Process for the preparation of cycloastragenol
By leveraging the synergistic effect of GSM inoculant and xylosidase, the problems of low yield and low purity of cycloastragalool in existing technologies have been solved, achieving efficient preparation of high-purity cycloastragalool.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN ANHUI BIOTECHNOLOGY CO LTD
- Filing Date
- 2023-03-23
- Publication Date
- 2026-07-07
AI Technical Summary
Existing methods for preparing cycloastragaloyl alcohol suffer from low yield and low purity.
Cycloastragaloside was prepared by conversion reaction using the synergistic effect of GSM inoculant and xylosidase. The specific steps included preparing a mixed solution of astragaloside A, xylosidase and GSM inoculant, controlling the pH value to 5.5-6.5, the temperature to 36-38℃, the reaction time to more than 20 hours, and the reaction under stirring conditions, followed by separation and purification.
The conversion of astragaloside A was achieved, and the resulting cycloastragalool had high yield and high purity.
Smart Images
Figure CN116334165B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing cycloastragaloyl alcohol. Background Technology
[0002] Astragaloside A is the main active ingredient of the traditional Chinese medicine Astragalus membranaceus, and cycloastragalool (CA) is the aglycone of astragaloside A. Cycloastragalool can extend telomeres by activating telomerase, thereby achieving the effects of anti-aging and improving cell proliferation.
[0003] The chemical formula of cycloastragaloyl alcohol is C 30 H 50 O5, with a relative molecular mass of 490.71, is a colorless needle-like crystal, readily soluble in methanol, n-butanol, etc. Cycloastragaloyl alcohol is present in very small amounts in natural plants; therefore, its production is primarily achieved through the hydrolysis of the xylosinolytic bond at the C3 position and the glucoside bond at the C6 position of astragaloyl A.
[0004] There are currently several methods for preparing cycloastragaloyl alcohol, but they all have certain drawbacks, such as low yield and low purity.
[0005] In view of this, the present invention is hereby proposed. Summary of the Invention
[0006] The first objective of this invention is to provide a method for preparing cycloastragaloyl alcohol to solve at least one of the above-mentioned problems.
[0007] The second objective of this invention is to provide a cycloastragalool product.
[0008] In a first aspect, the present invention provides a method for preparing cycloastragaloyl alcohol, comprising the following steps:
[0009] A mixed solution of astragaloside A, xylosidase and GSM bacterial agent was prepared, and cycloastragalool was obtained after conversion reaction.
[0010] By weight, the GSM bacterial agent mainly consists of 40-47 parts of Bacillus licheniformis, 38-43 parts of Bacillus subtilis, and 12-17 parts of Bifidobacterium.
[0011] As a further technical solution, the pH of the mixed solution is 5.5-6.5;
[0012] Preferably, the mixed solution is a buffer solution containing astragaloside A, xylosidase and GSM bacterial agent;
[0013] Preferably, the buffer solution comprises a phosphate buffer.
[0014] As a further technical solution, the temperature of the conversion reaction is 36-38℃, preferably 37℃;
[0015] Preferably, the conversion reaction takes 20 hours or more, and more preferably 30 hours.
[0016] Preferably, the conversion reaction is carried out under stirring conditions.
[0017] As a further technical solution, the concentration of astragaloside A in the mixed solution is 0.0009-0.012 g / mL, preferably 0.009 g / mL;
[0018] Preferably, the concentration of xylosidase in the mixed solution is 0.001-0.005 g / mL, more preferably 0.002 g / mL;
[0019] Preferably, the GSM bacterial agent is a solution containing bacterial cells, and the concentration of bacterial cells in the GSM bacterial agent is 5 × 10⁻⁶. 7 -5×10 8 cfu / mL;
[0020] Preferably, the volume percentage of GSM bacterial agent in the mixed solution is 0.1%-0.5%, more preferably 0.2%.
[0021] As a further technical solution, the preparation method of the astragaloside A includes the following steps:
[0022] Astragaloside A was prepared by sequentially extracting Astragalus membranaceus with ethanol, concentrating, extracting with an extractant, drying, and separating and purifying it.
[0023] As a further technical solution, the ethanol extraction is performed by soaking Astragalus membranaceus in 65%-75% ethanol.
[0024] Preferably, the temperature for ethanol extraction is 35-45°C;
[0025] Preferably, the concentration is carried out until the extract contains no ethanol;
[0026] Preferably, the extractant comprises ethyl acetate or n-butanol.
