CN110790811A - Method for preparing diosgenin by hydrolysis of aluminum chloride in two-phase system - Google Patents

Abstract

The invention discloses a method for preparing diosgenin by hydrolysis of biphasic system AlCl ₃ . React at ~180°C for 0.5~2.5h, filter the reaction solution, filter off the residue, separate the petroleum ether layer, carry out rotary evaporation, and recrystallize to obtain diosgenin. The present invention adopts the non-traditional acid AlCl ₃ to be acid hydrolyzed to prepare Dioscorea saponin in a two-phase system, which not only improves the yield of saponin, shortens the extraction period, but also greatly reduces the consumption of acid, and more importantly , in the case of not using concentrated sulfuric acid, the pollution to the environment will also become minimal, and it has a great application prospect.

Description

Method for preparing diosgenin by hydrolysis of aluminum chloride in two-phase system

technical field

The invention relates to the technical field of plant extraction, in particular to the extraction of saponin from Dioscorea officinalis resources, and more particularly, to a method for preparing diosgenin by hydrolysis of biphasic system AlCl ₃ .

Background technique

Due to the remarkable effects of steroid hormone drugs in anti-tumor, immune system regulation, treatment of cardiovascular system diseases, rheumatic diseases, bacterial encephalitis, skin diseases, etc., the research on steroid hormones in various countries in the world has developed rapidly (Chen et al., 2015; Wang et al., 2007). However, the industrial development of steroid hormones faces the defects of scarcity of drug sources, low content of raw materials, and high cost of artificial synthesis, so it cannot meet the needs of large-scale mass production. The discovery of diosgenin has created a precedent for the synthesis of steroid drugs using diosgenous plants as raw materials, and has promoted the research process of the development and utilization of diosgenous plant resources. The carbon skeleton structure of diosgenin is very similar to that of steroid hormone drugs, and it is the most ideal precursor for the synthesis of steroid hormone drugs. Diosgenin, known as saponin in industry, is linked with sugars at the C-3 and C-26 positions through glycosidic bonds, and exists in plants in the form of steroidal saponins.

Diosgenin and different types and numbers of sugars combine to form different diosgenin through glycosidic bonds. Therefore, the hydrolysis of diosgenin is the key to extracting and preparing diosgenin. The current technology for preparing diosgenin is mainly based on two methods, chemical hydrolysis and biological hydrolysis, from which some auxiliary extraction preparation technologies or technologies combining multiple methods are derived.

Chemical hydrolysis mainly refers to the extraction and preparation method of using traditional inorganic strong acids such as sulfuric acid and hydrochloric acid to hydrolyze glycosidic bonds to release diosgenin. The traditional process of extracting diosgenin by acid hydrolysis was proposed by Rothrock et al., that is, directly hydrolyzing the rhizome of diosgenin with inorganic acid, washing it with water to neutrality, drying and performing water bath cycle extraction with organic solvents such as petroleum ether, and finally crystallized to obtain the finished saponin. (Rothrock et al., 1957). Zhang Yuqing et al. investigated the effect of extracting Dioscorea saponin by hydrolysis with sulfuric acid, and the results showed that the yield of saponin was 1.28% after hydrolysis at a concentration of 2 mol/L for 4 hours and Soxhlet extraction for 8 hours (Zhang Yuqing et al., 2006 ). The direct acid hydrolysis method is simple in process and simple in operation, and is suitable for large-scale industrial production. However, the yield of saponin in the inorganic acid hydrolysis process is generally low, about 1.6%. When hydrolyzed with strong inorganic acid, it will not only cause equipment corrosion and water consumption during washing, but also the discharged acidic wastewater is difficult to be recycled and treated, and some of the waste residue will be The saponins could not be separated from the starch and cellulose substances present in a large amount after acid hydrolysis. This directly causes environmental pollution and waste of resources, and also affects the yield of diosgenin.

