CN110790811A

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

Info

Publication number: CN110790811A
Application number: CN201910957364.1A
Authority: CN
Inventors: 谢君; 张帅帅; 毕桂灿
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-10-10
Filing date: 2019-10-10
Publication date: 2020-02-14

Abstract

The invention discloses a biphase system AlCl₃The method for preparing diosgenin by hydrolysis comprises the steps of adding dried dioscorea powder into a closed reactor, sequentially adding an aluminum trichloride aqueous solution, uniformly mixing, adding petroleum ether, reacting at 130-180 ℃ for 0.5-2.5 hours, filtering reaction liquid, filtering out residues, separating out a petroleum ether layer, performing rotary evaporation, and recrystallizing to obtain the diosgenin. The invention uses Lewis acid AlCl of unconventional acid₃The method for preparing the dioscorea composita saponin by acidolysis in a two-phase system not only improves the saponin yield and shortens the extraction period, but also greatly reduces the consumption of acid, more importantly, minimizes the environmental pollution under the condition of not using concentrated sulfuric acid, and has a wide application prospect.

Description

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

Technical Field

The invention relates to the technical field of plant extraction, in particular to saponin extraction from dioscorea opposita resources, and more particularly relates to a biphase system AlCl₃A method for preparing diosgenin by hydrolysis.

Background

Due to the remarkable effects of steroid hormones in resisting tumors, regulating the immune system, treating cardiovascular diseases, rheumatic diseases, bacterial encephalitis, skin diseases and the like, the research on steroid hormones in various countries in the world is rapidly developed (Chen et al, 2015; Wang et al, 2007). However, the way of industrial development of steroid hormones faces the defects of scarce drug sources, low content of raw materials and high cost of artificial synthesis, so that the requirement of large-scale batch production cannot be met. The discovery of diosgenin creates a precedent for synthesizing steroid drugs by using dioscorea plants as raw materials, and promotes the research process of development and utilization of dioscorea plant resources. The carbon skeleton structure of diosgenin is very similar to that of steroid hormone drugs, and is the most ideal precursor for synthesizing steroid hormone drugs. Diosgenin, known as saponin in the industry, is linked to sugars at its C-3 and C-26 positions by glycosidic bonds and exists in the plant body in the form of steroid saponins.

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

The chemical hydrolysis method mainly refers to an extraction preparation method for releasing diosgenin by hydrolyzing glycosidic bonds with traditional inorganic strong acid such as sulfuric acid, hydrochloric acid and the like. The traditional acid hydrolysis process for extracting diosgenin is proposed by Rothrock et al, which comprises hydrolyzing rhizome of Dioscorea opposita directly with inorganic acid, washing with water to neutrality, oven drying, performing water bath circulation extraction with organic solvent such as petroleum ether, and crystallizing to obtain saponin product (Rothrock et al, 1957). The results of Zyuqing and the like investigating the effect of extracting dioscorea zingiberensis diosgenin by sulfuric acid hydrolysis show that: hydrolyzing at 2mol/L for 4h, and performing Soxhlet extraction for 8h to obtain saponin with yield of 1.28% (radix Cynanchi Margarizanii, etc., 2006). The direct acid hydrolysis method has simple process and simple and convenient 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 inorganic strong acid is used for hydrolysis, not only can equipment be corroded and the water consumption is large during washing, but also the discharged acidic wastewater is difficult to recycle, and a part of saponin in waste residues cannot be separated from a large amount of starch and cellulose substances after acidolysis. This directly causes environmental pollution and waste of resources, and also affects the yield of diosgenin.

