Application and preparation method of 5 alpha-hydroxyl laxogenin

Abstract

The embodiment of the invention discloses application and a preparation method of 5 alpha-hydroxyl laxogenin. Results of growth curve tests for inhibiting different florae, inhibition zone tests and long-time-range escherichia coli fluorescent staining comparison prove that the 5alpha-hydroxylaxogenin has certain antibacterial property on gram-negative bacteria, gram-positive bacteria and saccharomycetes, has obvious antibacterial and bactericidal effects, and can be used for preparing a novel antibacterial agent for preparing the 5-alpha-hydroxylaxogenin, and the application of the 5-alpha-hydroxylaxogenin in preparing the antibacterial agent for preparing the 5-alpha-hydroxylaxogenin. The compound can be applied to antibacterial drugs or health care products, and has good development and application prospects. The preparation method disclosed by the invention is simple to operate, good in chiral selectivity and mild in reaction condition, and large-scale preparation of the 5 alpha-hydroxyl laxogenin can be realized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application and preparation method of 5α-hydroxylasiosapogenin. Background Technology

[0002] Diosgenin is a steroidal compound, and steroids are an important class of bioactive substances in plants. Research on steroids has always held a significant position in natural product chemistry. Currently, among the derivatives of diosgenin, lasiogenin is a widely used plant growth regulator. However, there are still few reports in the literature regarding the application and synthesis methods of 5α-hydroxylasiogenin, which is obtained by further modifying the structure of lasiogenin.

[0003] In terms of applications, the paper "Polyphosphazene-based nanocarriers for the release of agrochemicals and potential anticancerdrugs" reports the potential application of 5α-hydroxylasiosperidin in inhibiting tumor cell growth; the paper "In Vitro Neuroprotective and Anti-Inflammatory Activities of Natural and Semi-Synthetic Spirosteroid Analogues" reports its application prospects as an anti-inflammatory drug. Currently, there are no reports on the antibacterial and antimicrobial applications of 5α-hydroxylasiosperidin.

[0004] Regarding the synthetic method, the literature "Synthesis of New Steroidal Carbamates with Plant-Growth-Promoting Activity: Theoretical and Experimental Evidence" describes the epoxidation of the diosgenin double bond using m-CPBA (m-chloroperoxybenzoic acid), followed by ring-opening oxidation with perchloric acid to synthesize 5α-hydroxylasiosuccinate. However, mCPBA and perchloric acid are explosive, making them difficult to control during scale-up production and posing safety management challenges. Furthermore, this method exhibits poor control over the chirality of intermediates, leading to a tendency for racemization in the product and affecting the purity and activity of the final product. There is significant room for optimization in the existing synthetic routes. Summary of the Invention

[0005] Therefore, this invention provides an application and preparation method of 5α-hydroxylasiosapogenin.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides the use of 5α-hydroxylactoside in the preparation of antibacterial drugs or health products, wherein the 5α-hydroxylactoside has the structure shown in formula (I):

[0008]

[0009] Furthermore, the bacteria include Gram-negative bacteria, Gram-positive bacteria, and yeasts.

[0010] Furthermore, the Gram-negative bacteria are Escherichia coli; the Gram-positive bacteria are Staphylococcus, such as Staphylococcus aureus and Staphylococcus epidermidis; and the yeast is Candida, such as Candida glabrata.

[0011] According to a second aspect of the present invention, the present invention provides a method for preparing 5α-hydroxylasiosapogenin, the reaction route of which is as follows:

[0012]

[0013] The method includes the following steps:

[0014] S1, diosgenin and acetic anhydride undergo an acetylation reaction in the presence of an acid-binding agent, acetic anhydride and solvent to obtain intermediate M1;

[0015] S2 and intermediate M1 undergo a dihydroxylation reaction with potassium osmium dihydrate in the presence of pyridine to obtain intermediate M2;

[0016] S3 and intermediate M2 undergo an oxidation reaction with an oxidant in the presence of a solvent to obtain intermediate M3;

[0017] S4 and intermediate M3 undergo hydrolysis with a base in the presence of a solvent to yield 5α-hydroxylasiosaccharide.

