A preparation method of indirubin

Abstract

This invention relates to a method for preparing indirubin, comprising the following steps: S1, pretreating indigo leaves containing indoside to prepare an indoside solution; S2, removing carbonyl compounds from the indoside solution obtained in step S1 to obtain indoside; S3, decomposing the indoside obtained in step S2 to obtain a solid mixture of indirubin and indigo; S4, separating the solid mixture of indirubin and indigo obtained in step S3 to obtain indigo and indirubin. Compared with the prior art, this invention improves the conversion rate of indirubin and reduces the conversion rate of indigo by removing carbonyl compounds to change the oxidation properties in the reaction system, thereby increasing the formation of the intermediate product indirubin and achieving a high-yield preparation of plant indirubin. It has the advantages of simple process, low cost, and high directional conversion efficiency of indirubin.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction, and in particular to a method for preparing indirubin. Background Technology

[0002] Indigo red has long been considered a byproduct of indigo biochemical production. However, in the 1960s, the Chinese Academy of Medical Sciences discovered that the traditional Chinese medicine "Dang Gui Long Hui Wan" (Angelica and Aloe Vera Pill) was effective in treating chronic myeloid leukemia (CML). To explore the effective components of this formula, indigo red in indigo naturalis was ultimately identified as the key ingredient through a drug reduction method. Subsequent clinical observations in over 400 CML patients across multiple hospitals confirmed that indigo red treatment achieved an efficacy rate of 87.3%, making it one of the first innovative anticancer drugs discovered in my country. Recent research indicates that indigo red inhibits cyclin-dependent kinases (CDKs) by binding to the ATP pocket. Furthermore, indigo red can inhibit glycogen synthase-3β (GSK-3β), bispecific tyrosine phosphorylation-regulated kinase 1A (Dyrk1A), and interfere with the Stat3 signaling pathway, thereby inhibiting cancer cell differentiation and ultimately inducing apoptosis. Indigo red has demonstrated great potential as a novel anticancer drug and has attracted global attention, with research areas continuously expanding.

[0003] Chemical synthesis is a commonly used method for preparing indirubin. However, due to the cumbersome reaction units, limited availability of starting materials, harsh reaction conditions, low overall yield of multi-step synthesis, and the potential health and environmental hazards of the raw materials required in chemical synthesis, its application in industrial production is limited. Based on the trend of modern biocatalytic synthesis of natural products, microbial indirubin production is considered an environmentally friendly alternative to chemical synthesis. However, biosynthesis uses expensive substrates, and due to their toxicity, the substrates often inhibit microbial cell growth. There are no reports of directly producing indirubin from simple carbon sources. Therefore, current research on biosynthesis is limited to academic reports and has not been applied to actual industrial production.

[0004] As early as the 1890s, researchers attempted to use tissue engineering techniques to culture root tissues of Polygonum tinctorium, hoping to increase the indirubin content by optimizing culture conditions. However, significantly increasing the indirubin content in plants is not easy, and due to the complexity of tissue culture, this research did not achieve substantial progress. On the other hand, researchers used orthogonal experimental design to investigate the effects of different factors and levels, such as ultrasonication and reflux methods, on the extraction conditions of indirubin in order to optimize the extraction process. However, the content of indirubin in natural products is extremely low. Currently, several traditional Chinese medicinal materials can be used to extract indirubin, including Isatis indigotica leaves, Polygonum tinctorium, and Isatis tinctoria root. Even in Isatis indigotica leaves, which have the highest content, the indirubin content is only about 0.03% of its dry weight. This makes the production cost of plant-based indirubin high, the product price expensive, and the production process consumes a large amount of organic solvents and energy, causing an environmental burden. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing indirubin to address the problem of low yield in existing traditional processes. This invention transforms indirubin into the main product of the process by removing carbonyl compounds from the reaction system, reducing the antioxidant capacity of the reaction system, promoting the formation of indirubin, and thus improving the conversion rate and yield of indirubin.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A method for preparing indigo red, the specific steps of which are as follows:

[0008] S1. Pre-treat indigo leaves containing indigo glycosides to prepare an indigo glycoside solution;

[0009] S2. Remove the carbonyl compound from the indigo glycoside solution obtained in step S1 to obtain indigo glycoside;

[0010] S3. Decompose the indigo glycoside obtained in step S2 to obtain a solid mixture of indirubin and indigo.

