CN100439511C - Method for Catalyzed Extraction of Diosgenin by Modified Cellulase - Google Patents

Abstract

The invention discloses a method for extracting diosgenin by using modified cellulase. The invention belongs to the technology of extracting diosgenin from yam plants. The method mainly includes the following steps: dissolving cyanuric chloride in benzene, and after recrystallization, reacting with polyethylene glycol treated with molecular sieves and anhydrous sodium carbonate in benzene according to a certain ratio to prepare activated polyethylene glycol. Ethylene glycol; activated polyethylene glycol reacts with natural cellulase in a certain ratio in citric acid-sodium citrate buffer solution, and finally uses ultrafiltration membrane to separate the modified cellulase; add the modified cellulase to the raw material The diosgenin product is obtained through catalysis, hydrolysis, neutralization, extraction and crystallization in the water soaking solution. The advantage of the present invention is that the ability of the modified cellulase to resist the environment and temperature is enhanced, it has higher catalytic activity in a long period of time, and the catalytic effect is obvious. The modified enzyme is especially suitable for the extraction of active ingredients in natural products rich in cellulose , has wide versatility.

Description

Method for Catalyzed Extraction of Diosgenin by Modified Cellulase

technical field

The invention relates to a method for extracting diosgenin by using modified cellulase. The invention belongs to the technology of extracting diosgenin from yam plants.

Background technique

Diosgenin, also known as diosgenin, is an important intermediate in the steroid pharmaceutical industry. It is obtained from the rhizome of the genus Dioscorea as raw material through acid hydrolysis and organic solvent extraction. The production of diosgenin was initially obtained by direct acid hydrolysis and extraction from the rhizome of yam plant, that is, the Rothrok method. This method has been used both at home and abroad. my country began to adopt this method to establish diosgenin factories in the late 1950s, and it is still widely used even in some small enterprises in my country. However, this method has the disadvantage that on the one hand the yield of diosgenin obtained is low, and only 45% of the diosgenin in the plant can be extracted; on the other hand, a large amount of active ingredients in the plant are discarded, resulting in resource serious waste and environmental pollution.

In order to increase the yield of diosgenin, people have carried out a lot of research work and a variety of process improvements, one of the important discoveries is that in the 1960s and 1970s, Hardman in Canada and Paseshnichenko in the Soviet Union et al. After the pre-fermentation is carried out for a period of time, the purpose of increasing the yield of diosgenin can be achieved. But this method takes a long time, and the fermentation process will produce other by-products.

In recent years, with the development of biocatalytic technology, some domestic scholars have used cellulase, amylase, pectinase, etc. to carry out biocatalytic treatment followed by synergistic acid hydrolysis for research, and the results can make 65% to 70% of yam Saponin components are extracted. But, along with the carrying out of biological enzyme catalyzed reaction, the product that substrate degradation generates can produce inhibitory action to biological enzyme, adds raw material to contain more impurity, makes biological enzyme activity greatly reduce, is unfavorable for the diosgenin combination in the plant. Effective degradation and fragmentation of polymer substances, so the resistance to acid hydrolysis is not effectively reduced, thereby limiting the yield and quality improvement of diosgenin.

Contents of the invention

The object of the present invention is to provide a method for catalyzing the extraction of diosgenin in yam plants by using a modified cellulase with higher activity. The method has the characteristics of high extraction rate of diosgenin, energy saving and low environmental pollution.

In order to achieve the above object, the present invention is achieved through the following technical solutions. A method for catalyzing the extraction of diosgenin by modifying cellulase, characterized in that it comprises the following process:

1. The purification of cyanuric chloride, preparation mass concentration is the cyanuric cyanuric benzene solution of 6%～15%, recrystallization twice, obtains the purified cyanuric chloride.

2. the activation of Polyethylene Glycol, by the mass ratio of Polyethylene Glycol 4000 and 4A molecular sieve and anhydrous sodium carbonate is 1: 2～6: 4～10 will polyethylene glycol 4000, 4A molecular sieve and anhydrous sodium carbonate Add in the solvent benzene, stir at room temperature for 30-60 minutes, filter, remove 4A molecular sieve and sodium carbonate to obtain the filtrate, then according to the molar ratio of polyethylene glycol 4000 and purified cyanuric chloride to be 2-2.5: 1, the step 1 The prepared purified cyanuric chloride is added to the filtrate, and reacted for 20 to 50 hours under normal temperature conditions. The solution is then concentrated, filtered, and dried at low temperature to obtain activated polyethylene glycol.

