Extraction method of diosgenin

Abstract

The invention provides an extraction method of diosgenin, which comprises the following steps: step 1, dioscorea zingiberensis rhizome and an extraction solvent are put into a crushing extraction device, the crushing extraction device comprises a feed port, a discharge port, a rotating shaft, crushing blades connected with the rotating shaft and a heater, and the rotating speed of the rotating shaft is 800-900 r / s; step 2, after the rhizome of dioscorea zingiberensis and the extraction solvent are extracted for 1-1.5 h in the crushing extraction device, discharging the mixture from a discharge port, filtering, concentrating the filtrate until no alcohol smell exists, adding distilled water, fully stirring, settling and centrifuging to obtain a precipitate, namely dioscin; 3, dioscin and helicase are put into a reaction container, and an acetic acid-sodium acetate buffer solution is added for hydrolysis; 4, after the hydrolysis reaction is finished, a hydrolysis product is inactivated, suspension is centrifuged, supernate is removed, sediment is washed with distilled water, residues are dried and recrystallized with an organic solvent, and diosgenin is obtained. According to the present invention, the dioscin is extracted from the yam plant by using the crushing extraction device, and the diosgenin is further obtained by using the enzymolysis, such that the extraction method can improve the diosgenin extraction amount, can avoid the use of a large amount of acid, can alleviate the environmental pollution problem, and has good industrial application prospects.

Description

Technical Field

[0001] The invention relates to the field of yam plant extraction, and in particular to a method for extracting diosgenin. Background Art

[0002] Dioscorea zingiberensis CH Wright, commonly known as yellow ginger and firehead root, is a perennial twining vine of the Dioscoreaceae family. It belongs to the rhizome group in the Dioscorea genus. It is a herbaceous vine with horizontal rhizomes, shallow roots, thick papery leaves, simple leaves that are alternate, triangular ovate, cordate or lanceolate, and shield-shaped petioles. Hence the name Dioscorea zingiberensis, which is the main feature that distinguishes it from other species in the Dioscorea genus.

[0003] Dioscorea zingiberensis has long been used as a Chinese herbal medicine for the treatment of diseases. Its medicinal value and artificial cultivation methods are recorded in monographs such as Shennong's Herbal Classic, Compendium of Materia Medica, Synopsis of the Golden Chamber, and Illustrated Records of Plant Names and Realities. Its rhizomes are used as medicine and have very high medicinal value. Chinese medicine believes that its nature and flavor are sweet, bitter, and cool, and it has the functions of clearing the lungs and relieving cough, promoting dampness and relieving stranguria, dredge meridians and relieving pain, and detoxifying and reducing swelling. It can be used to treat lung heat cough, damp-heat stranguria pain, rheumatic back pain, carbuncle, sprains, bee and insect bites, acute suppurative skin infection, appendicitis, etc. Dioscorea zingiberensis contains a large amount of starch and cellulose (about 40% each), as well as a small amount of xanthophyll, tannins, water-soluble glycosides, cardiac glycosides, alkaloids, flavonoid glycosides and other ingredients, which are mainly used to extract pharmacologically active dioscin and diosgenin. These steroidal saponin ingredients have been shown to have the effects of removing dampness, detoxifying and reducing swelling, anti-tumor, lowering blood lipids, and treating cardiovascular diseases such as hypertension, hyperlipidemia, thrombosis, coronary heart disease, angina pectoris, myocardial ischemia, etc. It can also be used to treat acute suppurative skin infections, soft tissue injuries, bee stings, insect bites and various surgical inflammations. Among them, diosgenin is an important raw material for synthetic contraceptives and steroid hormone drugs, and is known as the "mother of hormones."