The biological hydrolysis method to extract saponin is to use the biological enzymes produced by some mold microorganisms to directly convert saponin to generate saponin, which has the advantage of being environmentally friendly and pollution-free. Some researchers used Aspergillus oryzae, Trichoderma harzianum, and Aspergillus awamori to inoculate and cultivate Dioscorea splendens respectively to ferment and directly convert saponins to extract saponin. etc., the difference is significant, the process and mechanism of saponin hydrolysis in biotransformation are complex, the yield of saponin is low, and many by-products are generated (Chen et al., 2018; Liu et al., 2010; Dong Yuesheng et al., 2009 ). Although the bioconversion of saponins to diosgenin by the combination of biological methods and enzymatic hydrolysis has attracted the attention of many researchers, it has no pollution to the environment. However, compared with the traditional direct acid hydrolysis method, due to the uneconomical cost of enzyme preparation and biological The inefficiency of the transformation has led to its limited application in the diosgenin industry. Therefore, there is an urgent need to find a preparation method of diosgenin with high saponin yield, low acid consumption and small sewage discharge.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and provide a method for preparing diosgenin by hydrolysis of a two _- phase system AlCl .

The above-mentioned purpose of the present invention is achieved by the following technical solutions:

A method for preparing diosgenin by hydrolysis of biphasic system AlCl ₃ . The dried diosgenus powder is added into a closed reactor, followed by adding aluminum trichloride aqueous solution, mixing evenly, then adding petroleum ether, and reacting at 130-180 DEG C for 0.5 After ~2.5h, the reaction solution was filtered, the residue was filtered off, the petroleum ether layer was separated out and subjected to rotary evaporation, and diosgenin was obtained after recrystallization.

The method of the invention adopts the non-traditional acid aluminum trichloride of Lewis acid to be acidly hydrolyzed in a two-phase system of water/petroleum ether to prepare Dioscorea saponin. The Lewis acid aluminum trichloride is a water-soluble inorganic salt, and the aqueous solution is Acidic, safe, non-toxic and easy to operate, it can be used to replace inorganic strong acid sulfuric acid hydrolysis to prepare saponin. Compared with the traditional direct acid hydrolysis of Dioscorea glutinosa raw materials, the organic solvent is used to repeatedly reflux and extract the acid hydrolysis residue to extract the saponin. The two-phase system acid hydrolysis extraction method of the present invention utilizes the difference in the solubility of the saponin and the saponin to simultaneously extract the saponin while the saponin is hydrolyzed. Saponin is added to the organic phase of petroleum ether, and Lewis acid and petroleum ether are used to make the acidolysis of saponin and the extraction of saponin simultaneously, which not only simplifies the operation and saves time, but also greatly reduces the amount of acid used, and the extraction rate of saponin is higher. , the amount of sewage is small.

Preferably, the reaction temperature is 150-170°C; for example, 150°C, 155°C, 160°C, 165°C, 170°C.

More preferably, the reaction temperature is 160°C.

Preferably, the reaction time is 1-2.5h.

More preferably, the reaction time is 1-2h (preferably 2h).

Preferably, the concentration of the aluminum trichloride aqueous solution is 1.25g/L～7.5g/L.

When the AlCl ₃ concentration is too low, the low concentration AlCl ₃ solution is obviously not enough to completely hydrolyze the steroidal saponins, and can only break the partial glycosidic bonds of the steroidal saponins, so that the saponins are hydrolyzed into some intermediate products, such as diosgenin-triglycoside, Diosgenin-biglycoside and trillium glucoside, etc.; and high concentration of AlCl ₃ solution may cause side reactions, dehydration and cyclization of saponin, which affects the yield of saponin. Therefore, the concentration of the aluminum trichloride aqueous solution is preferably 2.5 to 5.0 g/L (more preferably 4.0 g/L).

Preferably, the volume ratio of the water/petroleum ether solution is 140/40, 120/60, 100/80, 80/100, 60/120 or 40/140 mL.

More preferably, the volume ratio of the water/petroleum ether solution is 80/100 mL.

Preferably, the addition amount of the diosgenus powder in the reaction system is 26-28 g/L (preferably 27.8 g/L).

Preferably, the Dioscorea chinensis is Dioscorea japonica; because Dioscorea japonica is a more ideal raw material for saponin production due to its high saponin content.