[0011] S4. Separate the solid mixture of indigo red and indigo obtained in step S3 to obtain indigo and indigo red.

[0012] Furthermore, in step S1, the bluegrass leaves include fresh leaves and dried leaves.

[0013] Furthermore, the dried leaves are prepared by methods such as baking, air drying, or microwave drying.

[0014] Furthermore, in step S1, the indigo leaves include leaves of strychnine, woad, and woad.

[0015] Further, in step S1, the method for pretreating the indigo leaves containing indigo glycosides is as follows:

[0016] Indigo glycoside solution is prepared by extracting indigo leaves containing indigo glycoside using organic solvents or water.

[0017] Furthermore, the extraction conditions are as follows: temperature 60-100℃, time 10-60 minutes, solid-liquid ratio 5%-30% (w / v).

[0018] Furthermore, the organic solvents mentioned above include methanol, ethanol, and acetone, etc.

[0019] Furthermore, in step S2, the methods for removing carbonyl compounds from the indigo glycoside solution obtained in step S1 include solvent extraction, adsorbent adsorption, coagulation precipitation, enzyme treatment, and redox reaction.

[0020] Furthermore, the adsorbent includes silica gel, activated alumina, activated carbon, molecular sieves, and macroporous resins, etc.

[0021] Furthermore, the enzyme used in the enzyme treatment method is an enzyme with polyphenol oxidase activity.

[0022] As a preferred technical solution, the enzymes include peroxidase and laccase, etc.

[0023] Furthermore, the precipitant used in the coagulation and precipitation method includes enzymes with polyphenol oxidase activity, calcium hydroxide, barium chloride, and ferrous salts, etc., and the precipitant reacts with carbonyl compounds to produce a precipitate.

[0024] As a preferred technical solution, the enzymes include peroxidase and laccase, etc.

[0025] Furthermore, the redox reaction method includes: oxidizing the polyphenols in the indigo glycoside solution with an oxidizing agent to form easily separable products; reducing the polyphenols in the indigo glycoside solution with a reducing agent to colorless or water-soluble products, and then removing them by separation technology, thereby removing carbonyl compounds.

[0026] As a preferred technical solution, the oxidant includes hydrogen peroxide and potassium permanganate.

[0027] As a preferred technical solution, the reducing agent is sodium bisulfite.

[0028] Further, in step S3, the concentrated solution containing indoside is enzymatically or photolyzed to generate an indophenol solution, the indophenol solution is oxidized to generate a mixed solution of indirubin and indigo, and after sedimentation, a solid mixture of indirubin and indigo is obtained.

[0029] Further, in step S4, the solid mixture of indigo and indigo obtained in step S3 is separated by acetone extraction to obtain crude extracts of indigo and indigo. After drying the precipitate, indigo is obtained, and acetone is removed by rotary evaporation to obtain purified indigo.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) In the preparation method of the present invention, the oxidation properties in the reaction system are changed by removing carbonyl compounds, thereby increasing the generation of intermediate product indigo, which in turn increases the conversion rate of indigo and reduces the conversion rate of indigo, thus realizing the preparation of high-yield plant indigo.

[0032] (2) The method of the present invention is simple and low in cost. The process of preparing indigo is also the process of purifying indigo, thus realizing the coupled preparation of high-yield indigo and high-purity indigo.

[0033] (3) Indirubin is a major byproduct in indigo production. Its formation principle is that indigo, an oxidation product of indolephenol, undergoes an aldol condensation reaction with indolephenol under alkaline conditions. This invention uses indole glycoside extract as raw material, reduces the content of carbonyl compounds in the reaction system, reduces the antioxidant capacity of the system, and improves the directional conversion of indole glycosides to indigo, thereby achieving high-yield production of indirubin. This invention affects the formation of indigo by changing the composition and chemical properties of the reaction system. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the effect of microwave drying on the extraction of indigo glycosides from *Indigofera tinctoria* leaves in Experiment Example 1.