[0027] As a further technical solution, the separation and purification includes silica gel column chromatography, activated carbon purification, and methanol recrystallization.
[0028] As a further technical solution, the conversion reaction is completed by a step of separating and purifying cycloastragalool;
[0029] The separation and purification process includes sequentially salting out, extracting with an extractant, drying, and recrystallizing the solution from the conversion reaction.
[0030] As a further technical solution, sodium chloride is used for salting out;
[0031] Preferably, the extractant comprises ethyl acetate;
[0032] Preferably, the extraction process further includes a step of washing the extract phase with saturated brine.
[0033] Preferably, the solvent for recrystallization includes ethanol.
[0034] Secondly, the present invention provides a cycloastragaloyl alcohol product, which is prepared by the above-described preparation method.
[0035] Compared with the prior art, the present invention has the following beneficial effects:
[0036] The method for preparing cycloastragaloside provided by this invention achieves 100% conversion of astragaloside A through the synergistic effect of GSM bacterial agent and xylosidase. This preparation method is simple and efficient, and the cycloastragaloside obtained is produced with high yield and high purity. Attached Figure Description
[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0038] Figure 1 This is a molecular structure diagram of the process from astragaloside A to cycloastragaloyl alcohol;
[0039] Figure 2 The 1H NMR spectrum of cycloastragalol provided in Example 1;
[0040] Figure 3 The results of TLC analysis of the reaction solution after 6 hours of reaction provided in Example 1;
[0041] Figure 4 The TLC results are for the reaction solution after 30 hours of reaction provided in Example 1. Detailed Implementation
[0042] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.
[0043] The reaction process for preparing cycloastragaloside from astragaloside A is as follows: Figure 1 As shown. Regarding the conversion of astragaloside A to cycloastragaloyl alcohol, from a molecular structure perspective, high temperature and acid treatment easily lead to the destruction of the three-membered ring; furthermore, the inventors' research found that alkali treatment is not very effective; enzymatic hydrolysis removes xylose and glucose, resulting in low yield and low purity; and bacterial treatment cannot completely convert the alcohol. Based on these findings, the present invention is proposed.
[0044] In a first aspect, the present invention provides a method for preparing cycloastragaloyl alcohol, comprising the following steps:
[0045] A mixed solution of astragaloside A, xylosidase and GSM bacterial agent was prepared, and cycloastragalool was obtained after conversion reaction.
[0046] The GSM microbial agent, by weight, mainly consists of Bacillus licheniformis, Bacillus subtilis, and Bifidobacterium. Specifically, the weight percentage of Bacillus licheniformis may be, for example, but not limited to, 40, 42, 44, 46, or 47 parts; the weight percentage of Bacillus subtilis may be, for example, but not limited to, 38, 40, 42, or 43 parts; and the weight percentage of Bifidobacterium may be, for example, but not limited to, 12, 14, 16, or 17 parts.
[0047] Features of GSM microbial agents: They can produce large amounts of amylase, protease, xylosidase, and cellulase; the fermentation process is simple, the microbial cells multiply rapidly, and no toxic substances are produced; anaerobic, aerobic, and facultative anaerobic bacteria grow in a stable state together, resulting in strong antibacterial and antitoxic capabilities of the bacterial solution and good environmental adaptability; the compound action mechanism leads to high degradation efficiency and a wide range of applications; multi-level and multi-site treatment of raw materials results in high extraction efficiency; the reaction is rapid, shortening the process flow.
[0048] The method for preparing cycloastragaloside provided by this invention achieves 100% conversion of astragaloside A through the synergistic effect of GSM bacterial agent and xylosidase. This preparation method is simple and efficient, and the cycloastragaloside obtained is produced with high yield and high purity.
[0049] In some preferred embodiments, the pH of the mixed solution may be, for example, but not limited to, 5.5, 5.7, 5.9, 6.1, 6.3 or 6.5, preferably 6.
[0050] Preferably, the mixed solution is a buffer solution containing astragaloside A, xylosidase and GSM bacterial agent to maintain pH stability for the conversion reaction.
[0051] Preferably, the buffer solution includes, but is not limited to, phosphate buffer, or other buffers known to those skilled in the art, to maintain the pH stability of the mixed solution.
[0052] In some preferred embodiments, the temperature of the conversion reaction can be, for example, but not limited to, 36°C, 37°C or 38°C, preferably 37°C;
[0053] Preferably, the conversion reaction takes 20 hours or more, and more preferably 30 hours.