3. the modification of cellulase, cellulase and the activated polyethylene glycol that step 2 makes is that 1: 2～6 joins pH in the citric acid-sodium citrate buffer solution of 5.0～7.0 according to mass ratio, control The temperature is 0°C-8°C, and the reaction is 1-5 hours. The cellulase is separated by an ultrafiltration membrane with a molecular weight cut-off of 5000-10000, and then freeze-dried at a low temperature to obtain the modified cellulase.

4. Transformation and extraction of diosgenin, the raw material of crushed Dioscorea scutellaria or Dioscorea punctatus is added into water according to the mass ratio of 1:3 and soaked for 30-60 minutes, and then according to the mass ratio of 2.0‰ of Dioscorea scutellaria or Dioscorea punctatus Proportion Add the modified cellulase prepared in step 3 into the soaking solution, control the temperature at 50°C-60°C for 4-7 hours of catalytic reaction, and then use sulfuric acid or hydrochloric acid to adjust the acid concentration of the system to 2mol/L-5mol/L , hydrolyzed at a temperature of 98-108°C, the hydrolyzate was washed with 5% Na ₂ CO ₃ aqueous solution until neutrally filtered, and the filter cake was dried in an oven at 60-80°C, and the dried solids were extracted with an extractant 120# The gasoline is extracted in a Soxhlet extractor, the extract is concentrated, filtered, and dried at low temperature to obtain the diosgenin product.

The invention has the advantages of improving the ability of cellulase to resist enzymatic inactivation caused by environmental conditions, thereby improving the catalytic activity of the enzyme, maintaining a high catalytic activity during the reaction process, reducing the resistance of the subsequent treatment of materials, and effectively The invention is beneficial to convert the diosgenin in the dioscorea plant into diosgenin, thereby improving the extraction rate of the diosgenin, increasing the yield, and having low waste discharge.

Detailed ways

Example 1: 20.0 g of cyanuric chloride was dissolved in 300 mL of benzene, and after recrystallization twice, 17.9 g of purified cyanuric chloride was obtained. Add 5.0g of 4A molecular sieves, 10.0g of anhydrous sodium carbonate, 500mL of benzene and 20.0g of polyethylene glycol 4000 in a 1000mL three-necked flask, stir at room temperature for 30 minutes, and filter through a Buchner funnel to remove 4A molecular sieves and sodium carbonate; Collected in a three-necked flask, then added 0.4613 g of purified cyanuric chloride, and reacted for 36 hours at normal temperature. The solution was concentrated, filtered through a Buchner funnel, and then dried at a temperature of -14°C to obtain activated polyethylene glycol 4000. Add 4.0g of activated polyethylene glycol 4000 and 100mL of citric acid-sodium citrate buffer solution with a pH of 6.8 into a 500mL flask, adjust the temperature to 4°C, and add 1.0g of cellulase after the activated polyethylene glycol 4000 is fully dissolved After reacting for 2 hours, the modified cellulase was obtained by separating the polysulfone ultrafiltration membrane with a molecular weight cut-off of 5000.