As a preferred embodiment, the method for preparing diosgenin by hydrolysis of the biphasic system AlCl ₃ is to add 5.0 g of dried Dioscorea officinalis powder into a closed reactor, and then add 4.0 g/L of aluminum trichloride solution in turn 80mL, mix well, then add 100mL petroleum ether, react at 160 ℃ for 2h, filter the reaction solution, filter off the residue, separate the petroleum ether layer for rotary evaporation, recrystallize to obtain diosgenin, and the yield of saponin can reach 1.92 %

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

In the invention, the non-traditional acid AlCl ₃ is acid hydrolyzed in the water/petroleum ether two-phase system to prepare Dioscorea saponin, which not only improves the yield of saponin, the highest yield of saponin can reach 1.92%, and shortens the time During the extraction cycle, the amount of acid consumed at the same time is greatly reduced, and more importantly, without the use of concentrated sulfuric acid, the pollution to the environment will also become minimal, and it has a great application prospect.

Description of drawings

Fig. 1 is the HPLC chromatogram of the saponin standard product (top) and the saponin sample (bottom) prepared by the present invention.

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Experimental materials: Triennial tubers of Dioscorea chrysanthemum were taken from the planting base of Dioscorea chrysanthemum in Wengyuan County, Guangdong Province. Then cut the rhizomes of Triennial Chrysanthemum Leaf Dioscorea into thin slices, freeze-dried in a freeze dryer for 24 hours to remove excess water, and then ground into powder with a grinder, put into a sealed bag, and stored in a constant temperature water-removing sealer for later use.

Dioscorea saponin standard (≥99.9%, HPLC grade), purchased from Fluka, USA; Methanol, chromatographically pure, purchased from Sigma-Aldrich, USA; Sulfuric acid, analytically pure, purchased from Guangzhou Chemical Reagent Factory; Petroleum ether (60- 90°C), analytically pure, purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; aluminum trichloride, analytically pure, purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

Example 1

Accurately weigh 5.0g of Dioscorea officinalis powder and put it into the reactor, followed by 0.5g of anhydrous AlCl ₃ and 100mL of distilled water, after stirring evenly, add 80mL of petroleum ether, set the speed of the reactor to be 150rpm, and react at an extraction temperature of 160°C for 2.0 h. After the end, the diosgenin extract in the petroleum ether organic phase was rotary-evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. Dioscorea saponin analysis and quantification were then performed.

Analysis method of diosgenin

其中W：菊叶薯蓣原料重量，g； C：皂素浓度，mg/mL；N：稀释倍数。高效液相色谱法测定参数：UV检测器； C18色谱柱(4.6mm×250mm,5μm)；柱温箱温度：40℃；流动相：甲醇/水；流速为1.0mL/min；上样体积为10μL。每次测定重复三次。皂素标准品和制备样品的HPLC色谱图如图1所示。The above-mentioned diosgenin extract dissolved in constant volume of methanol was diluted to a certain number, filtered with a 0.22 μm filter membrane, and collected into a 1 mL sample bottle, and the sample was prepared for use. The standard product of saponin (purity ≥99.9%) from Fluka Company was used as the control, and the detection by high performance liquid chromatography was carried out. The specific process is as follows: after dissolving the diosgenin standard in methanol, the concentrations of 4.0×10 ^-3 mg/mL, 2.0×10 ^-2 mg/mL, 1.0×10 ^-1 mg/mL and 0.5 mg/mL were analyzed by high performance liquid chromatography. The diosgenin standard product of mg/mL was measured. Based on the chromatographic peak area of the saponin standard product, a standard curve was drawn, and the peak area was used as the quantitative basis for the sample. The calculation of the saponin yield was calculated by the following formula:

Wherein W: raw material weight of Dioscorea japonica, g; C: saponin concentration, mg/mL; N: dilution ratio. Parameters determined by high performance liquid chromatography: UV detector; C18 chromatographic column (4.6mm×250mm, 5μm); column oven temperature: 40°C; mobile phase: methanol/water; flow rate is 1.0mL/min; sample volume is 10 μL. Each assay was repeated three times. The HPLC chromatograms of the saponin standard and prepared samples are shown in Figure 1.

