Method for synchronously, efficiently and directionally preparing indirubin and tryptanthrin by using blue grass raw material

Abstract

The invention discloses a method for synchronously, efficiently and directionally preparing indirubin and tryptanthrin by using a blue grass raw material, which comprises the following steps: pretreating blue grass to obtain blue grass powder or slurry; adding water into the blue grass powder or slurry, then adding a precursor substance, and carrying out ultrasonic or microwave treatment; according to different target products, static conversion or dynamic conversion can be carried out, or static conversion or dynamic conversion is carried out after microorganisms producing cellulase and oxygenase are inoculated, and the products are subjected to post-treatment such as drying and grinding. The method is simple to operate, high in processing efficiency, flexible in product scheme, low in production cost and suitable for large-scale production. The content of the indole alkaloid in the obtained material rich in the indole alkaloid is 1%-10%; the yield of indirubin reaches 5.64 mg / g, which is 21.7 times that of the traditional process; and the tryptanthrin yield reaches 7.68 mg / g, which is 10.8 times that of the traditional process.

Description

Technical Field

[0001] The present invention relates to a method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials, and belongs to the field of pretreatment of plant raw materials and processing of active substances. Background Art

[0002] Indigo plants refer to a general term for various plants that can produce indigo dyes and are used for cloth dyeing. Traditionally, indigo plants mainly refer to Baphicacanthus cusia (Nees) Bremek. of the family Acanthaceae, Indigofera tinctoria Linn of the family Leguminosae, Polygonum tinctorium Ait of the family Polygonaceae, etc.

[0003] Baphicacanthus cusia, a plant of the genus Baphicacanthus in the family Acanthaceae, is widely distributed in East Asia and Southeast Asian regions such as China, Bangladesh, India, and Myanmar (Zeng Meijuan, Diao Yong. Research progress on secondary metabolites of Baphicacanthus cusia [J]. Chinese Agricultural Science Bulletin, 2016, 32(20): 30 - 34). This plant is widely used as a traditional herb and dye in southwestern China. Its root is called "Southern Isatis Root" and is often used to treat viral hepatitis, influenza, inflammation, and snake venom bites (Chen Yilong. Quality standard research on Isatis indigotica Fort. var. woadwax leaf (Ⅰ) [D]. Guangzhou University of Chinese Medicine, 2014). It contains various active ingredients such as indoles, quinazolinones, benzoxazinones, triterpenoids, lignans, etc. Among them, the most widely used are indigo, indirubin, and tryptanthrin (Fan Li, Li Wanshu, Liu Wangcai, etc. Isolation and identification of chemical constituents from the leaves of Baphicacanthus cusia [J]. Journal of Chinese Medicinal Materials, 2023, 46(03): 648 - 651).

[0004] Indigofera tinctoria is a plant of the genus Indigofera in the family Leguminosae, also known as Sophora indigo and wild indigo. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding. It is mainly used to treat epidemic encephalitis B, mumps, acute pharyngitis, lymphadenitis, red eyes, mouth sores, carbuncles and furuncles, erysipelas, scabies, insect and snake bites, and hematemesis.

[0005] Polygonum tinctorium is an annual herb of the family Polygonaceae and the genus Polygonum. Polygonum tinctorium prefers warm and humid climates and has low requirements for soil. It is the raw material of some natural plant blue dyes in some areas. Polygonum tinctorium is also a commonly used medicinal plant, and its leaves have the effect of clearing heat and detoxifying.

[0006] Indigo, one of the oldest pigments known to humans, is widely used for dyeing and is made by fermenting the leaves of Baphicacanthus cusia or Isatis indigotica. Recently, studies have reported its anti-inflammatory effects, so it is widely used in the treatment of anti-inflammatory diseases and psoriasis (Lin Lin, Diao Yong, Zhou Xinyi, et al. Research progress on the biological activities of indigo [J]. Dyes and Pigments, 2019, 56(04): 16-18). Indirubin is a traditional Chinese medicine extract found in the Chinese herbal medicine Qingdai that has a therapeutic effect on chronic myeloid leukemia. It is a bis-indole drug with anti-inflammatory and anti-tumor effects, etc. It also has a fast onset, low toxicity, and few side effects. Therefore, it has been widely studied as an anti-tumor drug (Ma Mengxue, Ning Xia, Liu Huashi. Research progress on the extraction, separation and pharmacology of indirubin [J]. Chinese Journal of Ethnomedicine and Ethnopharmacy, 2023, 32(18): 64-69).