[0018] Furthermore, in step S1,

[0019] The acid-binding agent is selected from at least one of triethylamine, pyridine, N,N-diisopropylethylamine, N-methylmorphophallene, and 4-dimethylaminopyridine, preferably triethylamine;

[0020] The solvent is selected from at least one of tetrahydrofuran, N,N-dimethylformamide, acetone, and dioxane, preferably tetrahydrofuran;

[0021] The molar ratio of the acid-binding agent to diosgenin is 1.0 to 5.0:1, for example, it can be 1.0:1, 2.0:1, 3.0:1, 4.0:1, 5.0:1 or any value formed by any two of them, preferably 3.0:1;

[0022] The molar ratio of acetic anhydride to diosgenin is 1.0 to 3.0:1, for example, it can be 1.0:1, 1.5:1, 2.0:1, 2.5:1, 3.0:1 or any value formed by any two of them, preferably 2.5:1;

[0023] The volume ratio of the solvent to the mass ratio of diosgenin is 5 to 20 mL: 1 g, for example, it can be 5 mL: 1 g, 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g or any value between any two of them, preferably 10 mL: 1 g.

[0024] The acetylation reaction is carried out at a temperature of 30 to 70°C, for example, 30°C, 40°C, 50°C, 60°C, 70°C or any combination thereof, preferably 60°C, and for a time of 6 to 20 hours, for example, 6, 8, 10, 15, 20 hours or any combination thereof, preferably 10 hours.

[0025] Step S1 further includes: adding the acetylation reaction solution to water and stirring, then filtering to obtain the intermediate M1;

[0026] The volume ratio of water to diosgenin is 10-30 mL:1 g, for example, it can be 10 mL:1 g, 15 mL:1 g, 20 mL:1 g, 25 mL:1 g, 30 mL:1 g or any value formed by any two of them, preferably 20 mL:1 g.

[0027] Furthermore, in step S2,

[0028] The volume ratio of pyridine to diosgenin is 3 to 10 mL: 1 g, for example, it can be 3 mL: 1 g, 5 mL: 1 g, 8 mL: 1 g, 10 mL: 1 g or any value between any two of them, preferably 5 mL: 1 g;

[0029] The molar ratio of potassium osmium tetroxide dihydrate to diosgenin is 1.0 to 2.0:1, for example, it can be 1.0:1, 1.1:1, 1.2:1, 1.5:1, 2.0:1 or any value formed by any two of them, preferably 1.2:1;

[0030] The dihydroxylation reaction is carried out at a temperature of 20–30°C for a time of 1–12 h, for example, 1, 2, 4, 8, 12 h or any combination of these values, preferably 8 h.

[0031] Step S2 further includes: adding water and extraction solvent to the reaction solution of the dihydroxylation reaction, extracting and separating the liquid, concentrating and filtering the organic phase to obtain the intermediate M2;

[0032] The extraction solvent is selected from at least one of dichloromethane, ethyl acetate, and methyl tert-butyl ether, preferably dichloromethane;

[0033] The volume ratio of water to diosgenin is 5 to 20 mL: 1 g, for example, it can be 5 mL: 1 g, 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g or any value between any two of them, preferably 15 mL: 1 g.

[0034] The volume ratio of the extraction solvent to the mass ratio of diosgenin is 5 to 20 mL: 1 g, for example, it can be 5 mL: 1 g, 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g or any value formed by any two of them, preferably 15 mL: 1 g.