The results show that the yield of saponin prepared by the method described in Example 1 can reach 1.61%.

Example 2

In this example, the influence of temperature on the yield of saponin was investigated. Accurately weigh 5.0g of Dioscorea officinalis powder and put it into the reactor, add 0.5g _of anhydrous AlCl and 100mL of distilled water successively, stir evenly, add 80mL of petroleum ether, set the reactor speed to be 150rpm, and wait until the reactor temperature rises to a predetermined series After the temperature (130°C, 140°C, 150°C, 160°C, 170°C, 180°C), the time was started, and the reaction time was 1.5h. After the end, the diosgenin extract in the petroleum ether organic phase was rotary-evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the analysis method in Example 1, the yield of Dioscorea saponin was calculated, and the influence of temperature on the yield of saponin extracted by AlCl ₃ in the two-phase system was investigated. The results are shown in Table 1:

Table 1

It can be seen from Table 1 that the temperature has a great influence on the yield of Dioscorea saponin extracted by hydrolysis with anhydrous AlCl ₃ as the acid catalyst. When the reaction temperature was lower than 150℃, the saponin yield increased gradually with the increase of the reaction temperature, and at 150℃, the saponin yield reached about 1.3%. As the temperature continued to rise, the saponin yield showed a fluctuating trend of first decreasing, then increasing and then decreasing. In general, when the reaction temperature was 150 °C, 160 °C, and 170 °C, the saponin yield exceeded 100 °C. 1.2% and not much difference. In order not to affect the highest extraction rate of Dioscorea saponin, three temperatures will be optimized in the next time optimization experiment, so as to screen out the most suitable temperature for the extraction of saponin.

Example 3

In this example, the influence of temperature on the yield of saponin was investigated. Accurately weigh 5.0g of Chrysanthemum Leaf Dioscorea powder and put it into the reactor, add 0.5g _of anhydrous AlCl and 100mL of distilled water successively, stir evenly, add 80mL of petroleum ether, set the reactor speed to be 150rpm, and wait until the reactor temperature rises to 150°C , 160 ℃, 170 ℃ after reaction respectively (0.5h, 1.0h, 1.5h, 2.0h, 2.5h). After the end, the diosgenin extract in the petroleum ether organic phase was rotary-evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the analysis method in Example 1, the yield of Dioscorea saponin was calculated, and the influence of reaction time on the yield of saponin extracted by AlCl ₃ in the biphasic system was investigated. The results are shown in Table 2:

Table 2

It can be seen from Table 2 that when three different temperatures are optimized, with the extension of extraction time, when the extraction temperature is 150 °C, the yield of saponin is continuously increasing, and the highest reaction can be obtained for 2.5h. About 1.56% When the extraction temperature was 170 °C, the saponin yield decreased continuously with the increase of extraction time, and the maximum yield was 1.55% at 0.5 h; the difference was that when the temperature was set to 160 °C In the range of 1.0-2.5h, the saponin yield was higher than the other two groups and the relative fluctuation range was small. Comprehensive consideration, 160 ℃ was selected as the optimal extraction temperature, and the yield of 1.65% was obtained after 2.0 h of reaction. On this basis, continue to carry out optimization experiments.

Example 4

In this example, the influence of the concentration of AlCl ₃ solution on the yield of saponin was investigated. Weigh 5.0g of Dioscorea officinalis powder and put it into the reactor, add 100mL of anhydrous AlCl solutions _of different concentrations successively, stir evenly, add 80mL of petroleum ether, set the speed of the reactor to be 150rpm, and wait until the temperature of the reactor rises to 160 ℃. Start timing, the reaction time is 2h. After the end, the diosgenin extract in the petroleum ether organic phase was rotary-evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the analysis method in Example 1, the yield of Dioscorea saponin was calculated, and the effects of different concentrations of AlCl ₃ solutions on the yield of saponin extracted by AlCl ₃ in the two-phase system were investigated. The results are shown in Table 3:

table 3

By adding AlCl ₃ solutions of different concentrations, while keeping the raw material weight and solution volume unchanged, the saponin yield reflected in Table 3 showed a trend of sharply increasing first and then slowly decreasing with the increase of AlCl ₃ concentration. A yield of 1.83% saponin was obtained at an AlCl ₃ concentration of 4.0 g/L. It can be seen that the hydrolysis and extraction of Dioscorea saponin with AlCl ₃ has the best hydrolysis concentration range. When the AlCl ₃ concentration is below 4.0g/L, the low concentration AlCl ₃ solution is obviously not enough to completely hydrolyze the steroidal saponins, and can only break the partial glycosidic bonds of the steroidal saponins, so that the saponins are hydrolyzed into some intermediate products, such as diosgenin -Triglycoside, diosgenin-diglycoside and triglyceride, etc.; and high concentration AlCl ₃ solution may cause the occurrence of side reactions, which makes saponin dehydration and cyclization and affects the yield of saponin.

Example 5

In this example, the influence of the volume ratio of water/petroleum ether solution on the yield of saponin was investigated. Accurately weigh 5.0g of Dioscorea officinalis powder and put it into the reactor, add AlCl solution with a concentration _of 4.0g/L and water/petroleum ether in different volume ratios, keep the total volume of the biphasic solution at 180mL, stir evenly, set the reactor The rotation speed was 150 rpm, and the time was started after the temperature of the reaction kettle rose to 160 °C, and the reaction time was 2 h. After the end, the diosgenin extract in the petroleum ether organic phase was rotary-evaporated, concentrated, crystallized, and dissolved in chromatographic grade methanol to prepare a sample for later use. According to the method in Example 1, the yield of Dioscorea saponin was calculated, and the influence of different volume ratios of water/petroleum ether solutions on the yield _of saponin extracted by AlCl in the biphasic system was investigated. The results are shown in Table 4:

Table 4

A biphasic solution with a suitable volume ratio can completely dissolve the raw materials during the hydrolysis process, extract the produced saponin in a timely and effective manner, and ensure that the extraction rate reaches a saturated state to maximize the extraction rate. Table 4 represents the influence of different volume ratios of water/petroleum ether solution on the yield of saponin. It can be seen from Table 4 that with the decrease of the volume of the aqueous phase solution and the increase of the volume of the organic phase of petroleum ether, the yield of saponin is at Increase slowly and get the maximum value when the volume ratio is 80mL/100mL, which is 1.92%; continue to reduce the amount of water and increase the amount of petroleum ether, and the yield of saponin begins to decrease. Therefore, the optimal volume ratio of water/petroleum ether is 80mL/100mL.

The optimal extraction technical parameters optimized by the above examples are 5 g of Dioscorea officinalis powder, 4.0 g/L of AlCl ₃ solution and a water/petroleum ether volume ratio of 80 mL/100 mL to be reacted for 2.0 h at an extraction temperature of 160 °C, The highest yield of saponin can reach 1.92%.

Comparative Example 1

Method 1: Biphasic acid hydrolysis method: replace aluminum trichloride with 0.5mol/L concentrated sulfuric acid, hydrolyze at 140 °C for 1.5h under the optimal reaction conditions, and repeat the extraction of diosgenin from chrysanthemum leaves with aluminum trichloride. step.

Method 2: Direct acid hydrolysis method: hydrolyze 20.0 g of Dioscorea japonica powder in 200 mL of 2mol/L concentrated sulfuric acid aqueous solution for 2.0 h. After filtering the mixture, the solid residue was washed with 200 mL of hot water and dried at 80°C. The solid residue was then used to extract diosgenin through a Soxhlet extractor with petroleum ether at repeated reflux for 4 hours.