[0007] Tryptanthrin is an alkaloid of indoloquinazolinone type and has pharmacological activities in aspects such as anti-tumor, anti-leishmania and anti-parasite, anti-inflammatory and anti-allergic, antibacterial, and immunomodulation (Liao Wei, Song Jiao, Han Li, et al. Research progress on the natural product tryptanthrin [J]. Natural Product Research and Development, 2023, 35(11): 2003-2013).

[0008] In the traditional process of making indigo with indigo plants, after harvesting fresh indigo plants, they are soaked in an open trough with the water surface covering the indigo leaves, and heavy objects are used to press them down to ensure that all the indigo plants are soaked in water. They are fermented for several days or a week according to the temperature. The soaking liquid turns dark green. Then, the indigo plant residues are drained, and freshly prepared lime water is added to the filtrate and stirred quickly. The stirring speed has a direct relationship with the final product quality of indigo dye. Oxygen enters the whole system during the stirring process, oxidizing and polymerizing precursors such as indican into products such as indigo. Part of the products float up with the foam, and part precipitate with the lime. After standing, the clear liquid is removed, and the floating indigo and the sinking mud-like substances are collected and called indigo mud. After drying, it is called indigo or Qingdai (Liu Kun, Gong Jixian, Li Huiqin, Li Zheng, Li Qiujin, Zhang Jianfei. Traditional dyeing process of Bouyei plant indigo [J]. Knitting Industry, 2018, (03): 56-58).

[0009] Indigo and indirubin are isomers of each other. In the traditional process, indigo is the main product (with a small amount of indirubin and tryptanthrin as by-products). Since indoxyl is very easy to dimerize itself to form indigo, the yield of indirubin is low when produced by this method (CN112824385A). In the traditional method, wild bacteria and enzymes in the environment act on indican to form indoxyl, and the oxidation and transformation reaction is more inclined to produce indigo. Indirubin or tryptanthrin only accounts for a small amount in the product composition. If indirubin and tryptanthrin are the main target products, the traditional process of preparing indigo must be improved. For example, by screening better bacteria and multiplying them, changing the system pH, increasing the release of indoxyl, increasing the oxygen supply, etc. to improve the conversion rate.

[0010] CN114540442A discloses a method for increasing the production of indigo and indirubin by transforming indigo plants with screened microorganisms or enzymes. Based on the series of studies on Strobilanthes cusia, indigo, indirubin, and tryptanthrin, a method for increasing the production of indigo and indirubin by transforming indigo plants with screened excellent microorganisms (such as Bacteroides odoratus) and corresponding enzymes has been developed, and the materials in the system have been fully utilized and developed. For example, a method for preparing melanin from indigo plants or the residues after indigo plant processing has been developed (CN113549657A). CN113549657A and CN114540442 use screened microorganisms or enzymes to transform indigo plants to increase the production of indigo and indirubin, which can increase the yield of indirubin to a certain extent, but the strains used, the selectivity of the products, and the conversion rate still need to be further optimized and improved.

[0011] Transformation mechanism of indigo, indirubin, and tryptanthrin: These alkaloid compounds have a common precursor, indoxyl (obtained by catalyzing indole with oxygenase). Indoxyl is then converted into indoxolone or anthranilic acid, which are the precursor substances of indirubin and tryptanthrin ( Figure 1 ). In most cases, indoxyl will dimerize with itself to form indigo, and only a small amount of indoxyl will be converted into indoxolone or anthranilic acid, which reacts with the remaining indoxyl to be converted into indirubin or tryptanthrin. This is the reason why indigo accounts for most of the products in most cases.