The biological hydrolysis method for extracting saponin is to directly convert saponin into saponin by utilizing biological enzymes produced by some mildew microorganisms, and has the advantages of environmental friendliness and no pollution. Some researchers respectively use aspergillus oryzae, trichoderma harzianum and aspergillus awamori to inoculate and culture dioscorea zingiberensis so as to ferment and directly extract saponin by utilizing saponin conversion, and research results prove that the yield of the dioscorea zingiberensis saponin is different from 17.06 mg/g-74.26 mg/g, the difference is obvious, the process and mechanism of saponin hydrolysis by biotransformation are complex, the saponin yield is low, and the generated byproducts are more (Chen et al, 2018; Liu et al, 2010; Dongyeng et al, 2009). Although the bioconversion of saponin into diosgenin by using a combination of biological and enzymatic hydrolysis methods is of interest to many researchers without environmental pollution, it has limited application in the diosgenin industry due to the uneconomical cost of enzyme preparations and the inefficiency of bioconversion compared to conventional direct acid hydrolysis methods. Therefore, a method for preparing diosgenin with high saponin yield, low acid consumption and small discharge capacity is needed.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provide a two-phase system AlCl₃A method for preparing diosgenin by hydrolysis.

The above object of the present invention is achieved by the following technical solutions:

biphase system AlCl₃A method for preparing diosgenin by hydrolysis comprises the steps of adding dried yam powder into a closed reactor, sequentially adding an aluminum trichloride aqueous solution, uniformly mixing, adding petroleum ether, reacting at 130-180 ℃ for 0.5-2.5 h, filtering reaction liquid, filtering out residues, separating out a petroleum ether layer, performing rotary evaporation, and recrystallizing to obtain the diosgenin.

The method adopts the non-traditional acid Lewis acid aluminum trichloride to carry out acidolysis in a water/petroleum ether two-phase system to prepare the dioscorea composita saponin, the Lewis acid aluminum trichloride is a water-soluble inorganic salt, the aqueous solution is acidic, safe, nontoxic and easy to operate, and the Lewis acid aluminum trichloride can be used for replacing inorganic strong acid sulfuric acid hydrolysis to prepare the saponin. Compared with the traditional method of directly hydrolyzing the dioscorea opposita raw material by acid and extracting the acidolysis residue by repeated reflux of the organic solvent to extract the saponin, the diphasic system acidolysis extraction method of the invention utilizes the solubility difference of the saponin and the saponin to synchronously extract the saponin into the petroleum ether organic phase while hydrolyzing the saponin, and adopts the Lewis acid and the petroleum ether to synchronously perform the acidolysis of the saponin and the extraction of the saponin, thereby simplifying the operation, saving the time, greatly reducing the use amount of the acid, having higher saponin extraction rate and small discharge capacity.

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

More preferably, the reaction temperature is 160 ℃.

Preferably, the reaction time is 1-2.5 h.

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

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

When AlCl is present₃Low concentration of AlCl at too low a concentration₃The solution is obviously not enough to completely hydrolyze the steroid saponin, and can only break partial glycosidic bonds of the steroid saponin, so that the saponin is hydrolyzed into a plurality of intermediate products, such as diosgenin-triglycoside, diosgenin-diglucoside, trillin and the like; and too high concentration of AlCl₃The solution may cause side reaction, so that the dehydration and cyclization of saponin further affect the yield of saponin. Therefore, the concentration of the aluminum trichloride aqueous solution is preferably 2.5 to 5.0g/L (more preferably 4.0 g/L).

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

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

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

Preferably, the yam is dioscorea composita; because the dioscorea composita is based on the characteristic of high saponin content, the dioscorea composita is a more ideal saponin production raw material.

As a preferred embodiment, the biphasic system AlCl₃The method for preparing diosgenin by hydrolysis comprises adding dried Dioscorea composita powder 5.0g into sealed containerSequentially adding 4.0g/L aluminum trichloride solution 80mL into reactor, mixing, adding 100mL petroleum ether, reacting at 160 deg.C for 2 hr, filtering the reaction solution, filtering to remove residue, separating out petroleum ether layer, rotary evaporating, recrystallizing to obtain diosgenin with saponin yield of 1.92%

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

the invention uses Lewis acid AlCl of unconventional acid₃The dioscorea composita saponin is prepared by acidolysis in a water/petroleum ether two-phase system, so that the saponin yield is improved and can reach 1.92 percent, the extraction period is shortened, the consumption of acid is greatly reduced, more importantly, the environmental pollution is minimized under the condition of not using concentrated sulfuric acid, and the application prospect is wide.