[0004] The traditional production method of diosgenin is direct acid hydrolysis. Its main principle is that under the action of dilute acid, the glycosidic bond of diosgenin in Dioscorea zingiberensis is broken, thereby being hydrolyzed into diosgenin and monosaccharide residues, and the diosgenin released after acid hydrolysis is extracted by petroleum ether. However, a large amount of sulfuric acid is used in the traditional preparation method, which causes serious pollution. At present, about 60% of manufacturers in the country are forced to shut down, and there is a serious shortage of goods both at home and abroad. The demand for diosgenin is expanding year by year, and the gap between production and demand is very large. In addition, the amount of diosgenin extracted from Diosgena zingiberensis in the prior art is limited, which is not conducive to the extraction of diosgenin. Summary of the invention

[0005] In view of this, the present invention aims to provide a method for extracting diosgenin to solve the problems of pollution caused by large amount of acid used in the extraction process and low extraction yield.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows: A method for extracting diosgenin comprises the following steps: Step 1, putting the rhizome of Dioscorea zingiberensis and the extraction solvent into a crushing and extraction device, wherein the crushing and extraction device comprises a feed inlet, a discharge outlet, a rotating shaft, a crushing blade connected to the rotating shaft, and a heater, and the rotating speed of the rotating shaft is 800-900 r / s; Step 2: After the rhizome of Dioscorea zingiberensis and the extraction solvent are extracted in a crushing and extraction device for 1 to 1.5 hours, the mixture is discharged from the discharge port, filtered, and the filtrate is concentrated until there is no alcohol taste, distilled water is added and stirred, sedimented, and centrifuged to obtain a precipitate, which is dioscin; Step 3, adding dioscin and snail enzyme into a reaction container, and adding acetic acid-sodium acetate buffer for hydrolysis; Step 4: After the hydrolysis reaction is completed, the hydrolysis product is inactivated at 90-100° C. for 20-30 min, the suspension is centrifuged, the supernatant is discarded, the precipitate is washed with distilled water, the residue is dried and recrystallized with an organic solvent to obtain diosgenin.

[0007] Furthermore, in the step three, the conditions for hydrolysis using snail enzyme are: the usage ratio of snail enzyme to diosgenin is 1:1.2, the temperature is 45° C., the pH value is 6.5, and the hydrolysis time is 24 hours.

[0008] Furthermore, in step three, the mass of dioscin and the volume of acetic acid-sodium acetate buffer are 1:100-140.

[0009] Furthermore, in the step three, dioscin is added in stages. First, snail enzyme and acetic acid-sodium acetate buffer are put into the reaction container. In the first stage, 1 / 2 of the total amount of dioscin is added at the initial time. In the second stage, 1 / 4 to 1 / 3 of the total amount of dioscin is added after the reaction for 4 to 5 hours. In the third stage, the remaining dioscin is added after the reaction for 10 to 15 hours.

[0010] Furthermore, in step three, hot air at 45° C. is continuously introduced into the reaction container.

[0011] Furthermore, in step 1, the extraction solvent is ethanol with a volume fraction of 70-80%.

[0012] Furthermore, in the step 1, the amount of the extraction solvent is 10 to 12 times that of the rhizome of Dioscorea zingiberensis, and the extraction temperature is 60 to 70°C.

[0013] Furthermore, in step 4, the organic solvent used in the recrystallization process is petroleum ether, methanol or ethanol.

[0014] Compared with the prior art, the method for extracting diosgenin of the present invention has the following advantages: (1) Using snail enzymes to hydrolyze diosgenin from diosgenin can not only increase the extraction amount of diosgenin, but also avoid the use of large amounts of acid, alleviate the problem of environmental pollution, and has good industrial application prospects; (2) hot air is introduced into the reaction vessel to cause the dioscin and snail enzyme to tumble, thereby increasing the contact area between the two, promoting the enzyme reaction, and reducing the adhesion of the substrate and enzyme to the inner wall of the reaction vessel; (3) Adding dioscin in stages can better hydrolyze with snail enzymes and reduce incomplete contact with enzymes caused by adhesion and agglomeration between dioscin powders; (4) When extracting dioscin, a crushing and extraction device is used, which integrates crushing and extraction into one process. At the same time, the extraction solvent can form a strong cyclonic effect, impacting and destroying the cell wall of the Dioscorea plant, and with the help of the mechanical force of high-speed rotation, the effective ingredients are released better and faster, thereby improving the extraction rate of dioscin. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 It is a schematic structural diagram of the crushing and extraction device described in the present invention.

