A preparation method for obtaining 1-deoxynojirimycin from mulberry leaves

Abstract

The application discloses a preparation method for obtaining 1-deoxynojirimycin from mulberry leaves, and mainly solves the problem that the prior art only considers product content and ignores yield, and comprises the following steps: (1) drying and crushing mulberry leaves, adding water to make pulp, adding vitamin C after adjusting pH, stirring uniformly, extracting, filtering and centrifuging to obtain a centrifugal liquid; (2) purifying the centrifugal liquid through two-stage OSN organic membrane to obtain a cut-off membrane liquid; (3) sequentially flowing the cut-off membrane liquid through more than five cation resin exchange columns in series, first eluting with 1-3 % ml / 100g of ammonia water, then eluting with 4-5.5 % ml / 100g of ammonia water, concentrating the eluate collected from the second column to the penultimate column to no ammonia water to obtain a extract; (4) loading the extract into a mixed ion resin exchange column, eluting with pure water, and collecting the eluate; (5) making the eluate pass through a reverse osmosis membrane to obtain a cut-off liquid, concentrating and vacuum drying. The method provided by the application reduces production processes and cost, improves product content and guarantees the recovery rate.

Description

Technical Field

[0001] This invention relates to the field of extraction technology of active ingredients from mulberry leaves, and more specifically, to a method for preparing 1-deoxynojirimycin from mulberry leaves. Background Technology

[0002] Mulberry leaves contain a variety of functional components. 1-Deoxynojirimycin (DNJ) is one of the main functional active substances. Modern pharmacological studies have shown that DNJ has a strong competitive inhibitory effect on α-glucosidase activity in the small intestine and has anti-tumor and antiviral pharmacological activities, making it a research hotspot for the comprehensive utilization of sericulture. The content of plant-derived DNJ is relatively low, and the presence of other secondary metabolic interferences increases the difficulty of extraction technology. Currently, this technology is still under continuous improvement and refinement. 1-Deoxynojirimycin can be extracted by hot reflux extraction, macroporous resin extraction, ultrasonic-assisted extraction, and microwave-assisted extraction; separation and purification only use cation exchange resin for crude separation, followed by treatment with organic solvents, and the content is generally less than 20%. Chemically synthesized 1-deoxynojirimycin has many byproducts and is difficult to separate and purify. Its synthesis process is costly, has a long synthetic route, high technical requirements, and low DNJ yield, which currently prevents large-scale production. Therefore, the main way to obtain 1-deoxynojirimycin samples is through extraction from natural plants, but research on the extraction process of 1-deoxynojirimycin is still in its early stages.

[0003] Patent No. 201910660067.0 discloses a method for extracting 1-deoxynojirimycin from mulberry branches. The method involves pulverizing the raw material and sieving it through a 40-mesh sieve, extracting with an alcohol-water solution, eluting and purifying with a macroporous resin stepwise, concentrating and drying, and then further purifying and concentrating and drying. This patent is simple to operate, low in cost, and suitable for large-scale production. However, it also has certain drawbacks: the extraction process uses organic solvents, which are neither environmentally friendly nor safe; it still uses macroporous resin chromatography, a step that is energy-intensive and complex; and although the collected 1-deoxynojirimycin content is ≥95%, the recovery rate is not considered.

[0004] Patent No. 201210411230.8 discloses a process for extracting and preparing 1-deoxynojirimycin from mulberry leaves. The process involves pulverizing the raw material and sieving it through a 10-30 mesh sieve, extracting it by reflux with water, filtering with alcohol, centrifuging, concentrating, decolorizing and purifying with macroporous resin, concentrating again, adsorbing with cation exchange resin, decolorizing again, concentrating to a dry solid state, dissolving in methanol, filtering, adding silica gel to a dry powder state, adding ethylenediamine or triethylamine for gradient elution, and detecting by thin-layer chromatography. The patent yields a product with a content ≥70%, suitable for large-scale production. However, the extraction process uses a significant amount of organic solvents. While this method involves first adding multiple organic solvents for separation and purification, followed by recycling, which is more environmentally friendly, the operation is cumbersome. Furthermore, the organic solvents used in the process, such as ethylenediamine or triethylamine, are toxic and flammable. Although the product content is increased, the recovery rate of 1-deoxynojirimycin is not considered.