[0035] Furthermore, in step S3,

[0036] The oxidant is selected from at least one of pyridinium chlorochromate, pyridinium dichromate, chromium trioxide, and potassium dichromate, preferably pyridinium chlorochromate;

[0037] The solvent is selected from at least one of tetrahydrofuran, dioxane, dichloromethane, dichloroethane, chloroform, and carbon tetrachloride, preferably tetrahydrofuran;

[0038] The molar ratio of the oxidant to intermediate M2 is 1.0 to 2.0:1, for example, it can be 1.0:1, 1.1:1, 1.2:1, 1.5:1, 2.0:1 or any value formed by any two of them, preferably 1.5:1;

[0039] The volume ratio of the solvent to the mass ratio of intermediate M2 is 5 to 20 mL: 1 g, for example, it can be 5 mL: 1 g, 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g or any value formed by any two of them, preferably 10 mL: 1 g.

[0040] The oxidation reaction is carried out at a temperature of 20–30°C for a time of 1–8 hours, for example, 1, 2, 4, 8 hours or any combination of these values, preferably 2 hours.

[0041] Step S3 further includes: adding water dropwise to the reaction solution of the oxidation reaction, stirring to precipitate crystals, filtering, and obtaining the intermediate M3;

[0042] The volume ratio of water to intermediate M2 is 10-30 mL: 1 g, for example, it can be 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g, 25 mL: 1 g, 30 mL: 1 g or any value formed by any two of them, preferably 20 mL: 1 g.

[0043] The stirring and crystallization temperature is 20-30°C, and the time is 1-4 hours, for example, it can be 1, 2, 3, 4 hours or any value formed by any two of them, preferably 2 hours.

[0044] Furthermore, in step S4,

[0045] The solvent is selected from at least one of methanol, ethanol, isopropanol, n-butanol, and tetrahydrofuran, preferably methanol;

[0046] The alkali is selected from at least one of potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, and lithium carbonate, preferably potassium hydroxide;

[0047] The volume ratio of the solvent to the mass ratio of intermediate M2 is 5 to 20 mL: 1 g, for example, it can be 5 mL: 1 g, 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g or any value formed by any two of them, preferably 10 mL: 1 g.

[0048] The molar ratio of the alkali to intermediate M2 is 1.0 to 5.0:1, for example, it can be 1.0:1, 2.0:1, 3.0:1, 4.0:1, 5.0:1 or any value formed by any two of them, preferably 3.0:1;

[0049] The hydrolysis reaction is carried out at a temperature of 30–70°C for a time of 1–8 hours, for example, 1, 2, 4, 8 hours or any combination of these values, preferably 4 hours.

[0050] Step S4 further includes: adding water and extraction solvent to the reaction solution of the hydrolysis reaction, extracting and separating the liquid, concentrating the organic phase and adding a refining solvent, pulping, filtering and drying to obtain the 5α-hydroxylasiosaurin product.

[0051] The volume ratio of water to intermediate M2 is 10-30 mL: 1 g, for example, it can be 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g, 25 mL: 1 g, 30 mL: 1 g or any value formed by any two of them, preferably 20 mL: 1 g.

[0052] The extraction solvent is selected from at least one of dichloromethane, toluene, and methyl tert-butyl ether, preferably dichloromethane;

[0053] The volume ratio of the extraction solvent to the mass ratio of intermediate M2 is 5 to 20 mL: 1 g, for example, it can be 5 mL: 1 g, 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g or any value between any two of them, preferably 10 mL: 1 g.

[0054] The refining solvent is selected from at least one of methanol, ethanol, dichloromethane, ethyl acetate, acetone, and methyl tert-butyl ether, preferably ethanol;

[0055] The volume ratio of the refined solvent to the mass ratio of intermediate M2 is 3 to 10 mL: 1 g, for example, it can be 3 mL: 1 g, 5 mL: 1 g, 8 mL: 1 g, 10 mL: 1 g or any value formed by any two of them, preferably 5 mL: 1 g.

[0056] The pulping temperature is 20-30℃ and the time is 1-8h, for example, it can be 1, 2, 4, 8h or any value formed by any two of them, preferably 4h.

[0057] The embodiments of the present invention have the following advantages:

[0058] This invention has discovered that 5α-hydroxylasiosine has significant antibacterial activity. Through growth curve experiments, inhibition zone experiments, and long-term fluorescence staining comparisons of different bacterial groups, it has been confirmed that 5α-hydroxylasiosine exhibits antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, and yeast, demonstrating significant antibacterial and bactericidal effects. It can be applied to antibacterial drugs or health products and has promising development and application prospects.