[0035] Figure 2 The graph shows the effect of purification on the antioxidant activity of indigo glycosides in Experiment Example 2.

[0036] Figure 3 The effect of potassium permanganate oxidation of indophenol on the formation of indigo in Experiment Example 4 is shown in the figure.

[0037] Figure 4 The graph shows the effect of activated carbon purification on the hydrolysis of indoleglycoside in Experiment 5, where (A) represents the concentration of indoleglycoside and (B) represents the concentration of indophenol.

[0038] Figure 5 The diagram shows the effect of indigo glycoside purification on the formation of indigo and indirubin in Experiment Example 6. (A) Conversion rate of indigo and indirubin; (B) Purity of indigo and indirubin;

[0039] Figure 6 The diagram shows the separation and purification of indigo and indirubin in Experiment Example 7. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0041] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise defined, the following embodiments and features can be combined with each other. All technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art.

[0042] Example 1

[0043] This embodiment provides a method for preparing a crude indigo glycoside solution, the specific steps of which are as follows:

[0044] S1. Drying of Indigofera tinctoria leaves: Microwave drying was performed using a laboratory microwave oven (G70D20CN1P-D2(S0), Guangdong Galanz Group Co., Ltd., China) with a maximum output power of 700W. The internal dimensions of the microwave oven were 195×315×329mm (height×width×depth). The sample was laid in a single layer on a glass turntable and dried at a microwave power of 700W. Drying continued for 3 minutes until the moisture content of the sample dropped below 10%, resulting in microwave-dried Indigofera tinctoria leaves.

[0045] S2. Water extraction of Indigofera tinctoria leaves: The Indigofera tinctoria leaves obtained by microwave drying in step S1 were placed in preheated deionized water and boiled in a water bath for 10 min at a solid-liquid ratio of 1:10 (w:v); then filtered through 8 layers of gauze to obtain a crude indigo glycoside solution.

[0046] Example 2

[0047] This embodiment provides a method for preparing purified indigo glycosides, the specific steps of which are as follows:

[0048] S1. Drying of Indigofera tinctoria leaves: Microwave drying was performed using a laboratory microwave oven (G70D20CN1P-D2(S0), Guangdong Galanz Group Co., Ltd., China) with a maximum output power of 700W. The internal dimensions of the microwave oven were 195×315×329mm (height×width×depth). The sample was laid in a single layer on a glass turntable and dried at a microwave power of 700W. Drying continued for 3 minutes until the moisture content of the sample dropped below 10%, resulting in microwave-dried Indigofera tinctoria leaves.

[0049] S2. Water extraction of Indigofera tinctoria leaves: The Indigofera tinctoria leaves obtained by microwave drying in step S1 were placed in preheated deionized water and boiled in a water bath for 10 min at a solid-liquid ratio of 1:10 (w:v); then filtered through 8 layers of gauze to obtain a crude indigo glycoside solution.

[0050] S3. Removal of carbonyl compounds: 5% (w:v) of granular activated carbon was added to the crude indigo glycoside solution obtained in step S2, and then stirred at 600 rpm for 1 h and filtered to obtain the filtered activated carbon.

[0051] S4. Separation of indigo glycosides: First, add equal volumes of acetone and ethanol of different concentrations to the filtered activated carbon simultaneously, with three replicates per group. Stir at 600 rpm for 1 hour and filter. For the second elution, use 100% acetone, stir at 600 rpm for 1 hour, and filter a second time to obtain the eluent after two elutions.

[0052] S5. Remove the organic solvent from the two eluents obtained in step S4 in a rotary evaporator, and concentrate to obtain a concentrated solution containing indigo glycosides.

[0053] S6. Freeze the concentrated solution obtained in step S5 at -80°C for 4 hours, then use a freeze dryer to remove moisture to obtain solid purified indigo glycosides. Grind the solid purified indigo glycosides into powder and seal and store at 4°C.