[0005] Patent No. 201511020693.1 discloses a method for extracting 1-deoxynojirimycin from mulberry leaves, including steps such as pulverization, microwave extraction, centrifugation, water extraction and alcohol precipitation, concentration and drying, membrane separation, and drying to extract 1-deoxynojirimycin. Microwave extraction technology is used to improve extraction efficiency and ensure that the molecular structure of 1-deoxynojirimycin is not destroyed, resulting in a higher medicinal value of the product. However, the content of the product is <10%, and it cannot be mass-produced.

[0006] In summary, existing technologies are cumbersome, use a lot of organic solvents, and only consider product content while ignoring yield. Therefore, there is a need for a method that does not use organic solvents throughout the process, causes less environmental pollution, and improves product content while ensuring recovery rate to extract 1-deoxynojirimycin. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing 1-deoxynojirimycin from mulberry leaves, which reduces production steps and costs, increases product content while ensuring recovery rate. This invention can obtain 1-deoxynojirimycin with a content >50% and a recovery rate >90%, and does not use organic solvents throughout the process, resulting in less environmental pollution and meeting market requirements for mulberry leaf extracts.

[0008] The technical solution to achieve the objective of this invention is: a method for preparing 1-deoxynojirimycin from mulberry leaves, comprising the following steps:

[0009] (1) The dried mulberry leaves were pulverized to ≥200 mesh by ultra-fine grinding, pure water was added and pulped, the pH was adjusted with citric acid and vitamin C was added, the mixture was stirred evenly and then extracted, filtered and centrifuged to obtain centrifuged liquid.

[0010] (2) The centrifuged liquid was purified by passing it through two stages of OSN organic membrane to obtain the membrane-retained liquid;

[0011] (3) Pass the retentate membrane solution through 5 or more cation exchange resin columns connected in series. Stop feeding the drug when the 1-DNJ content in the effluent is detected to be >0.1%. Use ammonia water for fractional elution. First, use 1-3% ml / 100g of ammonia water for elution, and then use 4-5.5% ml / 100g of ammonia water for elution. Collect the eluent from the second to the penultimate column and concentrate it until there is no ammonia water to obtain the extract.

[0012] (4) The extract was loaded into a mixed ion exchange column, eluted with pure water, and the liquid was collected.

[0013] (5) Pass the drug solution through a reverse osmosis membrane to obtain a retentate, concentrate it, and vacuum dry it to obtain the final product.

[0014] In step (3), the cation exchange resin is any one of SQD-65, 002SC NA, SQ66, 001×7MB, D110, and D001TR. In one embodiment of the invention, the cation exchange resin is SQD-65, with 300g of resin per column, and a diameter-to-height ratio of 1:8-10.

[0015] In step (3), before fractionation and elution with ammonia, the effluent is washed with water until it is colorless and clear.

[0016] In step (1), the weight ratio of pure water to crushed mulberry leaves is 3-5:1; the concentration of citric acid is 2-5%; the pH is adjusted to 4.0-4.5; and the amount of vitamin C added is 0.1-1.0‰.

[0017] In step (1), the extraction temperature is 2-6℃, the extraction time is 3-4h, and the extraction is performed once.

[0018] In step (1), the centrifugation is carried out by a benchtop centrifuge for graded centrifugation, first by butterfly centrifugation at 1200 r / min for 60 min, and then by tube centrifugation at 12000 r / min for 2.5 h.

[0019] In step (2), the two-stage OSN organic membrane purification process involves first passing through an organic membrane with a density of 800-1200 Daltons, and then through an organic membrane with a density of 150-250 Daltons.

[0020] In one embodiment of the present invention, the inlet pressure of the first organic membrane is 290-870 PSi, and the inlet pressure of the second organic membrane is 500-850 PSi.

[0021] In step (4), the upper layer of the mixed ion exchange column is anion exchange resin, and the lower layer is cation exchange resin, with a mass ratio of anion exchange resin to cation exchange resin of 5-8:0.5-1. As an embodiment of the present invention, the anion exchange resin is any one of SQ-70A, D945, D296, D330, and D918; the cation exchange resin is any one of D113, D110, and D111.