1, extract diosgenin according to the optimal extraction technical parameter after the optimization of the present invention, and compare with the yield of diosgenin prepared by above-mentioned method one and method 2, the results are as shown in Table 5:

table 5

It can be seen from the results in Table 5 that the yield of saponin extracted by the dual _- phase AlCl method of the present invention is much higher (> 2 times) than that of the saponin extracted by hydrolysis of concentrated H ₂ SO ₄ in the dual-phase system under the same conditions. This not only shows that it is feasible to extract saponin with Lewis _acid AlCl ₃ , but also proves that H 2 SO 4 ( H ₂ SO ₄ ( 0.5mol/L=49.0g/L) more saponin production, the reason may be that Lewis acid AlCl3 hydrolyzes part _of hemicellulose and lignin under the action of high temperature water, destroying and loosening the structure of the cell wall, thus making The steroidal saponins attached to the cell wall are more easily hydrolyzed; but compared with the traditional method of direct acid hydrolysis with concentrated H ₂ SO ₄ , the yield of saponin extracted by biphasic AlCl ₃ method is far lower than its 2.59% saponin yield . The reason may be due to: On the one hand, the acid concentration used in the traditional sulfuric acid method is higher, which is 2.0mol/L, which can fully hydrolyze the steroidal saponins attached to the cell wall and the starch-encapsulated saponins, and at the same time release the saponins on the released saponins. The glycosidic bond is completely broken to generate saponin; on the other hand, compared with the traditional method of repeatedly extracting saponin with a Soxhlet extractor, the two-phase solution system is more likely to reach the saturated state of saponin dissolution. When separated, it was extracted into the organic layer without time to hydrolyze the glycosidic bond.

2, investigate the yield, solid residue, acid consumption and other relevant variables of the method of the present invention and above-mentioned method one and method two simultaneously, the results are shown in Table 6:

Table 6

Table 6 shows that lower acidity is beneficial to the formation of starch hydrolyzed into sugar, and the sugar content in the waste liquid of saponin extracted by traditional sulfuric acid method is the highest. The method of binary biphasic AlCl ₃ hydrolysis to extract saponin significantly improves the yield of saponin and shortens the acid hydrolysis time. Meanwhile, the absence of the use and consumption of concentrated H ₂ SO ₄ means that the environmental pollution will also become minimal. The above results show that the present invention is feasible to extract saponin with Lewis acid AlCl ₃ , and the non-traditional acid AlCl ₃ is used to prepare Dioscorea saponin by acid hydrolysis in a two-phase system, which not only improves the yield of saponin, but shortens the The extraction cycle is shortened, and the amount of acid consumed is greatly reduced. More importantly, the pollution to the environment will also become minimal without using concentrated H ₂ SO ₄ , and it has a great application prospect.

Claims (8)

1. a two _- phase system AlCl Hydrolysis prepares the method for diosgenin, it is characterized in that, the diosgenus powder after drying is added in the airtight reactor, adds aluminum trichloride aqueous solution successively, mixes, then adds sherwood oil, in The reaction is carried out at 130-180°C for 0.5-2.5 h, the reaction solution is filtered, the residue is filtered off, the petroleum ether layer is separated and subjected to rotary evaporation, and diosgenin is obtained after recrystallization. 2 . The method according to claim 1 , wherein the reaction temperature is 150-170° C. 3 . 3. The method according to claim 1, wherein the reaction time is 1-2.5h. 4. The method according to claim 1, wherein the concentration of the aluminum trichloride aqueous solution is 1.25g/L～7.5g/L. 5. The method according to claim 1, wherein the volume ratio of water/petroleum ether solution is 140/40, 120/60, 100/80, 80/100, 60/120 or 40/140. 6 . The method according to claim 1 , wherein the addition amount of the diosgenus powder in the reaction system is 26-28 g/L. 7 . 7. method according to claim 1, is characterized in that, adding 5.0g diosgenus powder after drying in closed reactor, adding 4.0g/L aluminum trichloride solution 80mL successively, mixing, then adding 100mL sherwood oil , reacted at 160 ° C for 2 h, filtered the reaction solution, filtered off the residue, separated the petroleum ether layer, carried out rotary evaporation, and obtained diosgenin after recrystallization. 8. The method according to any one of claims 1 to 7, characterized in that, the Dioscorea officinalis is Dioscorea japonica.

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