[0054] Preferably, the conversion reaction is carried out under stirred conditions. Stirring helps to increase the reaction rate of the conversion reaction.
[0055] By further optimizing and adjusting the conversion reaction conditions, the time required for the conversion reaction can be shortened, and the conversion rate can be improved.
[0056] In some preferred embodiments, the concentration of astragaloside A in the mixed solution may be, for example, but not limited to, 0.0009 g / mL, 0.001 g / mL, 0.003 g / mL, 0.006 g / mL, 0.008 g / mL, 0.010 g / mL or 0.012 g / mL, preferably 0.009 g / mL;
[0057] Preferably, the concentration of xylosidase in the mixed solution can be, for example, but not limited to, 0.001 g / mL, 0.002 g / mL, 0.003 g / mL, 0.004 g / mL or 0.005 g / mL, and is preferably 0.002 g / mL;
[0058] Preferably, the GSM bacterial agent is a solution containing bacterial cells, and the concentration of bacterial cells in the GSM bacterial agent can be, for example, but not limited to, 5 × 10⁻⁶. 7 cfu / mL, 7×10 7 cfu / mL, 9×10 7 cfu / mL, 1×10 8 cfu / mL, 3×10 8 cfu / mL or 5×10 8 cfu / mL;
[0059] Preferably, the volume percentage of GSM bacterial agent in the mixed solution can be, for example, but not limited to, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%, and is preferably 0.2%.
[0060] In some preferred embodiments, the preparation method of astragaloside A includes the following steps:
[0061] Astragaloside A was prepared by sequentially extracting Astragalus membranaceus with ethanol, concentrating, extracting with an extractant, drying, and separating and purifying it.
[0062] The preparation method is simple and convenient, and the yield and purity of astragaloside A are high.
[0063] In some preferred embodiments, the ethanol extraction is performed by extracting Astragalus membranaceus with 65%-75% ethanol, wherein the concentration of ethanol can be, for example, but not limited to, 65%, 67%, 69%, 71%, 73% or 75%.
[0064] Preferably, the temperature for ethanol extraction can be, for example, but not limited to, 35°C, 37°C, 39°C, 41°C, 43°C, or 45°C;
[0065] Preferably, the concentration is performed until the extract contains no ethanol. The concentration method can be, for example, evaporation concentration.
[0066] Preferably, the extractant comprises ethyl acetate or n-butanol.
[0067] By further optimizing and adjusting the preparation conditions of astragaloside A, the yield and purity of astragaloside A can be improved.
[0068] In some preferred embodiments, the separation and purification includes silica gel column chromatography, activated carbon purification, and methanol recrystallization.
[0069] In this invention, methanol recrystallization refers to the process of heating and dissolving the product with methanol, and then slowly cooling it to allow crystals to precipitate. The amount of methanol added can be, for example, 5-10 times the product mass.
[0070] In some preferred embodiments, the conversion reaction is further complicated by a step of separating and purifying cycloastragaloyl alcohol.
[0071] The separation and purification process includes sequentially salting out, extracting with an extractant, drying, and recrystallizing the solution from the conversion reaction.
[0072] In some preferred embodiments, sodium chloride is used for salting out. The addition of sodium chloride can reduce the solubility of ethyl acetate and the product in water, thereby extracting the product from the aqueous phase more efficiently and with greater capacity.
[0073] Preferably, the extractant comprises ethyl acetate;
[0074] Preferably, the extraction process further includes a step of washing the extract phase with saturated brine to remove residual impurities in the extract phase.
[0075] Preferably, the solvent for recrystallization includes ethanol.
[0076] Secondly, the present invention provides a cycloastragaloyl alcohol product, which is prepared by the above-described preparation method.
[0077] This cycloastragalool product has high purity.
[0078] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.
[0079] It should be noted that, by weight, the GSM bacterial agent in the following examples consists of 40-47 parts of Bacillus licheniformis, 38-43 parts of Bacillus subtilis, and 12-17 parts of Bifidobacterium. The concentration of bacteria in the bacterial agent is approximately 1×10⁻⁶. 8 The cfu / mL of this bacterial agent was cultivated by the inventor. Due to factors such as cultivation conditions, the content of each bacterial species in the agent may fluctuate. However, the bacterial agent with the above ratio, when combined with xylosidase, can achieve 100% conversion of astragaloside A.