[0022] In step (5), the reverse osmosis pressure is 0.8-1 MPa. After the original solution is permeated, pure water with 4 times the original solution is added for further permeation to obtain the retentate.

[0023] Beneficial effects

[0024] 1. This invention utilizes a series of cation exchange resins, combined with fractional elution with ammonia, to obtain a high content of 1-DNJ and improve the recovery rate. The use of a mixed ion exchange column achieves the combined effects of decolorization, purification, and pH neutralization, reducing production steps and improving production efficiency.

[0025] 2. This invention utilizes ultrafine grinding and sieving through a sieve of ≥200 mesh to improve the solubility, thereby greatly increasing the 1-DNJ content in the extract. Only one extraction is required, resulting in a high extraction rate and a fast and efficient method.

[0026] 3. This invention utilizes a combination of organic membranes to replace the traditional macroporous resin chromatography for decolorization and purification. It is convenient to operate, consumes less energy, and at the same time achieves good decolorization effect, reducing production costs and improving production efficiency.

[0027] 4. The method provided by this invention reduces production steps and costs, is simple and easy to control, and has good stability. It increases product content while ensuring recovery rate, making it suitable for large-scale production. The produced product has low solvent residue, high quality, and uses no organic solvents throughout the process, resulting in minimal environmental pollution. Attached Figure Description

[0028] Figure 1 The high-performance liquid chromatography (HPLC) chromatogram of 1-DNJ obtained in Example 1 of this invention is shown below.

[0029] Figure 2 The high-performance liquid chromatography (HPLC) chromatogram of 1-DNJ obtained in Example 2 of this invention is shown below.

[0030] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of 1-DNJ obtained in Example 3 of the present invention.

[0031] The 1-DNJ content detection result is taken from the peak at 14 min in the chromatogram. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments.

[0033] Example 1

[0034] (1) Take 1000g of dried mulberry leaves (total content is 0.12%) and pulverize them to a finer mesh than 200 mesh. Add 5000g of pure water to make a slurry. Adjust the pH to 4.0 with 5% citric acid. Add 1‰ vitamin C and stir evenly. Extract at 6℃ for 4h. Stir continuously during extraction. Extraction is only required once. After extraction, filter through a 200-mesh filter cloth and centrifuge using a benchtop centrifuge. First, centrifuge at 1200r / min disc speed for 60min, and then centrifuge at 12000r / min tube speed for 2.5h to obtain the centrifuged liquid.

[0035] (2) The centrifuged liquid was purified by passing it through two stages of OSN organic membranes. First, it was passed through an organic membrane with a pressure of 1000 Daltons and an inlet pressure of 870 PSi. After passing through the whole membrane, it was passed through an organic membrane with a pressure of 200 Daltons and an inlet pressure of 850 PSi to obtain the decolorized membrane retentate.

[0036] (3) The decolorized retentate membrane solution was passed sequentially through 5 cation exchange resin columns connected in series, model SQD-65, each column containing 300g of resin, with a diameter to height ratio of 1:10. The feeding was stopped when the 1-DNJ content in the effluent was detected to be >0.1%. The resin was first washed with water until the effluent was colorless and clear, then eluted with 3% ml / 100g ammonia water, with the elution volume being 4 times the column volume of each resin column. Finally, it was eluted with 5% ml / 100g ammonia water. The eluent from the second to the second to last column was collected and concentrated until no ammonia water was obtained to obtain the extract.

[0037] (4) The extract was directly placed into the mixed ion exchange resin with a resin amount of 900g. The upper layer of the mixed ion exchange resin column was 800g of anion exchange resin SQ-70A and the lower layer was 100g of cation exchange resin D113. The mass ratio of anion exchange resin to cation exchange resin was 8:1. After the extraction was completed, it was eluted with 1800g of pure water. The effluent and washing liquid were combined and collected. The solution was collected when the 1-DNJ content was detected in the effluent > 0.1% DNJ and was ready for use.