[0012] Bacillus subtilis, Bacillus megaterium, etc. can produce glycoside hydrolases. The P450 BM-3 enzyme is used to catalyze the conversion of indole to hydroxyindole, and the main product obtained by the conversion is indigo. After mutating the wild-type strain, mutations at three amino acid sites, Phe87, Leu188, and Ala74, can significantly improve the affinity of the enzyme for the substrate and the catalytic efficiency (1365 M-1S-1, kcat = 2.73 S-1), but do not significantly increase the yield of indirubin (Li, Q S, Schwaneberg, et al. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst [J]. Chemistry - A European Journal, 2000, 6(9): 1531 - 6). Hu et al. studied the role of P450 BM-3 in the synthesis of indirubin. They obtained a mutant strain D168W by modifying P450 BM-3, which mainly produced indirubin (about 90%, but took a long time and the concentration of the product indirubin was very low). The amount of indirubin produced by the mutant strain D168W in a 100 ml system with 0.5 mmol indole substrate after 150 hours of conversion was 0.0018 mmol (i.e., 0.004 mg / ml), while the parental enzyme mainly formed indigo (about 85%) (Hu S, Huang J, Mei L, et al. Altering the regioselectivity of cytochrome P450 BM-3 by saturation mutagenesis for the biosynthesis of indirubin [J]. Journal of Molecular Catalysis B: Enzymatic, 2010, 67(1): 29 - 35).

[0013] In the production of alkaloids from indigo plants, indigo, indirubin, and tryptanthrin have related precursors (Zhang Yiming, Huang Yuanzhen, Wan Huihua, etc. Research progress on the biosynthetic pathway of indigo in plants [J]. China Journal of Chinese Materia Medica, 2020, 45(03): 491 - 496). The traditional process only takes indigo as the target product. When it comes to producing higher-value-added products of indirubin and tryptanthrin (both are important drugs with much higher added value than indigo products), there is an urgent need to develop methods for the directed preparation of indirubin and tryptanthrin. Summary of the Invention

[0014] Object of the Invention: In order to overcome the deficiencies of the prior art, the present invention provides a method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials in a directed manner.

[0015] Technical solution: A method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to the present invention comprises the following steps:

[0016] (1) Pretreat the indigo plant: Dry the fresh leaves of the indigo plant in the shade or by microwave to obtain dry leaves, and then grind them after grinding or freezing, or make a slurry from the fresh leaves of the indigo plant;

[0017] (2) Add water to the indigo plant powder or slurry, and then add precursor substances, and perform ultrasonic or microwave treatment;

[0018] (3) Perform static conversion or dynamic conversion, or inoculate microorganisms producing cellulase and oxygenase and then perform static conversion or dynamic conversion, dry, and grind.

[0019] Further, in step (1), the indigo plant is one or more of Baphicacanthus cusia (Nees) Bremek. of the family Acanthaceae, Isatis indigotica Fortune of the family Brassicaceae, Indigofera tinctoria Linn of the family Leguminosae, Polygonum tinctorium Ait. of the family Polygonaceae, Clerodendrum cyrtophyllum Turcz. of the family Verbenaceae, or Wrightia laevis Hook.f. of the family Apocynaceae. The stems and / or leaves of the indigo plant are used.

[0020] Further, in step (1), the shade drying comprises the following steps:

[0021] Lay the fresh leaves of the indigo plant flat on a tray with a laying thickness of 0.5 cm to 1.5 cm, place them in a cool and ventilated place, and let them stand for 15 to 30 days until the water content of the dried leaves is 5% to 10%.

[0022] Further, in step (1), the microwave killing of green includes the following steps:

[0023] Lay the fresh leaves of the indigo plant flat on a tray with a laying thickness of 0.5 cm to 1.5 cm, dry them under 500 W microwave for 3 to 5 minutes, and dry them to a water content of 20% to 35% after killing the green.

[0024] Further, in step (1), when the target product is tryptanthrin, grind the dry leaves after freezing; when the target product is indirubin, use dry leaves for grinding or make a slurry from fresh leaves.

[0025] Further, in step (1), after freezing, grinding is carried out at low temperature using auxiliary cell wall-breaking machines such as a high-throughput grinder, a cutting crusher, and a tissue homogenizer to grind the dried bluegrass leaves.

[0026] Further, in step (2), 1 to 10 times the amount of water is added to the bluegrass powder or slurry.

[0027] Further, in step (2), when the target product is indirubin, the precursor substance is one or more of indol-3-one, indigoquinone, cysteine, or derivatives of the foregoing substances.

[0028] Further, in step (2), when the target product is tryptanthrin, the precursor substance is one or more of anthranilic acid, indol-3-one, indigoquinone, cysteine, indol-3-one-cysteine, or derivatives of the foregoing substances.

[0029] Further, in step (2), the addition amount of the precursor substance is 0.1% to 3.0% of the mass of the bluegrass leaves.

[0030] Further, in step (2), the ultrasonic treatment time is 0.1 to 3 h.