[0016] Description of reference numerals: 1. Feed port; 2. Discharge port; 3. Rotating axis; 4. Crushing blade; 5. Heater. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with specific embodiments. It should be noted that the data in the following experimental examples are obtained by the inventor through a large number of experiments. Due to space limitations, only a part of them is shown in the specification, and those of ordinary skill in the art can understand and implement the present invention under these data. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these changes or modifications also fall within the scope protected by the present application.

[0018] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0019] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0020] A method for extracting diosgenin of the present invention comprises the following steps: Step 1, putting the rhizome of Dioscorea zingiberensis and the extraction solvent into a crushing and extraction device, wherein the crushing and extraction device comprises a feed port 1, a discharge port 2, a rotating shaft 3, a crushing blade 4 connected to the rotating shaft 3, and a heater 5, and the rotating speed of the rotating shaft is 800-900 r / s; Specifically, in step 1, the extraction solvent is 70-80% ethanol by volume, the amount of the extraction solvent is 10-12 times that of the rhizome of Dioscorea zingiberensis, and the extraction temperature is 60-70°C.

[0021] Step 2: After the rhizome of Dioscorea zingiberensis and the extraction solvent are extracted in a crushing and extraction device for 1 to 1.5 hours, the mixture is discharged from the discharge port 2, filtered, and the filtrate is concentrated until there is no alcohol taste, distilled water is added, stirred sufficiently, settled, and centrifuged to obtain a precipitate, which is dioscin; Step 3, adding dioscin and snail enzyme into a reaction container, and adding acetic acid-sodium acetate buffer for hydrolysis; Specifically, in step 3, the conditions for hydrolysis using snail enzyme are: the dosage ratio of snail enzyme to diosgenin is 1:1.2, the temperature is 45°C, the pH value is 6.5, and the hydrolysis time is 24 hours. The mass of diosgenin and the volume of acetic acid-sodium acetate buffer are 1:100-140.

[0022] Step 4: After the hydrolysis reaction is completed, the hydrolysis product is inactivated at 90-100° C. for 20-30 min, the suspension is centrifuged, the supernatant is discarded, the precipitate is washed with distilled water, the residue is dried and recrystallized with an organic solvent to obtain diosgenin.

[0023] Specifically, in step 4, the organic solvent used in the recrystallization process is petroleum ether, methanol or ethanol.

[0024] Drugs and reagents: The rhizome of Dioscorea zingiberensis used in the present invention is from Henan Dubang Chinese Medicine Biotechnology Co., Ltd. and is identified as the rhizome of Dioscorea zingiberensis zingiberensis by Professor Fu Hongzheng of Peking University.

[0025] Ethanol (chromatographically pure, Tianjin Yongda Chemical Reagent Factory); glacial acetic acid (analytical pure, Beijing Tongguang Fine Chemical Company); sodium acetate (analytical pure, Beijing Tongguang Fine Chemical Company); snail enzyme (biochemical reagent, Beijing Bairuiji Biotechnology Co., Ltd.).

[0026] Preparation of buffer solution: 0.2mol / L sodium acetate solution: accurately weigh 162g of anhydrous sodium acetate, add water to dissolve and prepare 1000ml of 0.2mol / L sodium acetate solution; 0.2mol / L acetic acid solution: accurately measure 11.94ml of acetic acid, add water and shake well to prepare 1000ml of 0.2mol / L acetic acid solution. Prepare acetic acid-sodium acetate buffer with a pH of 6.5 according to the volume ratio of acetic acid to sodium acetate of 1.79:98.21.

[0027] The process of extracting dioscin of the present invention is completed in a crushing and extraction device, and the rhizome of Dioscorea zingiberensis and the extraction solvent are put in from a feed port 1. The motor of the crushing and extraction device drives the rotating shaft 3 to rotate, and drives the crushing blade 4 to rotate at a high speed. The rhizome of Dioscorea zingiberensis is divided into small pieces under the action of the crushing blade 4, thereby increasing the contact area between the rhizome and the extraction solvent. At the same time, the extraction solvent put in forms a strong cyclone effect under the action of high-speed rotation, which can impact and destroy the cell wall of the Dioscorea plant, and work together with the mechanical force of high-speed rotation to release the effective ingredients better and faster, thereby increasing the extraction rate of dioscin. At the same time, the crushing and extraction device integrates crushing and extraction into one process, which greatly saves the operation process for industrial large-scale production.

