Extraction of diosgenin from fenugreek using a biphasic Lewis acid hydrolysis method

Abstract

This invention discloses a biphasic combined Lewis acid hydrolysis method for extracting diosgenin from fenugreek, belonging to the field of natural product extraction technology. The extraction method disclosed in this invention includes: defatting fenugreek raw material, mixing it with a biphasic reaction system composed of Lewis acid, concentrated hydrochloric acid, methanol, water, and petroleum ether, and carrying out a hydrolysis reaction under heating conditions. After the reaction, the organic phase is separated, concentrated, and dried to obtain diosgenin. This invention innovatively introduces a Lewis acid catalyst into the biphasic acid hydrolysis system of fenugreek, achieving significant results such as a 25% reduction in reaction time, a 50% reduction in concentrated hydrochloric acid usage, and a 25% reduction in water usage while ensuring the extraction rate of diosgenin. This method has advantages such as mild conditions, low acid consumption, short processing time, and environmental friendliness, and is suitable for the large-scale green production of diosgenin.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to the extraction of diosgenin from fenugreek using a biphasic Lewis acid hydrolysis method. Background Technology

[0002] Diosgenin is a phytosteroid compound and a fundamental material for the synthesis of steroid drugs. It is widely used as an essential precursor in the partial synthesis of over 200 oral contraceptives, sex hormones, and other steroid drugs, earning it the titles of "mother of hormones" and "medicinal gold." In recent years, more research has focused on the pharmacological activity of diosgenin, yielding many significant discoveries, such as the prevention of cardiovascular and cerebrovascular diseases, the treatment of rheumatoid arthritis, tumor suppression, and the improvement of skin aging. These findings demonstrate the diverse medicinal value and potential of diosgenin in pharmaceutical applications. Global demand for hormonal drugs grows by more than 8% annually, requiring approximately 3,000 tons of diosgenin globally. China is a major producer of diosgenin, accounting for 50% of global production annually.

[0003] Fenugreek, the dried seed of the legume *Trigonella foenum-graecum* L., is a traditional Chinese medicinal and edible plant and spice. The *Chinese Pharmacopoeia* records that fenugreek is warm in nature and bitter in taste, entering the kidney meridian, warming the kidney and tonifying yang, dispelling cold and relieving pain, and is used for kidney yang deficiency. The main chemical components of fenugreek include trigonelline, saponins, flavonoids, dietary fiber, fenugreek oil, and 4-hydroxyisoleucine. Existing literature reports that the main product obtained after fenugreek hydrolysis is diosgenin. Acid hydrolysis of fenugreek can provide diosgenin for industrial production. With the development of fenugreek cultivation and the gum-making industry, the importance and necessity of extracting diosgenin from fenugreek after gum production are increasingly recognized. Currently, traditional methods for extracting diosgenin include acid hydrolysis, pre-fermentation, microbial transformation, and enzymatic hydrolysis. Direct acid hydrolysis, which uses strong acids to degrade organic matter such as starch, cellulose, and pigments in plants, is the most commonly used method. However, it suffers from drawbacks such as severe pollution, large wastewater volume, and low extraction efficiency. Microbial transformation is widely used due to its environmental friendliness, low cost, and high economic value; however, the conversion rates of currently screened microorganisms are generally low. Enzymatic hydrolysis can be further divided into single-enzyme hydrolysis, multi-enzyme hydrolysis, and stepwise enzymatic hydrolysis. It has advantages such as high specificity, few hydrolysis byproducts, mild reaction conditions, environmental friendliness, and simple operation. However, it suffers from drawbacks such as high enzyme or strain costs, strict control of reaction conditions (pH, temperature), complex processes, long processing times, and significant energy waste, making large-scale industrialization difficult.

[0004] In summary, existing extraction technologies generally face the challenge of synergistically optimizing extraction efficiency, product quality, environmental friendliness, and production costs. Therefore, developing a new, efficient, green, economical extraction process for fenugreek diosgenin suitable for large-scale production is of great significance and urgently needed. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a biphasic Lewis acid hydrolysis method for extracting diosgenin from fenugreek. The novel process for extracting diosgenin from fenugreek disclosed in this invention employs Lewis acid + biphasic acid hydrolysis conditions, innovatively applying Lewis acid to the production of diosgenin. Preliminary optimization of this process reveals that the reaction time is shortened, and the diosgenin produced by hydrolysis remains essentially stable in the reaction solution. This achieves the goal of efficient and highly selective extraction of diosgenin under mild conditions, significantly reducing energy consumption, strong acid reagent consumption, and environmental pollution while maintaining the extraction rate of existing processes.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses a method for extracting diosgenin from fenugreek, characterized by comprising the following steps: (1) Degreasing treatment: After crushing and drying the fenugreek raw material, it is degreased with petroleum ether to obtain degreased fenugreek powder; (2) Biphasic acid hydrolysis: The defatted fenugreek powder, Lewis acid, concentrated hydrochloric acid, alcohol solvent, water and petroleum ether are mixed to form a biphasic reaction system, and the hydrolysis reaction is carried out under heating and reflux conditions; (3) Product separation: After the reaction is completed, the petroleum ether phase is separated and collected, and then concentrated and dried to obtain diosgenin.