[0080] Example 1
[0081] A method for preparing cycloastragaloyl alcohol includes the following steps:
[0082] Preparation of astragaloside A: 10 kg of Astragalus membranaceus (Gansu) was extracted with 50 L of 70% ethanol at 60℃ for 1 h. The mixture was filtered, and the extraction was repeated three times. The extracts were combined and concentrated until there was no ethanol odor, approximately 20 L. The extracts were then extracted with 10 L-8 L-8 L of n-butanol (i.e., extraction was performed sequentially with 10 L, 8 L, and 8 L of n-butanol, and the extracts were then combined). The extracts were evaporated to dryness, passed through an AB-8 macroporous resin column, and crystallized from methanol to obtain 6.8 g of astragaloside A with a purity of 90.2%.
[0083] The conversion reaction of cycloastragaloside: 50 ml of 0.04 M NaH₂PO₄-NaOH pH 6.0 buffer solution was added sequentially to a 100 ml three-necked flask. Then, 0.50 g of 90.2% astragaloside A, 0.10 g of xylosidase, and 0.1 ml of GSM bacterial agent were added. The mixture was magnetically stirred at 37 °C for 30 h. Using astragaloside A and cycloastragaloside standards as controls, 0.6-0.8 ml of the solutions after 6 h and 30 h of reaction were taken respectively, and 1 ml of ethyl acetate was added. After shaking well, the organic phase was used for TLC. The developing solvent was dichloromethane:ethyl acetate:methanol = 5:0.8:0.5 (volume ratio). The results are as follows. Figure 3 ( Figure 3 From left to right: astragaloside A, the reaction solution after 6 hours of conversion, and the results of cycloastragaloyl alcohol standard. Figure 4 ( Figure 4 The results (from left to right in the image are astragaloside A, the reaction solution after 30 hours of conversion, and the standard of cycloastragaloyl alcohol) are shown in the image. Figure 3 It can be seen that after 6 hours of reaction, astragaloside A lost one sugar, but was not completely converted into cycloastragaloyl alcohol. From... Figure 4 As can be seen from the data, after the conversion reaction was completed, astragaloside A was completely reacted, and the product was cycloastragalool, achieving 100% conversion.
[0084] Preparation of cycloastragaloside: In a 100ml three-necked flask, 50ml of 0.04M NaH2PO4-NaOH pH 6.0 buffer solution, 0.50g of 90.2% astragaloside A, 0.10g of xylosidase, and 0.1ml of GSM bacterial agent were added sequentially. The mixture was magnetically stirred at 37℃ for 30h. 10g of sodium chloride was added, and the mixture was extracted five times with ethyl acetate, followed by washing the extract phase twice with saturated brine. The organic phase was evaporated to dryness and weighed to obtain a yellow powder. A sample was taken and the content was determined to be 90.5%. Recrystallization twice with 5-10 times anhydrous ethanol yielded 0.250g of pale yellow crystals. HPLC-ESLD analysis showed a content of 99.39%, with a total yield of 88.1%. The obtained cycloastragaloside was analyzed by 1H NMR spectroscopy as follows: Figure 2 .
[0085] Example 2
[0086] A method for preparing cycloastragaloyl alcohol includes the following steps:
[0087] Preparation of astragaloside A: 10 kg of Astragalus membranaceus (Gansu) was extracted with 50 L of 70% ethanol at 60℃ for 1 h. The mixture was filtered and extracted three times. The extracts were combined and concentrated until there was no ethanol odor, approximately 20 L. The extracts were then extracted with 10 L-8 L-8 L of n-butanol, evaporated to dryness, and passed through an AB-8 macroporous resin column. The resulting methanol crystallization yielded 6.8 g of astragaloside A with a content of 90.2%.
[0088] The conversion reaction of cycloastragaloside: 50 ml of 0.04 M NaH₂PO₄-NaOH pH 6.0 buffer solution, 0.050 g of 90.2% astragaloside A, 0.10 g of xylosidase, and 0.1 ml of GSM bacterial agent were added sequentially to a 100 ml three-necked flask. The mixture was magnetically stirred at 37°C for 20 h. TLC analysis using the method in Example 1 showed that astragaloside A reacted completely, and the product was cycloastragaloside, achieving 100% conversion.