[0028] In order to further improve the crushing effect, multiple layers of crushing blades 4 are distributed along the rotating shaft 3 from top to bottom. For each layer of crushing blades 4, multiple crushing blades 4 are distributed around the circumference of the rotating shaft 3. A heater 5 is used to maintain a certain extraction temperature inside the crushing and extraction device. Example 1

[0029] 100 kg of Dioscorea zingiberensis rhizomes and 1000 L of 75% ethanol were put into the crushing and extraction device from the feed port 1, and the rotating shaft 3 was started to rotate and drive the crushing blade 4 to rotate at a high speed, and the rotating speed of the rotating shaft 3 was 800 r / s. The Dioscorea zingiberensis rhizomes were divided into small pieces under the action of the crushing blade 4, and the ethanol formed a vortex under the action of the rotating shaft 3. The extraction temperature was set to 70°C.

[0030] After the rhizomes of Dioscorea zingiberensis and ethanol are extracted in the crushing extraction device for 1 hour, the obtained mixture is discharged from the discharge port 2. After filtration, the filtrate is concentrated until there is no alcohol taste, distilled water is added and stirred thoroughly, the mixture is settled, centrifuged, and dried to obtain a dioscin extract with a content of 14.61 kg. Example 2

[0031] 100 kg of Dioscorea zingiberensis rhizomes and 1200 L of 70% ethanol were put into the crushing and extraction device from the feed port 1, and the rotating shaft 3 was started to rotate and drive the crushing blade 4 to rotate at a high speed, and the rotating speed of the rotating shaft 3 was 900 r / s. The Dioscorea zingiberensis rhizomes were divided into small pieces under the action of the crushing blade 4, and the ethanol formed a vortex under the action of the rotating shaft 3. The extraction temperature was set to 70°C.

[0032] After the rhizomes of Dioscorea zingiberensis and ethanol are extracted in the crushing extraction device for 1.5 hours, the obtained mixture is discharged from the discharge port 2. After filtration, the filtrate is concentrated until there is no alcohol taste, distilled water is added and stirred thoroughly, and the mixture is settled, centrifuged, and dried to obtain a dioscin extract with a content of 15.28 kg. Example 3

[0033] 100 kg of Dioscorea zingiberensis rhizomes and 1200 L of 80% ethanol were put into the crushing and extraction device from the feed port 1, and the rotating shaft 3 was started to rotate and drive the crushing blade 4 to rotate at a high speed, and the rotating speed of the rotating shaft 3 was 850 r / s. The Dioscorea zingiberensis rhizomes were divided into small pieces under the action of the crushing blade 4, and the ethanol formed a vortex under the action of the rotating shaft 3. The extraction temperature was set to 60°C.

[0034] After the rhizomes of Dioscorea zingiberensis and ethanol are extracted in the crushing extraction device for 1.5 hours, the obtained mixture is discharged from the discharge port 2. After filtration, the filtrate is concentrated until there is no alcohol taste, distilled water is added and stirred thoroughly, and the mixture is settled, centrifuged, and dried to obtain a dioscin extract with a content of 15.22 kg.

[0035] Comparative Example 1 100 kg of Dioscorea zingiberensis rhizomes were crushed into coarse particles, and then extracted twice with 1200 L of 70% ethanol, with extraction time of 1 h and 0.5 h respectively, and the extraction temperature was 70 ° C. After the extraction, the extracts were combined, concentrated until there was no alcohol taste, and distilled water was added to stir well, settle, centrifuge, and dry to obtain dioscin extract with a content of 10.21 kg.

[0036] Comparative Example 2 100 kg of Dioscorea zingiberensis rhizomes were crushed into coarse particles, then soaked in 1200 L of 70% ethanol, and subjected to microwave action, with a microwave power of 500 W and an extraction time of 1 h. After the extraction, the extracts were combined, concentrated until there was no alcohol taste, and distilled water was added for full stirring, sedimentation, centrifugation, and drying to obtain a dioscin extract with a content of 12.60 kg.