[0008] Furthermore, in step (2), the Lewis acid is aluminum chloride.

[0009] Furthermore, in step (2), the volume ratio of Lewis acid, concentrated hydrochloric acid, alcohol solvent, water and petroleum ether in the biphase reaction system is 15:2:12:3:20.

[0010] Furthermore, in step (2), the ratio of defatted fenugreek powder to Lewis acid, concentrated hydrochloric acid, alcohol solvent, water, and petroleum ether is a mass-volume ratio, specifically 2:52.

[0011] Furthermore, in step (2), the hydrolysis reaction takes 3 hours.

[0012] Further, in step (1), the boiling range of the petroleum ether is 60-90℃, and the material-to-liquid ratio for the degreasing treatment is 1:10 (g / mL).

[0013] Furthermore, in step (2), the boiling range of the petroleum ether is 90-120°C.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0015] 1. Based on the traditional biphasic acid hydrolysis method, this invention adds Lewis acid, which improves the extraction speed while maintaining the yield of diosgenin. When applied to industrial production, it will significantly improve production efficiency and reduce production consumption such as industrial electricity, thereby reducing costs.

[0016] 2. This invention adds Lewis acid to the traditional biphasic acid hydrolysis method, which changes the overall acidity of the reaction system, reduces the amount of water and concentrated hydrochloric acid used, and makes steroidal saponins less damaged during hydrolysis. At the same time, it reduces resource consumption and makes the process more environmentally friendly. Attached Figure Description

[0017] Figure 1 Standard curve of diosgenin.

[0018] Figure 2 Chromatograms of diosgenin standard and sample: (A) Chromatogram of standard, (B) Chromatogram of fenugreek diosgenin sample. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0020] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0021] Example 1: A method for extracting diosgenin from fenugreek using a biphasic Lewis acid hydrolysis method.

[0022] This invention provides a method for extracting diosgenin, comprising the following steps.

[0023] Step (1): Degreasing: Take fenugreek medicinal material, crush it, pass it through a 40-mesh sieve, dry it at a constant temperature of 60℃ to constant weight, add petroleum ether with a material-to-liquid ratio of 1:10 (60-90℃), and ultrasonically degrease it twice at room temperature, 30min / time. Evaporate the petroleum ether and dry it at 60℃ for 3h for later use.

[0024] Step (2): Take the fenugreek powder obtained in step (1) and place it in a round-bottom flask. Add a certain proportion of solid aluminum chloride, concentrated hydrochloric acid, methanol, water and petroleum ether (90~120℃) (volume ratio of 15:2:12:3:20) (material-liquid ratio of 2:52). Heat and reflux in a boiling water bath for 3 hours. After the reaction is complete, filter, collect the filtrate, extract, separate the petroleum ether layer, and recover the solvent under reduced pressure. The residue obtained is diosgenin after drying.

[0025] Example 2: Evaluation of the process effect of biphasic Lewis acid hydrolysis for the extraction of diosgenin from fenugreek.

[0026] I. Experimental Materials.

[0027] Fenugreek was purchased from Hebei Jixintang Pharmaceutical Co., Ltd., batch number 231001414.

[0028] Anhydrous aluminum chloride (purity 99%, batch number: C16152511, Shanghai Maclean Biochemical Technology Co., Ltd.); concentrated hydrochloric acid (analytical grade, Tianjin Kemio Chemical Reagent Co., Ltd., content 36%~38%); methanol (analytical grade, Tianjin Kemio Chemical Reagent Co., Ltd.); petroleum ether (60~90℃) (analytical grade, Tianjin Kemio Chemical Reagent Co., Ltd.); petroleum ether (90~120℃) (analytical grade, Tianjin Kemio Chemical Reagent Co., Ltd.); purified water (Dalian Wahaha Drinking Water Co., Ltd.); edible salt (batch number: 20230224FXZ) was purchased from Shenyang Fengxing Salt Manufacturing Co., Ltd.