[0054] Example 3

[0055] This embodiment provides a method for preparing indirubin, the specific steps of which are as follows:

[0056] S1. Pretreated Stellera chasteum leaves

[0057] S1-1. Drying of Indigofera tinctoria leaves: Microwave drying was performed using a laboratory microwave oven (G70D20CN1P-D2(S0), Guangdong Galanz Group Co., Ltd., China) with a maximum output power of 700W. The internal dimensions of the microwave oven were 195×315×329mm (height×width×depth). The sample was laid in a single layer on a glass turntable and dried at a microwave power of 700W. Drying continued for 3 minutes until the moisture content of the sample dropped below 10%, resulting in microwave-dried Indigofera tinctoria leaves.

[0058] S1-2, Water extraction of Indigofera tinctoria leaves: The Indigofera tinctoria leaves obtained by microwave drying in step S1 were placed in preheated deionized water and boiled in a water bath for 10 min at a solid-liquid ratio of 1:10 (w:v); then filtered through 8 layers of gauze to obtain a crude indigo glycoside solution.

[0059] S2, Preparation of indigo glycosides

[0060] S2-1, Removal of carbonyl compounds: 5% (w:v) of granular activated carbon was added to the crude indigo glycoside solution obtained in step S2, and then stirred at 600 rpm for 1 h and filtered to obtain the filtered activated carbon.

[0061] S2-2, Separation of indigo glycosides: First, add equal volumes of acetone and ethanol of different concentrations to the filtered activated carbon simultaneously, with three replicates per group. Stir at 600 rpm for 1 hour and filter. For the second elution, use 100% acetone, stir at 600 rpm for 1 hour, and filter a second time to obtain the eluent after two elutions.

[0062] S2-3. Remove the organic solvent from the two eluents obtained in step S2-2 in a rotary evaporator, and concentrate to obtain a concentrated solution containing indigo glycosides.

[0063] S3. Preparation of a solid mixture of indigo and indirubin

[0064] S3-1. Add 0.05% β-glucosidase to the concentrated solution containing indoside, and enzymatically hydrolyze the solution in a bioreactor at 45°C for 2 hours to obtain an indophenol solution. Adjust the pH of the indophenol solution to 10 and continue stirring for 2 hours to oxidize and generate a mixed solution of indirubin and indigo.

[0065] S3-2. After adjusting the pH of the mixed solution of indigo and indigo to 2 using 72% (v:v) sulfuric acid, the mixture was allowed to settle overnight and then centrifuged at 11,000 rpm for 5 min to obtain a solid mixture of indigo and indigo.

[0066] S4, Separating indigo and indigo red.

[0067] Indigo and indigo in a solid mixture were separated by acetone extraction. The precipitate was indigo, and the supernatant was subjected to low-temperature crystallization to obtain high-purity indigo.

[0068] Comparative Example 1

[0069] This comparative example provides a commercial glycoside purchased from Shanghai Titan Technology.

[0070] Test Example 1

[0071] This experimental example provides a method for extracting indigo glycosides from leaves treated with different methods. The specific steps are as follows:

[0072] Fresh or microwave-dried leaves of *Indigofera tinctoria* were cut into slices approximately 1-2 cm in length and width and placed in preheated deionized water. The slices were then boiled in water for 10 min at a solid-liquid ratio of 1:10 (w:v). The solution was then filtered through eight layers of gauze, and the filtrate was immediately placed in an ice bath. Indigo glycosides and indophenol in the filtrate were immediately detected at 240 nm using an HPLC system equipped with a UV detector (EX1600, Exformma, USA). An Eclipse XDB C18 column (250 mm × 4.6 mm, Agilent, USA) was used for the determination of indigo glycosides and indophenol at 30 °C. Solvent A (water:acetic acid = 100:0.5) and solvent B (acetonitrile) were used as the mobile phases. The elution process was 0-15 min, 80% A-20% B.