[0037] By comparing the dioscin extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 2, the dioscin content of Comparative Example 1 is the lowest, indicating that the extraction degree of dioscin using only alcohol extraction is limited. The dioscin content extracted by the methods of Examples 1 to 3 is greatly improved, the high-speed rotating mechanical force crushes and stirs the Dioscorea zingiberensis, and the extraction solvent forms a high-speed cyclonic fluid to rub and impact the Dioscorea zingiberensis, destroying the cell structure of the Dioscorea zingiberensis and accelerating the separation and precipitation of dioscin.

[0038] In order to avoid using the traditional acid hydrolysis method to extract diosgenin, a green diosgenin extraction method is established. The present invention uses enzymatic hydrolysis of diosgenin instead of acid hydrolysis to achieve the purpose of efficient utilization of Dioscorea zingiberensis resources and reduce environmental pollution. The enzymatic hydrolysis of glycosidic bonds has the advantages of high selectivity, few by-products, and green environmental protection. The commercial enzyme conversion has good specificity and strong selectivity. At the same time, compared with acid, the reaction conditions are mild, and there is no need to protect and deprotect the functional groups, the aglycone structure is not destroyed, and the parent nucleus skeleton is kept unchanged; in addition, the operation is simple, the reaction time is short, and the repeatability is good.

[0039] Snail enzyme is a kind of mixed enzyme component obtained from the snail capsule and digestive tract, which is composed of more than 20 kinds of enzymes such as cellulase, α-amylase, protease, pectinase, etc. Therefore, the present invention uses snail enzyme to hydrolyze dioscin, and selects four factors of enzymatic hydrolysis time, enzyme addition amount, enzymatic hydrolysis pH value, and enzymatic hydrolysis temperature for orthogonal experiments to investigate the effect of their interaction on the yield of dioscin. L9(3 4 )Orthogonal design table was used for experimental design. The orthogonal experimental design and result analysis are shown in Tables 1 and 2.

[0040] Table 1

[0041] Table 2

[0042] It can be seen from Tables 1 and 2 that the enzymatic hydrolysis temperature B has a great influence on the hydrolysis rate of dioscin, but with the increase of enzymatic hydrolysis temperature, the hydrolysis rate of dioscin decreases. When the enzymatic hydrolysis temperature of snail enzyme is at level B2, the hydrolysis rate of dioscin is the highest, so the enzymatic hydrolysis temperature of snail enzyme is selected at level B2; the hydrolysis pH value C of snail enzyme has a great influence on the hydrolysis rate of dioscin, when the hydrolysis pH value of snail enzyme is at level C2, the hydrolysis rate of dioscin is the highest, so the hydrolysis pH value of snail enzyme is selected at level C2; the dosage ratio D of snail enzyme and dioscin extract has a great influence on the hydrolysis rate of dioscin. The influence of the rate on the hydrolysis rate is second only to the pH value of snail enzyme hydrolysis. The hydrolysis rate of dioscin at the D3 level is higher than that at the D2 and D1 levels, so the D3 level is selected as the liquid-to-solid ratio; while the hydrolysis time A has the least effect on the hydrolysis rate of dioscin. The hydrolysis rate of dioscin at the A2 level is higher than that at the A1 and A3 levels, so the A2 level is selected as the hydrolysis time. Finally, A2B2C2D3 is determined to be the optimal condition for snail enzyme hydrolysis of dioscin, that is, the dosage ratio of snail enzyme hydrolysis of dioscin is 1:1.2, the temperature is 45℃, the pH value is 6.5, and the hydrolysis time is 24h.

[0043] In order to increase the contact area between dioscin and snail enzyme, hot air is introduced into the reaction vessel, and the hot air is blown into the reaction vessel by a blower, so that dioscin and snail enzyme are in a tumbling state, the contact area between the two is increased, the reaction of the enzyme is promoted, and the adhesion of the substrate and the enzyme on the inner wall of the reaction vessel is reduced. The temperature of the introduced hot air is 45° C., which not only allows the hydrolysis reaction to be carried out at the most suitable temperature, but also promotes the contact reaction of the substrate and the enzyme. The flow rate of the hot air is not specifically limited, and those skilled in the art can set it according to actual operation, as long as it can ensure that dioscin and snail enzyme are in a tumbling state.