[0038] (5) The drug solution was permeated through the original solution under a pressure of 1 MPa, and then 4 times the volume of pure water was added for further permeation to obtain the retentate. The retentate was concentrated and vacuum dried to obtain 2.1 g of the product, which was analyzed by HPLC (e.g., ...). Figure 1 (As shown) The 1-DNJ content was 51.5%, and the recovery rate was 90.13%.

[0039] Example 2

[0040] (1) Take 1000g of dried mulberry leaves (total content is 0.12%) and pulverize them to a finer mesh than 200 mesh. Add 4000g of pure water to make a slurry. Adjust the pH to 4.0 with 4% citric acid. Add 0.5‰ vitamin C and stir evenly. Extract at 4℃ for 3.5h. Stir continuously during extraction. Extraction is only required once. After extraction, filter through a 200-mesh filter cloth and centrifuge using a benchtop centrifuge. First, centrifuge at 1200r / min disc speed for 60min, and then centrifuge at 12000r / min tube speed for 2.5h to obtain the centrifuged liquid.

[0041] (2) The extracted centrifuged liquid was purified by passing it through two stages of OSN organic membranes. First, it was passed through an organic membrane with a pressure of 1200 Daltons and an inlet pressure of 800 PSi. After passing through the whole membrane, it was passed through an organic membrane with a pressure of 250 Daltons and an inlet pressure of 780 PSi to obtain the decolorized membrane retentate.

[0042] (3) The decolorized membrane solution was passed sequentially through 5 cation exchange resin columns connected in series, model SQD-65, each containing 300g of resin, with a diameter to height ratio of 1:9. The feeding was stopped when the content of 1-DNJ was detected in the effluent > 0.1%. The resin was first washed with water until the effluent was colorless and clear, then eluted with 2% ml / 100g ammonia water, with the elution volume being 4 times the column volume of each resin. Finally, it was eluted with 4.5% ml / 100g ammonia water. The eluent from the second to the second to last column was collected and concentrated until no ammonia water was obtained to obtain the extract.

[0043] (4) The extract was directly placed into the mixed ion exchange resin. The amount of resin was 900g. The upper layer of the mixed ion exchange resin column was 700g of anion exchange resin SQ-70A and the lower layer was 50g of cation exchange resin D113. The mass ratio of anion exchange resin to cation exchange resin was 7:0.5. After the extraction was completed, it was eluted with 1800g of pure water. The effluent and washing liquid were collected together. The drug solution was collected when the effluent showed a DNJ content >0.1% DNJ and was ready for use.

[0044] (5) The drug solution was permeated through the original solution under a pressure of 0.9 MPa, and then 4 times the volume of pure water was added for further permeation to obtain the retentate. The retentate was concentrated and vacuum dried to obtain 2.11 g of product. HPLC analysis (e.g.) Figure 2 (As shown) The 1-DNJ content was 51.22%, and the recovery rate was 90.06%.

[0045] Example 3

[0046] (1) Take 1000g of dried mulberry leaves (total content is 0.12%), pulverize them into a fine powder of 200 mesh or higher, add 3000g of pure water to make a slurry, adjust the pH to 4.0 with 3% citric acid, add 0.2‰ vitamin C and stir evenly, extract at 2℃ for 3h, stirring continuously during extraction, and extract once. After extraction, filter through a 200-mesh filter cloth and centrifuge using a benchtop centrifuge. First, centrifuge at 1200r / min disc speed for 60min, and then centrifuge at 12000r / min tube speed for 2.5h to obtain the centrifuged liquid.

[0047] (2) The extracted centrifuged liquid was purified by passing it through two stages of OSN organic membranes. First, it was passed through an 800 Dalton organic membrane with an inlet pressure of 700 PSi. After passing through the whole membrane, it was passed through a 150 Dalton organic membrane with an inlet pressure of 680 PSi to obtain the decolorized membrane retentate.