[0059] The preparation method of the present invention is simple to operate, has good chiral selectivity, and mild reaction conditions, and can realize the large-scale preparation of 5α-hydroxylasiosapogenin. Attached Figure Description

[0060] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0061] Figure 1 The NMR spectrum of the 5α-hydroxylasiosapogenin product;

[0062] Figure 2 The growth inhibition curve of 5α-hydroxylactoside saponin against Escherichia coli;

[0063] Figure 3 The growth curve of 5α-hydroxylasiosperidin inhibiting Staphylococcus aureus growth;

[0064] Figure 4 The curve of 5α-hydroxylactoside's inhibition of Staphylococcus epidermidis growth;

[0065] Figure 5The growth curve of 5α-hydroxylactoside inhibitory effect on Candida glabrata;

[0066] Figure 6 Comparison of Escherichia coli inhibition zone test results;

[0067] Figure 7 Comparison of Staphylococcus aureus inhibition zone test results;

[0068] Figure 8 This is a comparison image of fluorescence staining of Escherichia coli over a long period of time. Detailed Implementation

[0069] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0071] Example 15: Preparation method of α-hydroxylasiosapogenin

[0072] Step S1, Preparation of intermediate M1

[0073]

[0074] 100 mL of tetrahydrofuran was added to a 500 mL three-necked flask, followed by diosgenin (10.0 g, 24.1 mmol). The mixture was stirred until a pale yellow clear solution was obtained. Then, triethylamine (7.3 g, 72.3 mmol) and acetic anhydride (6.2 g, 60.3 mmol) were added sequentially. The mixture was heated to 60 °C and stirred for 10 h. After the reaction was complete as monitored by TLC, the temperature was lowered to 25 °C, and the reaction solution was added dropwise to 200 mL of purified water. A solid precipitated, and after stirring for 1 h, the mixture was filtered. The filter cake was washed with 20 mL of purified water to obtain intermediate M1, a pale yellow solid with a wet weight of 12.8 g.

[0075] Step S2, Preparation of intermediate M2

[0076]

[0077] Add 50 mL of pyridine to a 500 mL three-necked flask, then add 12.8 g of wet intermediate M1 obtained in step 1, and stir to disperse evenly; then add potassium osmium tetroxide dihydrate (10.6 g, 28.9 mmol), and stir at 25 °C for 8 h. After the reaction is complete as monitored by TLC, add 150 mL of water and 150 mL of dichloromethane, extract and separate the liquid, transfer the organic phase to a rotary evaporator flask, heat to 60 °C and concentrate under reduced pressure to dryness to obtain intermediate M2, which is a pale yellow powder and lumpy solid with a dry weight of 8.9 g.

[0078] Step S3, Preparation of intermediate M3

[0079]

[0080] Add 89 mL of tetrahydrofuran to a 500 mL three-necked flask, then add intermediate M2 (8.9 g, 18.1 mmol) obtained in step 2, and stir until dissolved. Then add pyridinium chlorochromate (5.9 g, 27.15 mmol), and stir at 25 °C for 2 h. After the reaction is complete as monitored by TLC, add 178 mL of purified water dropwise to crystallize. After stirring for 2 h, filter, and wash the filter cake with 36 mL of purified water to obtain intermediate M3, a pale yellow solid with a wet weight of 10.8 g.

[0081] Step S4: Preparation of the 5α-hydroxylasiosapogenin product

[0082]

[0083] Add 89 mL of methanol to a 500 mL three-necked flask, then add 108 g of wet intermediate M3 obtained in step 3, and stir to disperse evenly; then add potassium hydroxide (3.0 g, 54.3 mmol), heat to 50 °C, and stir for 4 h. After the reaction is complete as monitored by TLC, cool to 25 °C, add 178 mL of water and 89 mL of dichloromethane, extract and separate the liquid, transfer the organic phase to a rotary evaporator flask, heat to 50 °C and concentrate under reduced pressure to dryness to obtain crude 5α-hydroxylasiosapogenin.