[0044] Further, in the step 3, dioscin is added in stages, and snail enzyme and acetic acid-sodium acetate buffer are first put into the reaction container. In the first stage, 1 / 2 of the total amount of dioscin is added at the initial time. In the second stage, 1 / 4 to 1 / 3 of the total amount of dioscin is added after 4 to 5 hours of reaction. In the third stage, the remaining dioscin is added after 10 to 15 hours of reaction. Since the enzymatic reaction speed is fast in the early stage of the reaction, it is ensured that all the snail enzyme is put into the reaction container before the second stage, so that there is sufficient enzyme and dioscin to contact and react.

[0045] Furthermore, in the step three, acetic acid-sodium acetate buffer is first put into the reaction container, and then dioscin is added in stages, and snail enzyme is introduced at a rate of snail enzyme dosage / 5h to ensure that all snail enzyme is put into the reaction container before the second stage. In the first stage, 1 / 2 of the total amount of dioscin is added at the initial time. In the second stage, 1 / 4 to 1 / 3 of the total amount of dioscin is added after 4 to 5 hours of reaction. In the third stage, the remaining dioscin is added after 10 to 15 hours of reaction.

[0046] A temperature sensor is arranged at the bottom center of the reaction container to monitor the temperature in the reaction container in real time. Example 4

[0047] Weigh 12 kg of dioscin prepared by the method of Example 2 and 10 kg of snail enzyme. Add dioscin into a reaction container, add 1200 L of acetic acid-sodium acetate buffer with a pH of 6.5 into the reaction container, and blow hot air at 45° C. into the reaction container with a blower. When the temperature in the reaction container reaches 45° C., add snail enzyme into the reaction container. During the entire reaction process, continue to pass hot air at 45° C.

[0048] After 24 hours of reaction, the hydrolysis product was inactivated at 90-100°C for 20-30 minutes, the suspension after enzymatic hydrolysis was centrifuged, the supernatant was discarded, the precipitate was washed three times with distilled water, the residue was dried and recrystallized with petroleum ether to obtain 1.81 kg of diosgenin. Example 5

[0049] Weigh 12kg of dioscin and 10kg of snail enzyme prepared by the method of Example 2. Add 6kg of dioscin and 1200L of acetic acid-sodium acetate buffer with a pH of 6.5 into a reaction vessel, and introduce hot air at 45°C. When the temperature in the reaction vessel reaches 45°C, add 10kg of snail enzyme into the reaction vessel. After 5h of reaction, add 4kg of dioscin, and after 15h of reaction, add 2kg of diosgenin. During the entire reaction process, continue to introduce hot air at 45°C.

[0050] After 24 hours of reaction, the hydrolysis product was inactivated at 90-100°C for 20-30 minutes, the suspension after enzymatic hydrolysis was centrifuged, the supernatant was discarded, the precipitate was washed three times with distilled water, the residue was dried and recrystallized with petroleum ether to obtain 2.03 kg of diosgenin. Example 6

[0051] Weigh 12kg of dioscin and 10kg of snail enzyme prepared by the method of Example 2. Add 6kg of dioscin and 1200L of acetic acid-sodium acetate buffer with a pH of 6.5 into the reaction vessel, and continue to pass hot air at 45°C. When the temperature in the reaction vessel reaches 45°C, add snail enzyme at a rate of 2kg / h until all the snail enzyme is added into the reaction vessel. After 5h of reaction, add 4kg of dioscin, and after 15h of reaction, add 2kg of dioscin. During the entire reaction process, continue to pass hot air at 45°C.

[0052] After 24 hours of reaction, the hydrolysis product was inactivated at 90-100°C for 20-30 minutes, the suspension after enzymatic hydrolysis was centrifuged, the supernatant was discarded, the precipitate was washed three times with distilled water, the residue was dried and recrystallized with petroleum ether to obtain 2.34 kg of diosgenin. Example 7

[0053] Weigh 12kg of dioscin and 10kg of snail enzyme prepared by the method of Example 2. Add 6kg of dioscin and 1200L of acetic acid-sodium acetate buffer with a pH of 6.5 into a reaction vessel, and continue to introduce hot air at 45°C. When the temperature in the reaction vessel reaches 45°C, add snail enzyme at a rate of 2kg / h until all the snail enzyme is added into the reaction vessel. After 5h of reaction, add 4kg of dioscin, and after 15h of reaction, add 2kg of dioscin.