[0073] like Figure 1As shown, the quantitative analysis results of indoside in the extract by HPLC showed that the indoside extraction rates of fresh leaves and microwave-dried leaves were 1.60% and 1.87%, respectively. The indoside content in microwave-dried leaves increased slightly, which may be due to the loss of some indoside during the heat conduction process in fresh leaves, even though the reflux liquid was preheated.

[0074] Microwave drying can rapidly raise the internal temperature of leaves, reducing enzyme activity and thus minimizing indoside loss. These experimental results suggest that microwave drying could potentially create an indigo production process that does not rely on fresh leaves, thereby solving the industry challenge of seasonal indigo production.

[0075] Experimental Example 2

[0076] This experimental example provides a method for verifying the effect of activated carbon purification on the antioxidant properties of indoleglycoside extracts. The specific steps are as follows:

[0077] Equal volumes of 2.5 mM potassium persulfate (K₂S₂O₈) solution and 7 mM ABTS solution were mixed and allowed to stand for 24 hours in the dark at room temperature. The mixture was then diluted with 80% methanol to a specific absorbance (OD). 735 The absorbance was 0.7 ± 0.005. Subsequently, 0.1 mL of diluted commercial glycoside solution (Comparative Example 1), crude indigo glycoside solution (Example 1), or purified indigo glycoside solution (Example 2) was added to 3.9 mL of the prepared reaction solution. The mixture was thoroughly mixed and reacted in the dark for 15 minutes. After the reaction was complete, the absorbance (OD) was measured. 735 The experiment was repeated three times. Antioxidant activity was expressed as ascorbic acid (vitamin C) equivalents.

[0078] Redox reactions play a central role in the formation of indigo and indirubin. Antioxidants can slow down or prevent the oxidation process by capturing free radicals and inhibiting their reactions with other molecules. The difference in the indigo and indirubin ratio between crude indigo glycosides (Example 1) and commercial glycosides (Comparative Example 1) under weakly alkaline and microaerobic conditions is due to the antioxidant properties of carbonyl compounds in the crude glycosides.

[0079] like Figure 2 As shown, the antioxidant analysis of the three glycosides revealed that the antioxidant activity of the crude indigo glycoside solution (Example 1) was almost three times that of the commercial glycoside (Comparative Example 1) and the purified indigo glycoside (Example 2). The higher antioxidant activity in the crude indigo glycoside solution may limit the oxidation of indophenol to indigo, thereby inhibiting the formation of indirubin. Conversely, the commercial glycoside solution lacked antioxidants, which favored the formation of indigo and the accumulation of indirubin.

[0080] Therefore, the final yield and purity of indigo and indirubin can be affected by controlling the composition and chemical properties of the glycoside solution.

[0081] Test Example 3

[0082] This experimental example provides a method for verifying the effect of purification on the content of carbonyl compounds. The specific steps are as follows:

[0083] Add 2 mL of commercial glycoside solution (Comparative Example 1), crude indigo glycoside solution (Example 1), or purified indigo glycoside solution (Example 2) to a 25 mL volumetric flask. Add 2 mL of 1 g / L 2,4-dinitrophenylhydrazine solution and react at room temperature for 30 min. Dilute to the mark with 100 g / L potassium hydroxide-methanol solution, stopper, shake well, and let stand for 12 min. Measure the absorbance at 480 nm. The carbonyl compound content is expressed as butanone equivalent.

[0084] Based on the reaction mechanism of indophenol reacting with indigo, a quinone compound, under weakly alkaline conditions to form indirubin through aldol condensation, this experimental example suggests that other carbonyl compounds present in the reaction system, such as ketones, aldehydes, and quinones, may undergo similar aldol reactions, generating monoindole byproducts, thus leading to differences in the conversion rate of indole glycosides.

[0085] The determination of carbonyl compound content supported the hypothesis of this experimental example. The carbonyl compound content in the crude indole extract solution (Example 1) was much higher than that in the purified indole glycoside (Example 2) and the commercial glycoside (Comparative Example 1) (Table 1). This suggests that carbonyl compounds may compete with indophenol for the same redox pathway in the crude indole extract, thus affecting the formation of bisindole compounds. Activated carbon purification reduced the content of these carbonyl compounds, thereby reducing the non-bisindole compounds formed by the reaction of indophenol and carbonyl compounds.