[0089] Preparation of cycloastragaloside: In a 100ml three-necked flask, 50ml of 0.04M NaH2PO4-NaOH pH 6.0 buffer solution, 0.050g of 90.2% astragaloside A, 0.10g of xylosidase, and 0.1ml of GSM bacterial agent were added sequentially. The mixture was magnetically stirred at 37℃ for 20h. 10g of sodium chloride was added, and the mixture was extracted five times with ethyl acetate, followed by washing the extract phase twice with saturated brine. The organic phase was evaporated to dryness and weighed to obtain a yellow powder. A sample was taken and the content was determined to be 90.25%. Recrystallization twice with 5-10 times anhydrous ethanol yielded 0.026g of pale yellow crystals. HPLC-ESLD analysis showed a content of 99.18%, with a total yield of 91.1%.
[0090] Example 3
[0091] A method for preparing cycloastragaloyl alcohol includes the following steps:
[0092] Preparation of astragaloside A: 10 kg of Astragalus membranaceus (Gansu) was extracted with 50 L of 70% ethanol at 60℃ for 1 h. The mixture was filtered and extracted three times. The extracts were combined and concentrated until there was no ethanol odor, approximately 20 L. The extracts were then extracted with 10 L-8 L-8 L of n-butanol, evaporated to dryness, and passed through an AB-8 macroporous resin column. The resulting methanol crystallization yielded 6.8 g of astragaloside A with a content of 90.2%.
[0093] The conversion reaction of cycloastragaloside: 100 ml of 0.04 M NaH₂PO₄-NaOH pH 6.0 buffer solution, 0.50 g of 90.2% astragaloside A, 0.08 g of xylosidase, and 0.1 ml of GSM inoculant were added sequentially to a 250 ml three-necked flask. The mixture was magnetically stirred at 37°C for 30 h. TLC analysis using the method in Example 1 showed that astragaloside A reacted completely, and the product was cycloastragaloside, achieving 100% conversion.
[0094] Preparation of cycloastragaloside: In a 250ml three-necked flask, 100ml of 0.04M NaH2PO4-NaOH pH 6.0 buffer solution was added sequentially, along with 0.50g of 90.2% astragaloside A, 0.08g of xylosidase, and 0.1ml of GSM bacterial agent. The mixture was magnetically stirred at 37℃ for 30h until complete conversion. 25g of sodium chloride was added, and the mixture was extracted five times with ethyl acetate, followed by washing the extract phase twice with saturated brine. The organic phase was evaporated to dryness and weighed to obtain a yellow powder. A sample was taken and the content was determined to be 90.20%. Recrystallization twice with 5-10% anhydrous ethanol yielded 0.242g of pale yellow crystals. HPLC-ESLD analysis showed a content of 99.26%, with a total yield of 85.2%.
[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing cycloastragaloyl alcohol, characterized in that, Includes the following steps: A mixed solution of astragaloside A, xylosidase and GSM bacterial agent was prepared, and cycloastragalool was obtained after conversion reaction. By weight, the GSM bacterial agent consists of 40-47 parts of Bacillus licheniformis, 38-43 parts of Bacillus subtilis, and 12-17 parts of Bifidobacterium; the concentration of bacterial cells in the agent is 1×10⁻⁶. 8 cfu / mL; The conversion reaction was carried out in a 0.04M NaH2PO4-NaOH buffer solution with pH=6.
0. 0.5g or 0.05g of 90.2% astragaloside A was added, the amount of xylosidase added was 0.08g or 0.1g, and the amount of GSM bacterial agent added was 0.1ml. The reaction was carried out under magnetic stirring at 37°C for 20h or 30h. Then, 10g or 25g of sodium chloride was added, and the mixture was extracted 5 times with ethyl acetate. The extract phase was washed twice with saturated brine. The organic phase was evaporated to dryness and recrystallized twice with 5-10 times anhydrous ethanol to obtain the final product crystals.
2. The preparation method according to claim 1, characterized in that, The preparation method of the astragaloside A includes the following steps: Astragaloside A was prepared by sequentially extracting Astragalus membranaceus with ethanol, concentrating, extracting with an extractant, drying, and separating and purifying it.
3. The preparation method according to claim 2, characterized in that, The ethanol extraction is performed by soaking Astragalus membranaceus in 65%-75% ethanol. The ethanol extraction temperature is 35-45℃; The concentration is carried out until the extract contains no ethanol; The extractant includes ethyl acetate or n-butanol.
4. The preparation method according to claim 2, characterized in that, The separation and purification process includes macroporous resin chromatography, activated carbon purification, and methanol recrystallization.