[0054] During the whole hydrolysis reaction, hot air at 45°C was continuously introduced, and the temperature in the reaction vessel was monitored throughout the process. When the temperature was monitored to be lower than 45°C, snail enzyme was added at a rate of 4kg / h, and when the temperature continued to maintain 45°C, snail enzyme was added at a rate of 2kg / h.

[0055] After 24 hours of reaction, the hydrolysis product was inactivated at 90-100°C for 20-30 minutes, the suspension after enzymatic hydrolysis was centrifuged, the supernatant was discarded, the precipitate was washed three times with distilled water, the residue was dried and recrystallized with petroleum ether to obtain 3.51 kg of diosgenin. Example 8

[0056] Weigh 12kg of dioscin and 10kg of snail enzyme prepared by the method of Example 2. Add 6kg of dioscin and 1680L of acetic acid-sodium acetate buffer with a pH of 6.5 into a reaction vessel, and continue to introduce hot air at 45°C. When the temperature in the reaction vessel reaches 45°C, add snail enzyme at a rate of 2kg / h until all the snail enzyme is added into the reaction vessel. Add 3kg of dioscin after 4h of reaction, and add 3kg of dioscin after 10h of reaction.

[0057] During the whole hydrolysis reaction, hot air at 45°C was continuously introduced, and the temperature in the reaction vessel was monitored throughout the process. When the temperature was monitored to be lower than 45°C, snail enzyme was added at a rate of 4kg / h, and when the temperature continued to maintain 45°C, snail enzyme was added at a rate of 2kg / h.

[0058] After 24 hours of reaction, the hydrolysis product was inactivated at 90-100°C for 20-30 minutes, the suspension after enzymatic hydrolysis was centrifuged, the supernatant was discarded, the precipitate was washed three times with distilled water, the residue was dried and recrystallized with petroleum ether to obtain 3.65 kg of diosgenin.

[0059] Comparative Example 3 Weigh 12kg of dioscin and 10kg of snail enzyme prepared by the method of Example 2. Add 6kg of dioscin and 1200L of acetic acid-sodium acetate buffer with a pH of 6.5 into the reaction vessel, and continue to pass hot air at 45°C. When the temperature in the reaction vessel reaches 45°C, add snail enzyme at a rate of 1kg / h until all the snail enzyme is added into the reaction vessel. After 5h of reaction, add 4kg of dioscin, and after 15h of reaction, add 2kg of dioscin. During the entire reaction process, continue to pass hot air at 45°C.

[0060] After 24 hours of reaction, the hydrolysis product was inactivated at 90-100°C for 20-30 minutes, the suspension after enzymatic hydrolysis was centrifuged, the supernatant was discarded, the precipitate was washed three times with distilled water, the residue was dried and recrystallized with petroleum ether to obtain 1.78 kg of diosgenin.

[0061] The diosgenin content extracted by the method of embodiment 4 is the lowest. It is speculated that the possible reason is that although hot air is introduced to make diosgenin tumble, due to the adhesion and reunion between the tiny diosgenin powders, all diosgenin and snail enzyme are mixed at one time, and only part of diosgenin can fully contact with snail enzyme. Therefore, diosgenin is added in stages, and enzymolysis with snail enzyme can be better. The diosgenin extracted by the method of embodiment 5 reaches 2.03kg.

[0062] The inventor further found that, because dioscin was added in stages, if all the snail enzymes were put into the initial stage, it would be unfavorable for the interaction between enzyme and substrate. Therefore, the snail enzymes were continuously introduced at a certain speed, and all the snail enzymes were put into the reaction vessel before the second stage, which not only ensured that sufficient enzymes and dioscin were in contact and reacted, but also avoided the situation that the amount of enzyme was too large and the amount of substrate was less in the initial stage. 2.34 kg of diosgenin can be extracted by the method of embodiment 6. In comparative example 3, due to the slow feeding rate of the snail enzymes, in the initial stage of hydrolysis, i.e., the stage with faster reaction speed, the amount of enzyme put into was less, and the effect of the enzyme was not in place, which affected the extraction of diosgenin.