[0084] Add 44 mL of ethanol to a 250 mL three-necked flask, then add the crude product, and stir and slurry at 25 °C for 4 h; filter, wash the wet product twice with 18 mL of ethanol, and dry it in a forced-air dryer at 50 °C to obtain 6.4 g of 5α-hydroxylasiosaurin, which is a white powder, with a total yield of 59.42%.

[0085] The NMR spectrum of the 5α-hydroxylasiosapogenin product prepared in this embodiment is as follows: Figure 1 As shown, the NMR data are as follows:

[0086] 1H NMR (400MHz, DMSO-d6): δ = 5.33 (s, 1H), 4.34 (d, J = 5.6Hz, 1H), 4.28 (q, J = 7.2H z,1H),3.69(qd,J=11.2,10.2,4.5Hz,1H),3.46–3.37(m,1H),3.26–3.13(m,2 H),2.68(t,J=12.3Hz,1H),1.92–1.66(m,7H),1.66–1.43(m,7H),1.40–1.09( m,8H),0.90(d,J=6.9Hz,3H),0.74(d,J=6.3Hz,3H),0.69(s,3H),0.67(s,3H).

[0087] Example 2 Antibacterial Test

[0088] Experimental materials:

[0089] Reagent: 5α-hydroxylasiosapogenin (prepared from Example 1)

[0090] Strains: Escherichia coli ATCC 13706, Staphylococcus aureus subsp. aureus Rosenbach ATCC 6538, Staphylococcus epidermidis ATCC 12228, Candidageochares ATCC 36852. All were purchased from the American Type Culture Collection (ATCC).

[0091] Experimental methods:

[0092] 1. Growth curve

[0093] Taking *Escherichia coli* 13706 as an example, *E. coli* was cultured on LB medium, which was purchased from Shanghai Sangon Biotech Co., Ltd. The preparation method followed the instructions. The culture method for *E. coli* was overnight in a water bath with shaking at 37°C. After sterilizing all equipment, the overnight culture was inoculated into fresh LB medium at an inoculation rate of 1%, with an inoculation concentration of 5 × 10⁻⁶. 4 cell·mL -1 The experimental group culture medium was supplemented with 5α-hydroxylasiosapogenin solution to a final concentration of 5 mg / ml, while the control group was supplemented with an equal volume of sterile double-distilled water. After inoculation, the culture was incubated in the dark at 25℃ for 12 hours. Then, two sterile bottles of LB nutrient solution were transferred to the two samples respectively and incubated in a 37℃ water bath shaking incubator for 20 hours until the bacteria reached the physiological death phase. During the incubation process, samples were taken every hour, 5 mL each time; after sampling, the samples were allowed to stand for 5 min, and the supernatant was centrifuged (8000 r·min). -1The bacterial cells were then collected and resuspended in 5 mL of distilled water; using distilled water as a control, the absorbance (OD) at 600 nm was measured using an ELISA reader. 600 Finally, using time as the x-axis, the OD values ​​at different time points are... 600 Growth curves were plotted with numerical values ​​on the ordinate. Three replicates were performed at each time point. The t-test was used for significance analysis, and p < 0.05 was considered statistically significant.

[0094] 2. Inhibition zone test

[0095] Taking *Escherichia coli* 13706 as an example, the test strain was cultured in liquid medium, and the bacterial suspension concentration was adjusted to 0.5 McFarland turbidity with physiological saline. A sterile cotton swab was used to apply the bacterial suspension evenly to the surface of a Muller-Hinton agar plate, ensuring the entire plate was covered. The tablets were placed on the agar plates coated with the bacterial suspension. 50 μl of 5α-hydroxylactoside aglycone solution (final concentration 5 mg / ml) was added to the experimental group tablets (A), and 50 μl of sterile double-distilled water was added to the control group tablets (B). Four tablets (C, D, E, F) containing different concentrations of 5α-hydroxylactoside aglycone solution (50–200 μg / ml) were placed on another plate. After drying, the inoculated plates were inverted and incubated at 37°C for 24 hours. Finally, the plates were observed and photographed.