[0086] Table 1. Effect of purification on carbonyl compound content in Example 2.

[0087]

[0088] Test Example 4

[0089] This experimental example provides a method for verifying the effect of oxidants on indigo formation. The specific steps are as follows:

[0090] Prepare 100 mL of crude indigo glycoside extraction solution (1 g / L) using 0.05 M citrate buffer (Example 1), adjust pH to 5, add enzyme at 1 U / mg, and react in a water bath at 45°C and 100 rpm, with continuous N2 bubbling at 20 mL / min for 1 h. Add 0, 0.1, 0.5, 1.0, and 2.0 mM potassium permanganate-citrate buffer solutions, respectively, with three replicates for each group. Continue the reaction at 45°C under N2 bubbling conditions for 2 h. After the reaction, measure the indigo content in the solution and the indigo content of the precipitate.

[0091] This experimental example suggests that the oxidizing properties of the system play a crucial role in the formation of indigo, an intermediate product of indirubin. To verify the effect of changes in the redox level of the reaction system on the formation of indigo and indirubin, this experimental example investigated the influence of changes in the oxidizing properties of the reaction system on the formation of indigo by adding potassium permanganate to the crude indigo glycoside extraction solution (Example 1).

[0092] like Figure 3 As shown, under weakly acidic and micro-oxygen conditions, a small concentration of potassium permanganate promoted the formation of indigo, which is attributed to the oxidizing effect of indophenol radicals in the presence of the oxidant. With increasing oxidant concentration, the conversion rate of indigo increased continuously, while the total conversion rate of indole glycosides decreased significantly, indicating that increased oxidizing power in the reaction system led to the conversion of indophenol towards indigo. However, excessively high antioxidant capacity may have caused over-oxidation of indigo, resulting in a decrease in the total conversion rate of indole glycosides. This result demonstrates that the directional conversion of indole glycosides to indigo and indirubin can be regulated by altering the components in the reaction system.

[0093] Test Example 5

[0094] This experimental example provides a method for verifying the effect of purified indigo glycosides on the formation of indophenol. The specific steps are as follows:

[0095] Prepared commercial glycoside solutions (Comparative Example 1), crude indigo glycoside solutions (Example 1), or purified indigo glycoside solutions (Example 2) were prepared to a concentration of 1 g / L. 100 mL of each solution was then adjusted to pH 5 and transferred to a self-made bioreactor. 1 U / mg of Acremonium cellulase was added, and the reaction was carried out in a 45°C water bath shaker for 2 hours. During the reaction, N2 was continuously bubbled through at a rate of 20 mL / min. Every 30 minutes, 2 mL of the enzymatic hydrolysate was collected for the quantitative determination of indigo glycosides and indophenol. After enzymatic hydrolysis, the solution was adjusted to pH 10 and reacted under different physicochemical conditions to produce indigo and indirubin.

[0096] Figure 4 Data showed a significant difference in the total indole glycoside conversion rate between crude indole glycosides (Example 1) and commercial glycosides (Comparative Example 1). This experimental example hypothesizes that changes in the composition of the reaction system are the main reason affecting the total indole glycoside conversion rate. Figure 4 As shown in Figure A, the purified indigo glycoside obtained by activated carbon purification (Example 2) significantly accelerated the enzymatic hydrolysis rate of indigo glycoside and the amount of indophenol generated, with almost no difference compared to the commercial glycoside (Comparative Example 1). This indicates that the purification treatment effectively improved the efficiency of the reaction system.

[0097] Within 20 minutes, the indigo glycoside consumption rates of crude indigo glycoside (Example 1), purified indigo glycoside (Example 2), and commercial glycoside (Comparative Example 1) showed a significant difference. After 40 minutes, the enzymatic hydrolysis of purified indigo glycoside (Example 2) and commercial glycoside (Comparative Example 1) was almost complete, while crude indigo glycoside (Example 1) still had residues. This result indicates that activated carbon purification may remove certain components that inhibit enzymatic hydrolysis, making the enzymatic hydrolysis reaction more complete.