[0063] With the addition of substrate dioscin and snail enzyme, and the continuation of the two reactions, the heat transfer of the reaction system will be affected. Therefore, the present invention introduces the judgment condition of temperature. In the whole hydrolysis reaction process, when the temperature is monitored to be lower than 45 ℃, it is shown that the conditions in this case are not conducive to the hydrolysis reaction. Therefore, the speed of the enzyme is increased by 50%, and on the one hand, 45 ℃ of hot air is continuously introduced to ensure the temperature of the reaction, and on the other hand, the adverse effect of the temperature reduction on the hydrolysis is compensated by increasing the feeding amount of the enzyme. The diosgenin extracted by the method of embodiment 7-8 has a content of 3.51-3.65kg.

[0064] The diosgenin extraction method of the present invention uses a crushing extraction device to extract diosgenin from a yam plant, and further uses enzymatic hydrolysis to obtain diosgenin. The extraction method can not only increase the extraction amount of diosgenin, but also avoid the use of a large amount of acid, alleviate the problem of environmental pollution, and has good industrial application prospects.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting diosgenin, characterized in that: The steps include: Step 1: putting the rhizomes of Dioscorea zingiberensis and an extraction solvent into a crushing and extraction device, wherein the crushing and extraction device comprises a feed port (1), a discharge port (2), a rotating shaft (3), a crushing blade (4) connected to the rotating shaft (3), and a heater (5), wherein the rotating shaft has a rotation speed of 800-900 r / s; Step 2: After the rhizomes of Dioscorea zingiberensis and the extraction solvent are extracted in a crushing and extraction device for 1 to 1.5 hours, the mixture is discharged from the discharge port (2), filtered, and the filtrate is concentrated until there is no alcohol taste, distilled water is added, stirred thoroughly, settled, and centrifuged to obtain a precipitate, which is dioscin; Step 3, adding dioscin and snail enzyme into a reaction container, and adding acetic acid-sodium acetate buffer for hydrolysis; Step 4: After the hydrolysis reaction is completed, the hydrolysis product is inactivated, the suspension is centrifuged, the supernatant is discarded, the precipitate is washed with distilled water, the residue is dried and then recrystallized with an organic solvent to obtain diosgenin.

2. The method for extracting diosgenin according to claim 1, wherein In the step 3, the conditions for hydrolysis using snail enzyme are: the usage ratio of snail enzyme to dioscin is 1:1.2, the temperature is 45° C., the pH value is 6.5, and the hydrolysis time is 24 hours.

3. The method for extracting diosgenin according to claim 1, characterized in that: In the step 3, the mass of dioscin and the volume of acetic acid-sodium acetate buffer are 1:100-140.

4. The method for extracting diosgenin according to claim 1, characterized in that: In the step three, dioscin is added in stages. First, snail enzyme and acetic acid-sodium acetate buffer are put into the reaction container. In the first stage, 1 / 2 of the total amount of dioscin is added at the initial time. In the second stage, 1 / 4 to 1 / 3 of the total amount of dioscin is added after the reaction for 4 to 5 hours. In the third stage, the remaining dioscin is added after the reaction for 10 to 15 hours.

5. The method for extracting diosgenin according to claim 1, characterized in that: In the step 3, hot air at 45° C. is continuously introduced into the reaction container.

6. The method for extracting diosgenin according to claim 1, characterized in that: In the step 1, the extraction solvent is 70-80% ethanol by volume.

7. The method for extracting diosgenin according to claim 1, characterized in that: In the step 1, the amount of the extraction solvent is 10-12 times that of the rhizome of Dioscorea zingiberensis, and the extraction temperature is 60-70°C.

8. The method for extracting diosgenin according to claim 1, characterized in that: In the step 4, the organic solvent used in the recrystallization process is petroleum ether, methanol or ethanol.

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