[0048] (3) The decolorized membrane solution was passed sequentially through 5 cation exchange resin columns connected in series, model SQD-65, each containing 300g of resin, with a diameter to height ratio of 1:8. The feeding was stopped when the content of 1-DNJ was detected in the effluent > 0.1%. The resin was first washed with water until the effluent was colorless and clear, then eluted with 1% ml / 100g ammonia water, with the elution volume being 4 times the column volume of each resin. Finally, it was eluted with 4% ml / 100g ammonia water. The eluent from the second to the penultimate column was collected and concentrated until no ammonia water was obtained to obtain the extract.

[0049] (4) The extract was directly placed into a mixed ion exchange resin column with a resin volume of 900g. The upper layer of the mixed ion exchange column consisted of 600g of anion exchange resin SQ-70A, and the lower layer consisted of 50g of cation exchange resin D113. The mass ratio of anion exchange resin to cation exchange resin was 6:0.5. After the extraction was complete, the column was eluted with 1800g of pure water. The effluent and washing solution were collected together. The solution was collected starting from the point where the effluent showed a DNJ content > 0.1% for later use.

[0050] (5) The drug solution was permeated through the original solution under a pressure of 0.8 MPa, and then 4 times the volume of pure water was added for further permeation to obtain the retentate. The retentate was concentrated and then vacuum dried to obtain 2.12 g of product. HPLC analysis (e.g.) Figure 3 (As shown) The 1-DNJ content was 51.38%, and the recovery rate was 90.77%.

[0051] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention, and these will not affect the effectiveness and practicality of the present invention.

Claims

1. A method for preparing 1-deoxynojirimycin from mulberry leaves, characterized in that, Includes the following steps: (1) The dried mulberry leaves are pulverized to ≥200 mesh by ultra-fine grinding, pure water is added and pulped, the pH is adjusted with citric acid and vitamin C is added, and after stirring evenly, the mixture is extracted at 2-6℃ for 3-4 hours, filtered and centrifuged to obtain centrifuged liquid; the centrifugation is carried out by a benchtop centrifuge for fractional centrifugation, first centrifuged at 1200r / min for 60min, and then centrifuged at 12000r / min for 2.5h. (2) The centrifuged liquid is purified by passing it through two stages of OSN organic membranes to obtain the membrane-retained liquid; the two-stage OSN organic membrane purification is to first pass it through an organic membrane with a pressure of 800-1200 Daltons, and then through an organic membrane with a pressure of 150-250 Daltons; the inlet pressure of the first organic membrane is 290-870 PSi, and the inlet pressure of the second organic membrane is 500-850 PSi. (3) Pass the retentate membrane solution through 5 or more cation exchange resin columns connected in series. Stop feeding the drug when the 1-DNJ content in the effluent is detected to be >0.1%. Use ammonia water for fractional elution. First, use 1-3% ml / 100g of ammonia water for elution, and then use 4-5.5% ml / 100g of ammonia water for elution. Collect the eluent from the second to the penultimate column and concentrate it until there is no ammonia water to obtain the extract. (4) The extract is loaded into a mixed ion exchange column, eluted with pure water and the liquid is collected; the upper layer of the mixed ion exchange column is anion exchange resin and the lower layer is cation exchange resin, and the mass ratio of anion exchange resin to cation exchange resin is 5-8:0.5-1. (5) Pass the drug solution through a reverse osmosis membrane at a pressure of 0.8-1 MPa to obtain a retentate, concentrate it, and vacuum dry it to obtain the final product.

2. The method for preparing 1-deoxynojirimycin from mulberry leaves according to claim 1, characterized in that, In step (3), the cationic resin is any one of SQD-65, 002SC NA, SQ66, 001×7MB, D110 and D001TR.

3. The method for preparing 1-deoxynojirimycin from mulberry leaves according to claim 1, characterized in that, In step (3), before eluting with ammonia, the effluent is washed with water until it is colorless and clear.

4. The method for preparing 1-deoxynojirimycin from mulberry leaves according to claim 1, characterized in that, In step (1), the weight ratio of pure water to crushed mulberry leaves is 3-5:1; the concentration of citric acid is 2-5%; the pH is adjusted to 4.0-4.5; and the amount of vitamin C added is 0.1-1.0‰.

5. The method for preparing 1-deoxynojirimycin from mulberry leaves according to claim 1, characterized in that, In step (1), the extraction is performed once.

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