Drawings

FIG. 1 shows HPLC chromatograms of saponin standard (top) and saponin sample prepared according to the present invention (bottom).

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

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

Experimental materials: the three-year-old dioscorea composita tuber is obtained from dioscorea composita planting base in Wenyuan county of Guangdong province. Cutting the three-year-old dioscorea composita rhizome into slices, freeze-drying for 24h by using a freeze dryer to remove excessive water, grinding into powder by using a grinder, filling into a sealing bag, and storing in a constant-temperature dewatering sealer for later use.

Diosgenin standard (greater than or equal to 99.9%, HPLC grade) from Fluka corporation; methanol, chromatographically pure, available from Sigma-Aldrich, usa; sulfuric acid, analytically pure, purchased from Guangzhou chemical reagent works; petroleum ether (60-90 ℃), analytically pure, purchased from Tianjin Fuyu Fine chemical Co., Ltd; aluminum trichloride, analytically pure, was purchased from Shanghai national pharmaceutical group chemical reagents, Inc.

Example 1

Accurately weighing 5.0g of Dioscorea composita powder, putting into a reaction kettle, and sequentially weighing 0.5g of anhydrous AlCl₃And 100mL of distilled water, stirring uniformly, adding 80mL of petroleum ether, setting the rotating speed of the reaction kettle at 150rpm, and reacting for 2.0h at the extraction temperature of 160 ℃. After the reaction is finished, the diosgenin extract in the petroleum ether organic phase is subjected to rotary evaporation, concentration and crystallization, and is dissolved by chromatographic grade methanol for preparing a sample for later use. Then analyzing and quantifying diosgenin.

Method for analyzing diosgenin

Diluting the dissolved diosgenin extract with methanol to a certain volume, filtering with 0.22 μm filter membrane, collecting into 1mL sample bottle, and preparing. High performance liquid chromatography detection is carried out by taking a standard saponin product (the purity is more than or equal to 99.9%) of Fluka company as a reference. The specific process is as follows: dissolving diosgenin standard with methanol, and performing high performance liquid chromatography to obtain solution with concentration of 4.0 × 10^-3mg/mL、2.0×10^-2mg/mL、1.0×10^-1measuring the mg/mL and 0.5mg/mL diosgenin standard, drawing a standard curve by taking the chromatographic peak area of the diosgenin standard as the basis, taking the peak area as the sample quantitative basis, and calculating the saponin yield by the following formula:

wherein W: weight of Dioscorea composita raw material, g; c: the concentration of saponin is mg/mL; n: dilution factor. High performance liquid chromatography measurement parameters: a UV detector; c18 column (4.6 mm. times.250 mm,5 μm); temperature of the column oven: 40 ℃; mobile phase: methanol/water; the flow rate is 1.0 mL/min; the loading volume was 10. mu.L. Each assay was repeated three times. The HPLC chromatograms of saponin standard and preparation sample are shown in FIG. 1.

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

Example 2

This example examines the effect of temperature on saponin yield. Accurately weighing 5.0g of Dioscorea composita powder, putting into a reaction kettle, and sequentially adding 0.5g of anhydrous AlCl₃And 100mL of distilled water, stirring uniformly, adding 80mL of petroleum ether, setting the rotating speed of the reaction kettle at 150rpm, starting timing after the temperature of the reaction kettle rises to a predetermined series of temperatures (130 ℃, 140 ℃, 150 ℃, 160 ℃, 170 ℃ and 180 ℃), and reacting for 1.5 h. After the reaction is finished, the diosgenin extract in the petroleum ether organic phase is subjected to rotary evaporation, concentration and crystallization, and is dissolved by chromatographic grade methanol for preparing a sample for later use. The yield of dioscorea composita saponin was calculated according to the analysis method of example 1, and the temperature was examined for AlCl in a biphasic system₃Influence of saponin yield. The results are shown in table 1:

TABLE 1

As can be seen from Table 1, the temperature is adjusted to the anhydrous AlCl₃The yield impact of extracting dioscorea composita saponin for acid catalyst hydrolysis is very large. When the reaction temperature is lower than 150 ℃, the saponin yield is gradually improved along with the rise of the reaction temperature, and at 150 ℃, the saponin yield reaches about 1.3 percent. The yield of saponin is fluctuated along with the continuous increase of the temperature, and the yield of saponin is firstly decreased and then increased and then decreased, but generally speaking, the yield of saponin exceeds 1.2% and has little difference when the reaction temperature is 150 ℃, 160 ℃ and 170 ℃. In order to not influence the maximum extraction rate of dioscorea composita saponin, the three temperatures are optimized in the next time optimization experiment, so that the most suitable temperature is selected for extracting saponin.

Example 3

This example examines the effect of temperature on saponin yield. Accurately weighing 5.0g of Dioscorea composita powder, putting into a reaction kettle, and sequentially adding 0.5g of anhydrous AlCl₃And 100mL of distilled water, stirring uniformly, adding 80mL of petroleum ether, setting the rotating speed of the reaction kettle at 150rpm, and reacting respectively (0.5h, 1.0h, 1.5h, 2.0h and 2.5h) after the temperature of the reaction kettle rises to 150 ℃, 160 ℃ and 170 ℃. After the reaction is finished, the diosgenin extract in the petroleum ether organic phase is subjected to rotary evaporation, concentration and crystallization, and is dissolved by chromatographic grade methanol for preparing a sample for later use. Dioscorea composita soap was analyzed according to example 1Calculating the yield of the elements, and investigating the reaction time on AlCl in a two-phase system₃Influence of saponin yield. The results are shown in table 2:

TABLE 2

As can be seen from Table 2, when three different temperatures are optimized, the yield of saponin is continuously increased along with the extension of the extraction time when the extraction temperature is 150 ℃, and the yield of 1.56 percent can be obtained at the maximum after 2.5 hours of reaction; when the extraction temperature is 170 ℃, the saponin yield is continuously reduced along with the increase of the extraction time, and the maximum yield is 1.55% in 0.5 h; different from the above, when the temperature is set to 160 ℃, the saponin yield is higher than that of the other two groups and the relative fluctuation range is small within the range of 1.0-2.5 h. Comprehensively considering, selecting 160 ℃ as the optimal extraction temperature, and reacting for 2.0h to obtain the yield of 1.65%. On this basis, the optimization experiment was continued.

Example 4

This example examines AlCl₃Influence of solution concentration on saponin yield. Weighing 5.0g Dioscorea composita powder, putting into a reaction kettle, and sequentially adding anhydrous AlCl with different concentrations₃And (3) stirring 100mL of the solution uniformly, adding 80mL of petroleum ether, setting the rotating speed of the reaction kettle to be 150rpm, starting timing after the temperature of the reaction kettle rises to 160 ℃, and reacting for 2 hours. After the reaction is finished, the diosgenin extract in the petroleum ether organic phase is subjected to rotary evaporation, concentration and crystallization, and is dissolved by chromatographic grade methanol for preparing a sample for later use. The yield of dioscorea composita saponin was calculated according to the analysis method of example 1, and AlCl was examined at different concentrations₃AlCl in solution-to-biphasic system₃Influence of saponin yield. The results are shown in Table 3:

TABLE 3

By adding different concentrations of AlCl₃Solution, keeping the weight of the raw material and the volume of the solutionThe saponin yields reflected in Table 3 follow AlCl without change₃The concentration increase shows a tendency of first sharp rise and then slow decline, and AlCl at 4.0g/L₃The saponin with the yield of 1.83% is obtained under the concentration. Thus, use of AlCl₃The hydrolysis extraction of dioscorea composita saponin has the best hydrolysis concentration range. When AlCl is present₃Low concentration AlCl at concentration below 4.0g/L₃The solution is obviously not enough to completely hydrolyze the steroid saponin, and can only break partial glycosidic bonds of the steroid saponin, so that the saponin is hydrolyzed into a plurality of intermediate products, such as diosgenin-triglycoside, diosgenin-diglucoside, trillin and the like; while high concentration AlCl₃The solution may cause side reaction, so that the dehydration and cyclization of saponin further affect the yield of saponin.

Example 5

This example examines the effect of water/petroleum ether solution volume ratio on saponin yield. Accurately weighing 5.0g of Dioscorea composita powder, adding into a reaction kettle, and adding AlCl with concentration of 4.0g/L₃Keeping the total volume of the two-phase solution to be 180mL by using the solution and water/petroleum ether with different volume ratios, uniformly stirring, setting the rotating speed of the reaction kettle to be 150rpm, starting timing after the temperature of the reaction kettle rises to 160 ℃, and keeping the reaction time to be 2 h. After the reaction is finished, the diosgenin extract in the petroleum ether organic phase is subjected to rotary evaporation, concentration and crystallization, and is dissolved by chromatographic grade methanol for preparing a sample for later use. The yield of dioscorea composita saponin was calculated according to the method of example 1, and different volume ratios of water/petroleum ether solution versus AlCl in the biphasic system were examined₃Influence of saponin yield. The results are shown in Table 4:

TABLE 4

The proper volume ratio of the two-phase solution can completely dissolve the raw materials in the hydrolysis process, timely and effectively extract the produced saponin and ensure that the extraction rate reaches the maximum saturated state, and is of great importance. Table 4 represents the effect on saponin yield under different volume ratios of water/petroleum ether solution conditions, and it can be seen from table 4 that: with the reduction of the volume of the aqueous phase solution and the increase of the volume of the petroleum ether organic phase, the yield of the saponin is slowly increased and reaches the maximum value of 1.92 percent when the volume ratio is 80mL/100 mL; the water consumption and petroleum ether consumption are continuously reduced, and the saponin yield begins to decrease. Thus, the optimal water/petroleum ether volume ratio is 80mL/100 mL.

The optimized extraction technical parameter of the embodiment is 5g of dioscorea composita powder, and 4.0g/L of AlCl is used₃The volume ratio of the solution to water/petroleum ether is 80mL/100mL, the reaction is carried out for 2.0h at the extraction temperature of 160 ℃, and the highest yield of the saponin can reach 1.92 percent.

Comparative example 1

The method comprises the following steps: and (3) a two-phase acid hydrolysis method: 0.5mol/L concentrated sulfuric acid is used to replace aluminum trichloride, hydrolysis is carried out for 1.5h under the optimal reaction condition, namely 140 ℃, and the step of extracting dioscorea composita saponin by aluminum trichloride is repeated.

The second method comprises the following steps: direct acid hydrolysis: 20.0g of Dioscorea composita powder is hydrolyzed in 200mL of 2mol/L concentrated sulfuric acid aqueous solution for 2.0 h. After filtration of the mixture, the solid residue was washed with 200mL of hot water and dried at 80 ℃. The solid residue was then used to extract diosgenin by repeated reflux extraction with petroleum ether for 4 hours using a soxhlet extractor.