[0096] 3. Long-term range of fluorescence staining comparison of Escherichia coli

[0097] Taking *Escherichia coli* 13706 as an example, *E. coli* was cultured on LB medium using a water bath at 37°C with shaking overnight. After sterilizing the glassware, the overnight culture was inoculated into fresh LB medium at an inoculation rate of 1% and a concentration of 5 × 10⁻⁶. 4 cell·mL -1 Incubate at 37℃ with shaking for 3 hours, OD 600 The concentration was approximately 0.5. The control group was treated with sterile double-distilled water (A1–A4), while the experimental group was treated with two concentrations of 5α-hydroxylasiosapogenin solution: MIC (101 μg / ml, B1–B4) and MBC (225 μg / ml, C1–C4). The cultures were incubated at 37°C with shaking for 1–8 hours. Samples were taken at 1, 2, 4, and 8 hours during the incubation period for staining and observation of the bacterial culture.

[0098] Based on the above, experiments were conducted to investigate the antibacterial activity against representative Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, and Candida glabrata. These were all routine experimental procedures and will not be detailed here.

[0099] Experimental results:

[0100] 1. For example Figures 2-5As shown, the addition of 5α-hydroxylasiodin during cultivation significantly inhibited the normal growth of *Escherichia coli*, *Staphylococcus aureus*, *Staphylococcus epidermidis*, and *Candida geochares*, with statistically significant differences. 600 Experimental results showed that 5α-hydroxylasiosapogenin has certain antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, and yeast.

[0101] 2. The results of the inhibition zone test showed that, Figure 6 , 7 As shown, after adding 5α-hydroxylasiosine tablets, the bacterial density around the tablets was significantly reduced compared to the control group, and this reduction showed a certain concentration dependence.

[0102] 3. Long-term fluorescence staining results for E. coli showed that, Figure 8 As shown, after adding 5α-hydroxylacto saponin solution, the number of bacteria in the field of view was significantly reduced compared with the control group; and the bacterial growth rate in the 5α-hydroxylacto saponin group could be effectively controlled over a longer period of time, indicating that 5α-hydroxylacto saponin has good antibacterial activity and achieves its antibacterial and bactericidal effect by affecting the integrity of the bacterial cell membrane.

[0103] The above experimental results show that 5α-hydroxylasiosapogenin has significant antibacterial and bactericidal effects and can be applied to antibacterial drugs or health products.

[0104] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

The application of 1,5α-hydroxylasiosapogenin in the preparation of antibacterial drugs or health products is characterized by, 5α-hydroxylasiosapogenin has the structure shown in formula (I):

2. The application according to claim 1, characterized in that, The bacteria include Gram-negative bacteria, Gram-positive bacteria, and yeasts.

3. The application according to claim 2, characterized in that, The Gram-negative bacteria are Escherichia coli; the Gram-positive bacteria are Staphylococcus aureus; and the yeast is Candida albicans.

4. A method for preparing 5α-hydroxylasiosapogenin, characterized in that, The reaction route is as follows: The method includes the following steps: S1, diosgenin and acetic anhydride undergo an acetylation reaction in the presence of an acid-binding agent, acetic anhydride and solvent to obtain intermediate M1; S2 and intermediate M1 undergo a dihydroxylation reaction with potassium osmium dihydrate in the presence of pyridine to obtain intermediate M2; S3 and intermediate M2 undergo an oxidation reaction with an oxidant in the presence of a solvent to obtain intermediate M3; S4 and intermediate M3 undergo hydrolysis with a base in the presence of a solvent to yield 5α-hydroxylasiosaccharide.