Then put 100g of 60-80 mesh Dioscorea scutellaria powder into a 500mL three-necked flask, add 300mL of water and stir at a temperature of 30°C for 45 minutes, fully soak and expand, add 0.2g of modified cellulase, and control the reaction temperature at 50°C. Modify the cellulase to catalyze the reaction for 5 hours; then measure the volume of the reaction solution to 330mL, add 64.3mL of 98% concentrated sulfuric acid to adjust the acid concentration of the hydrolysis system to 3mol/L, and carry out acid hydrolysis at 101°C for 2 hours; the hydrolyzed Dioscorea scutellaria The powder was washed with 5% Na ₂ CO ₃ aqueous solution to neutrality, and then dried in an oven at 60°C, and turned from time to time to prevent the material from being overcrowded; 21.6g of the dried hydrolyzate was extracted with 500mL of 120# gasoline in Soxhlet Extract in a vacuum cleaner for 7 hours, then use a rotary evaporator to recover gasoline, concentrate the extract, and crystallize the product at a low temperature of -4°C, filter it with a sand core funnel, and finally dry it in a freeze-drying oven at -15°C to obtain 2.2537 g of colorless needle crystals , The extraction rate can reach more than 96%. The product was determined by infrared spectroscopy, and the results (IR(KBr): v cm ^-1 3452, 1049, 978, 918, 898, 866) were consistent with the literature (IR(KBr): v cm ^-1 3450, 1050, 980, 918 , 898,864); melting point is 197°C-198°C; elemental analysis results are C: 77.98%, H: 10.19% consistent with theoretical calculation values (78.21% and 10.21%); HPLC results show that the product purity is as high as 98.6%.

Example 2: 10.0 g of cyanuric chloride was dissolved in 150 mL of benzene, and recrystallized twice to obtain 8.7 g of purified cyanuric chloride. Add 2.5g of 4A molecular sieves, 8.0g of anhydrous sodium carbonate, 400mL of benzene and 10.0g of polyethylene glycol 4000 in a 1000mL three-necked flask, stir at room temperature for 40 minutes, and filter through a Buchner funnel to remove 4A molecular sieves and sodium carbonate; Collected in a three-necked flask, then added 0.2306 g of purified cyanuric chloride, and reacted for 38 hours at normal temperature. The solution was concentrated, filtered through a Buchner funnel, and dried at -14°C to obtain activated polyethylene glycol 4000. Add 8.0g of activated polyethylene glycol 4000 and 200mL of citric acid-sodium citrate buffer solution with a pH of 7.0 into a 500mL flask, adjust the temperature to 4°C, and add 2.0g of cellulase after the activated polyethylene glycol 4000 is fully dissolved After reacting for 2 hours, the modified cellulase was obtained by separating with a polysulfone ultrafiltration membrane with a molecular weight cut-off of 4000.

Put 50.0g of 80-100 mesh Dioscorea scutellaria powder in a 250mL three-neck flask, add 150mL of water and stir for 45 minutes at a temperature of 25°C, fully soak and expand, add 0.1g of modified cellulase, control the reaction temperature at 50°C, and modify the fiber Sulfase catalyzed for 6 hours; then follow the operation methods of hydrolysis, neutralization and extraction according to Example 1, concentrate the extract, crystallize the product at a low temperature of -4°C, filter it with a sand core funnel, and finally dry it in a freeze-drying oven at -15°C 1.2365 g of colorless needle-like crystals were obtained, and the extraction rate could reach over 97%. The product was determined by infrared spectroscopy, and the results (IR(KBr): v cm ^-1 3450, 1050, 979, 918, 897, 867) were consistent with the literature (IR(KBr): v cm ^-1 3450, 1050, 980, 918 , 898, 864); melting point is 198°C-200°C; elemental analysis results are C: 77.95%, H: 10.20% consistent with theoretical calculation values (78.21% and 10.21%); HPLC results show that the purity is as high as 97.9% %.

Example 3: 20.0 g of cyanuric chloride was dissolved in 300 mL of benzene, and recrystallized twice to obtain 18.3 g of purified cyanuric chloride. Add 8.0g of 4A molecular sieves, 12.0g of anhydrous sodium carbonate, 600mL of benzene and 30.0g of polyethylene glycol 4000 in a 1000mL three-necked flask, stir at room temperature for 60 minutes, and filter through a Buchner funnel to remove 4A molecular sieves and sodium carbonate; Collected in a three-necked flask, then added 0.5535 g of purified cyanuric chloride, and reacted for 40 hours under normal temperature conditions. The solution was concentrated, filtered through a Buchner funnel, and dried at -14.2°C to obtain activated polyethylene glycol 4000. Add 8.0 g of activated polyethylene glycol 4000 and 200 mL of citrate sodium citrate buffer solution with a pH of 6.8 in a 500 mL flask, adjust the temperature to 4°C, and add 2.0 g of cellulase after the activated polyethylene glycol 4000 is fully dissolved. After reacting for 2 hours, the modified cellulase was separated by polysulfone ultrafiltration membrane with a molecular weight cut-off of 4000.

