is 25 μg / ml. The mother liquor is discharged for environmental protection treatment.

[0111] The titer of the mother liquor recovered in the above examples is very low, which basically indicates that the mother liquor is almost water and can be directly discharged for environmental protection treatment. The process flow of the present invention is simple, safe and environmentally friendly, has a large processing capacity, is very suitable for industrial production, and the content of the gibberellin GA7 crystalline powder obtained can reach more than 90%, and the yield of recrystallization purification can also reach more than 85%.

[0112] The specific embodiments described herein are merely illustrative of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, but will not deviate from the scope of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for recrystallization purification of gibberellin GA7, characterized in that, It includes the following steps: (1) Dissolve the crude gibberellin GA7 with an organic solvent to obtain a GA7 solution; (2) Decolorize the GA7 solution; (3) Filter the GA7 solution after the decolorization treatment in step (2) and collect the filtrate; (4) Adjust the pH of the filtrate to obtain a GA7 feed solution; (5) Add a certain amount of deionized water to the GA7 feed solution and let it stand until the GA7 feed solution is completely crystallized; (6) After the GA7 feed solution is completely crystallized, filter it to obtain GA7 fine powder and dry it; (7) Recover the solvent from the mother liquor after filtration in step (6); In step (1), the organic solvent is ethanol; In step (4), the pH adjustment range is between 1.4 and 1.7; In step (5), the volume ratio of the deionized water to the feed solution volume is 2 - 4:

1.

2. The method for recrystallization purification of gibberellin GA7 according to claim 1, wherein In step (1), the volume - mass ratio of the organic solvent to the crude gibberellin GA7 is 5 - 8:

1.

3. A method for recrystallization purification of gibberellin GA7 according to claim 1, characterized in that, In step (2), the decolorization treatment uses activated carbon, and the addition amount of activated carbon is 0.25 - 0.5% of the volume of the GA7 solution.

4. A method for recrystallization purification of gibberellin GA7 according to claim 1, characterized in that, In step (3), the titer of the filtrate is 100000 - 200000 ug / mL.

5. A method for recrystallization purification of gibberellin GA7 according to claim 1, characterized in that, In step (6), the drying is vacuum drying, the temperature is controlled at 50 - 60°C, and the vacuum degree is controlled at - 0.07 - - 0.10 MPa.

6. A method for recrystallization purification of gibberellin GA7 according to claim 1, characterized in that, In step (7), vacuum concentration is used for solvent recovery, the temperature is controlled at 55 - 65°C, the vacuum degree is controlled at - 0.08 - - 0.10 MPa, and the solvent recovery time is 3 - 5 hours.

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