1. The diosgenin is extracted according to the optimized extraction technical parameters of the invention, and compared with the yield of the dioscorea composita saponin prepared by the first method and the second method, the result is shown in table 5:

TABLE 5

As can be seen from the results in Table 5, the biphasic AlCl of the present invention₃The yield of saponin extracted by the method is far higher (more than 2 times) than that of the saponin extracted by the method in a two-phase system under the same condition₂SO₄The yield of saponin extracted by hydrolysis is not only proved by using Lewis acid AlCl₃It is feasible to extract saponinIt is also proved that in AlCl₃With a lower acid concentration (4.0g/L), a higher H concentration than the acid concentration is obtained₂SO₄(0.5mol/L ═ 49.0g/L) higher saponin yields, probably due to the Lewis acid AlCl₃Hydrolyzing part of hemicellulose and lignin under the action of high-temperature water to destroy and loosen the structure of the cell wall, so that the steroid saponin connected to the cell wall is easier to hydrolyze; but is at the same concentration of H₂SO₄Biphasic AlCl compared to the conventional method of direct acid hydrolysis₃The yield of the saponin extracted by the method is far lower than that of the saponin with the yield of 2.59 percent. The cause of this may be due to: on one hand, the acid concentration used by the traditional sulfuric acid method is higher and is 2.0mol/L, so that the steroid saponin connected to the cell wall and the saponin wrapped by starch can be fully hydrolyzed, and meanwhile, the glycosidic bond on the released saponin is thoroughly broken to generate the saponin; on the other hand, compared with the traditional method of repeatedly extracting saponin by refluxing with a Soxhlet extractor, a two-phase solution system is easier to reach the saturated state of dissolved saponin, and meanwhile, a part of fat-soluble saponin is extracted to an organic layer without time for hydrolyzing glycosidic bonds while being separated.

2. The yields, residual solids, acid consumption and other relevant variables of the process of the invention and of the first and second processes described above were also investigated, the results of which are shown in Table 6:

TABLE 6

Table 6 shows that lower acidity favors the formation of sugars by hydrolysis of starch, with the highest sugar content in the waste liquid from the traditional sulfuric acid process for saponin extraction. Binary biphase AlCl₃The method for extracting saponin by hydrolysis improves saponin yield remarkably and shortens acid hydrolysis time without concentrated H₂SO₄The use and consumption of (a) means that environmental pollution will also become minimal. The above results show that the present invention uses a Lewis acid AlCl₃The extraction of saponin is feasible by using Lewis acid AlCl of non-traditional acid₃The acidolysis in a two-phase system for preparing dioscorea composita saponin can not only improve the saponin yield and shorten the extraction period, but also can be used for preparing dioscorea composita saponinThe consumption of acid is greatly reduced, and more importantly, concentrated H is not used₂SO₄In the case of (2), the pollution to the environment is minimized, and the method has a wide application prospect.

Claims

1. Biphase system AlCl₃The method for preparing the diosgenin by hydrolysis is characterized by adding dried dioscorea powder into a closed reactor, sequentially adding an aluminum trichloride aqueous solution, uniformly mixing, adding petroleum ether, reacting at 130-180 ℃ for 0.5-2.5 h, filtering a reaction solution, filtering out residues, separating out a petroleum ether layer, performing rotary evaporation, and recrystallizing to obtain the diosgenin.

2. The method according to claim 1, wherein the reaction temperature is 150 to 170 ℃.

3. The method according to claim 1, wherein the reaction time is 1-2.5 h.

4. The method of claim 1, wherein the concentration of the aqueous solution of aluminum trichloride is from 1.25g/L to 7.5 g/L.

5. The method of claim 1, wherein the water/petroleum ether solution volume ratio 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 yam powder in the reaction system is 26-28 g/L.

7. The method as claimed in claim 1, wherein the dioscin is obtained by adding dried 5.0g of dioscin powder into a closed reactor, sequentially adding 80mL of 4.0g/L aluminum trichloride solution, mixing, adding 100mL of petroleum ether, reacting at 160 ℃ for 2h, filtering the reaction solution, filtering to remove residues, separating out a petroleum ether layer, performing rotary evaporation, and recrystallizing.

8. The method according to any one of claims 1 to 7, wherein the yam is Dioscorea composita.