Put 50.0g of 80-100 mesh Dioscorea chinensis powder in a 250mL three-necked flask, add 150mL of water and stir at a temperature of 30°C for 60 minutes, fully soak and expand, add 0.1g of modified cellulase, control the reaction temperature at 55°C, and modify the fiber The enzyme was catalyzed for 5.5 hours; then the subsequent hydrolysis, neutralization, extraction and other operating methods were according to the examples, and finally the extract was concentrated, and the product was crystallized at a low temperature of -4°C, filtered through a funnel with a sand core, and finally placed in a freeze-drying oven at -15°C. After drying, 1.3684 g of colorless needle-like crystals were obtained, and the extraction rate could reach more than 97%. The product was determined by infrared spectroscopy, and the results (IR(KBr): vcm ^-1 3450, 1050, 979, 918, 897, 867) were consistent with the literature (IR(KBr): vcm ^-1 3450, 1050, 980, 918, 898,864); the melting point is 197°C to 199°C; the elemental analysis results are C: 78.14%, H: 10.20% are consistent with the theoretical calculation values (78.21% and 10.21%); the high performance liquid chromatography results show that the purity is as high as 95.6% .

Example 4: 20.0 g of cyanuric chloride was dissolved in 300 mL of benzene, and recrystallized twice to obtain 17.6 g of purified cyanuric chloride. Add 4.0g of 4A molecular sieves, 9.0g of anhydrous sodium carbonate, 500mL of benzene and 16.0g of polyethylene glycol 4000 in a 1000mL three-necked flask, stir at room temperature for 40 minutes, and filter through a Buchner funnel to remove 4A molecular sieves and sodium carbonate; Collected in a three-necked flask, then added 0.2952 g of purified cyanuric chloride, and reacted for 40 hours at normal temperature. The solution was concentrated, filtered through a Buchner funnel, and dried at -14.2°C to obtain activated polyethylene glycol 4000. Add 12.0g of activated polyethylene glycol and 100mL of citric acid-sodium citrate buffer solution with a pH of 7.0 in a 500mL flask, adjust the temperature to 4.0°C, add 3.0g of cellulase after the activated polyethylene glycol 4000 is fully dissolved, and react After 2 hours, the modified cellulase was separated by polysulfone ultrafiltration membrane with a molecular weight cut-off of 5000.

Put 100g of 60-80 mesh Dioscorea chinensis powder into a three-necked flask, add 300mL of water and stir for 45 minutes at a temperature of 25°C, fully soak and expand, add 0.2g of cellulase modified with polyethylene glycol 4000, and control the reaction temperature 60°C, catalyzed by cellulase for 5 hours; then follow the operation methods of hydrolysis, neutralization, extraction, etc. according to Example 1, and finally concentrate the extract, crystallize the product at a low temperature of -4°C, filter it with a sand core funnel, and finally filter it at -60 Dry in a freeze-drying oven at ℃ to obtain 2.6957 g of colorless needle crystals, and the extraction rate can reach more than 95%. The product was determined by infrared spectroscopy, and the results (IR(KBr): v cm ^-1 3450, 1050, 980, 920, 897, 866) were consistent with the literature (IR(KBr): v cm ^-1 3450, 1050, 980, 918 , 898, 864); the melting point is 197 ° C ~ 199 ° C; the elemental analysis results are C: 78.18%, H: 10.23% consistent with the theoretical calculation values (78.21% and 10.21%); the results of high performance liquid chromatography are as high as 97.8 %.

Example five: 15.0 g of cyanuric chloride was dissolved in 250 mL of benzene, and recrystallized twice to obtain 13.6 g of purified cyanuric chloride. Add 5.0 g of 4A molecular sieves, 10.0 g of anhydrous sodium carbonate, 600 ml of benzene and 16.0 g of polyethylene glycol 4000 in a 1000 mL three-necked flask, stir at room temperature for 40 minutes, and filter through a Buchner funnel to remove 4A molecular sieves and sodium carbonate; Collected in a three-necked flask, then added 0.2952g of purified cyanuric chloride, and reacted for 40 hours at room temperature. The solution was concentrated, filtered through a Buchner funnel, and dried at a low temperature at -14.2°C to obtain activated polyethylene glycol 4000 . Add 14.0g of activated polyethylene glycol 4000 and 120mL of citric acid-sodium citrate buffer solution with a pH of 7.0 into a 500mL flask, adjust the temperature to 5.0°C, and add 4.0g of cellulase after the activated polyethylene glycol 4000 is fully dissolved , after 2 hours of reaction, the modified cellulase was separated by a polysulfone ultrafiltration membrane with a molecular weight cut-off of 5000.