5. The method for preparing 5α-hydroxylasiosapogenin according to claim 4, characterized in that, In step S1, The acid-binding agent is selected from at least one of triethylamine, pyridine, N,N-diisopropylethylamine, N-methylmorphophallene, and 4-dimethylaminopyridine; The solvent is selected from at least one of tetrahydrofuran, N,N-dimethylformamide, acetone, and dioxane; The molar ratio of the acid-binding agent to diosgenin is 1.0–5.0:1; The molar ratio of acetic anhydride to diosgenin is 1.0–3.0:1; The volume ratio of the solvent to the mass ratio of diosgenin is 5–20 mL: 1 g; The acetylation reaction is carried out at a temperature of 30–70°C for a time of 6–20 h. Step S1 further includes: adding the acetylation reaction solution to water and stirring, filtering, to obtain the intermediate M1; The volume ratio of water to diosgenin is 10–30 mL: 1 g.

6. The method for preparing 5α-hydroxylasiosapogenin according to claim 4, characterized in that, In step S2, The volume ratio of pyridine to diosgenin is 3-10 mL: 1 g; The molar ratio of potassium osmium tetroxide dihydrate to diosgenin is 1.0–2.0:1; The dihydroxylation reaction is carried out at a temperature of 20–30°C for 1–12 hours. Step S2 further includes: adding water and extraction solvent to the reaction solution of the dihydroxylation reaction, extracting and separating the liquid, concentrating and filtering the organic phase to obtain the intermediate M2; The extraction solvent is selected from at least one of dichloromethane, ethyl acetate, and methyl tert-butyl ether; The volume ratio of water to diosgenin is 5–20 mL: 1 g; The volume ratio of the extraction solvent to the mass ratio of diosgenin is 5–20 mL: 1 g.

7. The method for preparing 5α-hydroxylasiosapogenin according to claim 4, characterized in that, In step S3, The oxidant is selected from at least one of pyridine dichlorochromate, pyridine dichromate, chromium trioxide, and potassium dichromate. The solvent is selected from at least one of tetrahydrofuran, dioxane, dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; The molar ratio of the oxidant to intermediate M2 is 1.0–2.0:1; The volume ratio of the solvent to the mass ratio of intermediate M2 is 5–20 mL: 1 g; The oxidation reaction is carried out at a temperature of 20–30°C for 1–8 hours. Step S3 further includes: adding water dropwise to the reaction solution of the oxidation reaction, stirring to precipitate crystals, filtering, and obtaining the intermediate M3; The volume ratio of the water to the mass ratio of intermediate M2 is 10-30 mL: 1 g; The stirring crystallization temperature is 20–30°C, and the time is 1–4 hours.

8. The method for preparing 5α-hydroxylasiosapogenin according to claim 4, characterized in that, In step S4, The solvent is selected from at least one of methanol, ethanol, isopropanol, n-butanol, and tetrahydrofuran; The alkali is selected from at least one of potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, and lithium carbonate; The volume ratio of the solvent to the mass ratio of intermediate M2 is 5–20 mL: 1 g; The molar ratio of the alkali to intermediate M2 is 1.0–5.0:1; The hydrolysis reaction is carried out at a temperature of 30–70°C for a time of 1–8 hours. Step S4 further includes: adding water and extraction solvent to the reaction solution of the hydrolysis reaction, extracting and separating the liquid, concentrating the organic phase and adding a refining solvent, pulping, filtering and drying to obtain the 5α-hydroxylasiosaurin product. The volume ratio of the water to the mass ratio of intermediate M2 is 10-30 mL: 1 g; The extraction solvent is selected from at least one of dichloromethane, toluene, and methyl tert-butyl ether; The volume ratio of the extraction solvent to the mass ratio of intermediate M2 is 5-20 mL: 1 g; The refining solvent is selected from at least one of methanol, ethanol, dichloromethane, ethyl acetate, acetone, and methyl tert-butyl ether; The volume ratio of the refined solvent to the mass ratio of intermediate M2 is 3-10 mL: 1 g; The pulping temperature is 20–30°C, and the time is 1–8 hours.

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