[0098] In terms of indophenol production, purified indole glycosides (Example 2) and commercial glycosides (Comparative Example 1) also showed higher production efficiency than crude indole glycosides (Example 1). Figure 4 B). At 20 min, the indophenol production of purified indole (Example 2) and commercial glycoside (Comparative Example 1) reached 0.25 g / L and 0.26 g / L, respectively, while that of crude indole (Example 1) was 0.21 g / L. At 80 min, the indophenol production of all three indole glycosides reached their maximum values ​​of 0.42 g / L, 0.41 g / L, and 0.35 g / L, respectively. As time progressed to 120 min, the indophenol levels of purified indole (Example 2) and commercial glycoside (Comparative Example 1) remained relatively stable, while the indophenol level of crude indole (Example 1) decreased to 0.33 g / L. Based on the theoretical maximum indophenol production of 0.45 g / L, the commercial glycoside (Comparative Example 1) and purified indole (Example 2) achieved 93.3% and 91.1%, respectively, while the crude indole (Example 1) only achieved approximately 77.8%. This may be because certain components in the indole extract react with indophenol, reducing the concentration of indophenol.

[0099] Experimental Example 6

[0100] This experimental example provides a method for verifying the effect of purification on indirubin formation. The specific steps are as follows:

[0101] Prepare 100 mL of a 1 g / L commercial glycoside solution (Comparative Example 1), a crude indigo glycoside solution (Example 1), or a purified indigo glycoside solution (Example 2), adjust the pH to 5, add 1 U / mg of enzyme, and react in a water bath at 45°C and 100 rpm for 1 h, continuously purging with N2 at 20 mL / min. After the reaction, adjust the pH to 10 and add indigo red at 5 times the molar concentration of indophenol. Continue the reaction at room temperature and 200 rpm for 2 h under sealed conditions. After the reaction, add 5 g / L NaCl to promote sedimentation, let stand in a refrigerator at 4°C for 12 h, centrifuge at 10000 rpm for 5 min, collect the solid, and dry it overnight at 105°C.

[0102] 0.5 g of crude indigo (prepared under optimal conditions for indigo glycoside purification) was dissolved in 1 L of acetone and extracted for 30 min at 50 °C and 100 rpm in a water bath. After cooling, the mixture was centrifuged at 10,000 rpm for 5 min. The precipitate was the solid phase. The supernatant (acetone phase) was placed at 4 °C for 48 h, centrifuged again, and the acetone was evaporated. The indirubin precipitate was collected and dried overnight at 105 °C. The purity of indirubin was determined by HPLC.

[0103] This study investigated the yield and purity of indigo and indirubin produced from crude indigo glycosides (Example 1) and indigo glycosides purified by activated carbon (Example 2) under specific conditions. The results showed that the degree of indigo glycoside purification significantly affected the formation of indirubin. Figure 5 Under weakly alkaline and micro-oxygen conditions, especially at 45°C, activated carbon purification of indigo glycosides (Example 2) showed a significant advantage in producing indirubin compared to crude indigo glycoside extraction (Example 1). This experimental result not only reveals the preferred formation of indirubin but also demonstrates the feasibility of a co-production process of high-purity indigo and indirubin.

[0104] like Figure 5 As shown in Figure A, the crude glycosides (Example 1) yielded only 16.8% indirubin, accounting for 23.4% of the total indigo, while the activated carbon purified indirubin glycosides (Example 2) achieved a yield of 53.2% indirubin, accounting for 59.4% of the total indigo. This result indicates that the purified glycosides have reduced antioxidant properties, which is beneficial for the synthesis of indirubin.

[0105] Meanwhile, the yield and purity of indirubin purified by activated carbon (Example 2) were similar to those of the commercial glycosides (Comparative Example 1). Figure 5 A and 5B) indicate that the co-production process of indigo and indirubin does not require complex purification processes. The process requirements can be met by simply using simple separation methods such as activated carbon adsorption to remove carbonyl compounds and reduce the antioxidant properties of the reaction system, thereby significantly reducing the purification cost of indigo glycosides.