Put 100g of 80-100 mesh Dioscorea chinensis powder into a three-necked flask, add 300mL of water and stir for 60 minutes at a temperature of 30°C. After fully soaking and swelling, add 0.2g of cellulase modified with polyethylene glycol 4000 to control the reaction temperature 55°C, catalyzed by cellulase for 6 hours; then follow the operation methods of hydrolysis, neutralization, extraction, etc. according to Example 1, and finally concentrate the extract, crystallize the product at a low temperature of -4°C, filter it with a sand core funnel, and finally filter it at -60°C ℃ freeze-drying oven to obtain 2.7153 g of colorless needle-like crystals, and the extraction rate can reach more than 96%. The product was determined by infrared spectroscopy, and the results (IR(KBr): v cm ^-1 3450, 1049, 980, 920, 897, 866) were consistent with the literature (IR(KBr): v cm ^-1 3450, 1050, 980, 918 , 898, 864); the melting point is 197°C to 199°C; the elemental analysis results are C: 78.20%, H: 10.20% are consistent with the theoretically calculated values (78.21% and 10.21%); the high performance liquid chromatography results show that the purity is as high as 96.9% %.

Claims (1)

1. the method for a catalytic extraction of yam saponin by using modified cellulase is characterized in that comprising following process:

1). the purifying of cyanuric chloride, the preparation mass concentration is 6%～15% cyanuric chloride benzole soln, recrystallization twice obtains the cyanuric chloride of purifying;

2). the activation of polyoxyethylene glycol, by the mass ratio of Macrogol 4000 and 4A molecular sieve and anhydrous sodium carbonate is 1: 2～6: 4～10 with Macrogol 4000,4A molecular sieve and anhydrous sodium carbonate add in the solvent benzol, after the stirring at normal temperature 30～60 minutes, filter, remove 4A molecular sieve and yellow soda ash and obtain filtrate, be that the cyanuric chloride of 2～2.5: 1 purifying that step 1) is made joins in the filtrate according to the mol ratio of the cyanuric chloride of Macrogol 4000 and purifying then, normal temperature condition reacted 20～50 hours down, solution is again through concentrating, filter, cryodrying obtains activated polyglycol;

3). the modification of cellulase, with cellulase and step 2) activated polyethylene glycol that makes is that 1: 2～6 to join pH be in 5.0～7.0 citric acid-sodium citrate buffer according to mass ratio, controlled temperature is 0 ℃～8 ℃, reacted 1～5 hour, adopting molecular weight cut-off is that 5000～10000 ultra-filtration membrane is isolated cellulase, and frozen drying obtains using modified cellulase;

4). the conversion of diosgenin is extracted, is the Rhizome of Peltate Yam of pulverizing or Dioscorea nipponica Mak. Ningpo Yam Rhizome raw material to add at 1: 3 to soak 30～60 minutes in the entry according to mass ratio, the using modified cellulase that step 3) is made according to the ratio with Rhizome of Peltate Yam or Dioscorea nipponica Mak. Ningpo Yam Rhizome mass ratio 2.0 ‰ adds in the soak solution again, 50 ℃～60 ℃ following katalysis reactions of controlled temperature 4～7 hours, acid concentration with sulfuric acid or hydrochloric acid regulation system is 2mol/L～5mol/L then, temperature is 98～108 ℃ of following hydrolysis, and hydrolyzed solution adopts 5% Na ₂CO ₃Solution washing places 60～80 ℃ of baking ovens dry filter cake to neutral filtration, and dried solids utilizes extraction agent 120# gasoline to extract in cable type extractor according, and extraction liquid filters through concentrating, and gets the diosgenin product after the cryodrying.