[0029] II. Experimental Methods.

[0030] 1. The biphasic combined Lewis acid hydrolysis method is operated according to the steps described in Specific Example 1.

[0031] 2. Traditional biphasic acid hydrolysis method: Degreasing: Take fenugreek medicinal material, crush it, pass it through a 40-mesh sieve, dry it at a constant temperature of 60℃ to constant weight, add petroleum ether at a material-to-liquid ratio of 1:10 (60-90℃), and ultrasonically degrease it twice at room temperature, 30 minutes each time. Evaporate the petroleum ether and dry it at 60℃ for 3 hours for later use.

[0032] Defatted fenugreek powder was placed in a round-bottom flask and a two-phase mixture of concentrated hydrochloric acid, methanol, water and petroleum ether (90~120℃) (HCl:CH3OH:H2O:PE=1:3:1:5) (solid-to-liquid ratio 1:20) was added. The mixture was heated under reflux in a boiling water bath for 4 hours. After the reaction was complete, the mixture was filtered, the filtrate was collected, extracted, the petroleum ether layer was separated, the solvent was recovered under reduced pressure, and the residue obtained after drying was diosgenin.

[0033] 3. Statistical analysis methods.

[0034] All experiments were performed in triplicate. The independent samples t-test in SPSS 26.0 software was used to analyze the data. P <0.05 indicates a significant difference. All results are expressed as mean ± standard deviation. ) is used to represent this.

[0035] III. Experimental Results.

[0036] The experimental results are shown in Tables 1 and 2.

[0037] Table 1. Comparison between traditional and new processes.

[0038] Table 2. Statistical analysis of diosgenin content extracted by traditional and new processes.

[0039] The results above show that the new process for extracting diosgenin from fenugreek yields the highest diosgenin yield after 3 hours of extraction, which is 25% shorter than the traditional process. The highest diosgenin yield is achieved with 3 mL of water. The highest and similar yields are achieved with 2 mL and 4 mL of concentrated hydrochloric acid. Further increasing the amount of concentrated hydrochloric acid slightly decreases the yield, presumably due to increased degradation of diosgenin by high concentrations of hydrogen ions. The content of diosgenin extracted using the new process is slightly lower than that extracted using the traditional process, but the difference is not statistically significant. P Since the concentration of concentrated hydrochloric acid is greater than 0.05, the optimal amount of concentrated hydrochloric acid is 2 mL. Compared with the traditional process, the amount of concentrated hydrochloric acid and water used is reduced by 50% and 25%, respectively.

[0040] In summary, the new process with the addition of Lewis acid reduces overall extraction time by 25%, water consumption by 25%, and concentrated hydrochloric acid consumption by 50%. Applying this process to the industrial production of diosgenin will significantly reduce energy consumption and the use of concentrated hydrochloric acid, resulting in substantial cost savings and reduced waste disposal difficulties, making it more environmentally friendly.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting diosgenin from fenugreek, characterized in that, Includes the following steps: (1) Degreasing treatment: After crushing and drying the fenugreek raw material, it is degreased with petroleum ether to obtain degreased fenugreek powder; (2) Biphasic acid hydrolysis: The defatted fenugreek powder, Lewis acid, concentrated hydrochloric acid, alcohol solvent, water and petroleum ether are mixed to form a biphasic reaction system, and the hydrolysis reaction is carried out under heating and reflux conditions; (3) Product separation: After the reaction is completed, the petroleum ether phase is separated and collected, concentrated and dried to obtain diosgenin.

2. The method according to claim 1, characterized in that: In step (2), the Lewis acid is aluminum chloride.

3. The method according to claim 1, characterized in that: In step (2), the volume ratio of Lewis acid, concentrated hydrochloric acid, alcohol solvent, water and petroleum ether in the biphase reaction system is 15:2:12:3:

20.

4. The method according to claim 1, characterized in that: In step (2), the ratio of defatted fenugreek powder to Lewis acid, concentrated hydrochloric acid, alcohol solvent, water, and petroleum ether is a mass-volume ratio, specifically 2:

52.

5. The method according to claim 1, characterized in that: In step (2), the hydrolysis reaction takes 3 hours.

6. The method according to claim 1, characterized in that: In step (1), the boiling range of the petroleum ether is 60-90℃, and the material-to-liquid ratio for the degreasing treatment is 1:10 (g / mL).

7. The method according to claim 1, characterized in that: In step (2), the boiling range of the petroleum ether is 90-120℃.

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