[0106] Experimental Example 7

[0107] This experimental example provides a method for separating indigo and indirubin, the specific steps of which are as follows:

[0108] 0.5 g of crude indigo (prepared under the optimal conditions for glycoside purification in Example 2) was dissolved in 1 L of acetone and extracted in a water bath at 50 °C and 100 rpm for 30 min. After cooling, the mixture was filtered. The precipitate was the solid phase. The filtrate (acetone phase) was placed at 4 °C for 48 h, filtered again, and the acetone was removed by rotary evaporation. The indigo precipitate was collected and dried overnight at 105 °C. The purity of the indigo was determined by HPLC.

[0109] like Figure 6As shown, the solid phase represents the solid residue after acetone treatment, mainly composed of indigo. Acetone treatment increased the purity of indigo to 51.3%. The acetone phase was dominated by indirubin, with a purity of 97.7% and an actual yield of 94.8%.

[0110] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing indirubin, characterized in that, The specific steps are as follows: S1. Pre-treat indigo leaves containing indigo glycosides to prepare an indigo glycoside solution; S2. Remove the carbonyl compound from the indigo glycoside solution obtained in step S1 to obtain indigo glycoside; S3. Decompose the indigo glycoside obtained in step S2 to obtain a solid mixture of indirubin and indigo. S4. Separate the solid mixture of indigo red and indigo obtained in step S3 to obtain indigo and indigo red.

2. The method for preparing indigo red according to claim 1, characterized in that, In step S1, the bluegrass leaves include fresh leaves and dried leaves; The indigo leaves include leaves of strychnine, woad, and woad.

3. The method for preparing indigo red according to claim 1, characterized in that, In step S1, the method for pretreating the indigo leaves containing indigo glycosides is as follows: Indigo glycoside solution is prepared by extracting indigo leaves containing indigo glycoside using organic solvents or water.

4. The method for preparing indigo red according to claim 3, characterized in that, The extraction conditions are: temperature 60-100℃, time 10-60 minutes, solid-liquid ratio 5%-30% (w / v); The organic solvents include methanol, ethanol, and acetone.

5. The method for preparing indigo red according to claim 1, characterized in that, In step S2, the methods for removing carbonyl compounds from the indigo glycoside solution obtained in step S1 include solvent extraction, adsorbent adsorption, coagulation precipitation, enzyme treatment, and redox reaction.

6. The method for preparing indigo red according to claim 5, characterized in that, The adsorbents include silica gel, activated alumina, activated carbon, molecular sieves, and macroporous resins.

7. The method for preparing indigo red according to claim 5, characterized in that, The enzyme used in the enzyme treatment method is an enzyme with polyphenol oxidase activity; The precipitant used in the coagulation and precipitation method includes enzymes with polyphenol oxidase activity, calcium hydroxide, barium chloride, and ferrous salts. The precipitant reacts with carbonyl compounds to produce a precipitate.

8. The method for preparing indigo red according to claim 5, characterized in that, The redox reaction method includes: oxidizing the polyphenols in the indigo glycoside solution with an oxidizing agent to form easily separable products; reducing the polyphenols in the indigo glycoside solution with a reducing agent to colorless or water-soluble products, and then removing them by separation technology, thereby removing carbonyl compounds.

9. The method for preparing indigo red according to claim 1, characterized in that, In step S3, the concentrated solution containing indoside is enzymatically or photolyzed to generate an indophenol solution. The indophenol solution is oxidized to generate a mixed solution of indirubin and indigo. After sedimentation, a solid mixture of indirubin and indigo is obtained.

10. The method for preparing indigo red according to claim 1, characterized in that, In step S4, the solid mixture of indigo and indigo obtained in step S3 is separated by acetone extraction to obtain crude extracts of indigo and indigo. After drying the precipitate, indigo is obtained, and acetone is removed by rotary evaporation to obtain purified indigo.

Images