[0031] Further, in step (3), when the target product is indirubin, microorganisms producing cellulase and oxygenase or the produced enzymes are inoculated for static transformation or dynamic transformation. When the target product is tryptanthrin, direct static transformation or dynamic transformation is carried out.

[0032] Further, in step (3), the microorganisms producing cellulase and oxygenase are one or more of Bacillus subtilis YR08, Bacillus megaterium, and Bacillus natto

[0033] Furthermore, Bacillus subtilis YR08 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 24, 2023, with the deposit number CGMCC No. 29125 and the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] Further, in step (3), the inoculation form of the microorganisms producing cellulase and oxygenase can be in the form of strains, bacterial solutions, bacterial powders, enzyme solutions, and / or enzyme powders.

[0035] Further, in step (3), the concentration of the bacterial solution of the microorganisms producing cellulase and oxygenase is 10 8 -10 9CFU / ml, and the inoculation amount is 0.1 - 1 mL / g of fresh Strobilanthes cusia.

[0036] Furthermore, in step (3), the static conversion is carried out by standing still at 15 - 60 °C for 0.5 - 120 h

[0037] Furthermore, in step (3), the dynamic conversion is carried out by oscillating at 15 - 60 °C and 180 - 220 r / min for 0.5 - 120 h.

[0038] The applicant screened three strains of microorganisms that can promote the yield of indirubin in the previous research. Among them, the yield of indirubin obtained by treating Strobilanthes cusia materials with Bacteroides odorivorans is 0.26 mg / g, which is 10.3 times higher than the traditional method. The yield of indirubin obtained by treating materials with Pseudomonas reinekei is 0.23 mg / g, which is 8.85 times higher than the traditional method. The yield of indirubin obtained by treating materials with Bacillus altitudinis is 0.20 mg / g, which is 7.76 times higher than the traditional method (Zeng Qian. Optimization of the indigo-making process from Strobilanthes cusia and preparation of Strobilanthes cusia melanin [D], 2022; CN114540442A A method for increasing the production of indigo and indirubin by transforming Strobilanthes cusia with screened microorganisms or enzymes). In the present invention, Bacillus subtilis, Bacillus megaterium, and Bacillus natto are used to treat Strobilanthes cusia materials, and the increase in the yield of indirubin is many times higher than that of the above three strains. The highest yield of indirubin reaches 5.64 mg / g, which is 21.7 times that of the indirubin yield (0.26 mg / g) of the previous improved process; the yield of tryptanthrin reaches 7.68 mg / g, which is 8.7 times that of the tryptanthrin yield (0.88 mg / g) of the traditional process (Xiao Chunxia, Yang Wanxia, Tu Jiangtao, et al. Determination of the contents of six active ingredients in Strobilanthes cusia from different producing areas and different parts by RP-HPLC [J]. Natural Product Research and Development, 2018, 30(07): 1188 - 94).

[0039] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0040] (1) The method of the present invention shortens the time for treating materials to 1 / 5 of the traditional indigo-making method, has simple operation, high production efficiency, flexible product schemes, low cost, and is suitable for large-scale production.

[0041] (2) The traditional indigo-making method takes indigo (dye, a small amount as traditional Chinese medicine) as the target product, and hopes to produce less indirubin and tryptanthrin. However, in the method of the present invention, the main target is drugs with higher added value (indirubin or tryptanthrin), and the reaction direction can be controlled by controlling the types of precursors added, and the high-value indole alkaloid products required can be precisely synthesized.

[0042] (3) In the material rich in indole alkaloids obtained by the method of the present invention, the indole alkaloid content is 1% - 10%; the yield of indirubin reaches 5.64 mg / g, which is 21.7 times that of the previous process; the yield of tryptanthrin reaches 7.68 mg / g, which is 10.8 times that of the traditional indigo-making process. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of the synthesis pathways of three alkaloids;

[0044] Figure 2 It is the HPLC chart of indirubin reference standard;

[0045] Figure 3 It is the HPLC chart of tryptanthrin reference standard;

[0046] Figure 4 It is the HPLC chart of the sample solution when the target product is indirubin;

[0047] Figure 5 It is the HPLC chart of the sample when the target product is tryptanthrin. DETAILED DESCRIPTION OF THE INVENTION

[0048] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0049] Bacillus subtilis YR08 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 24, 2023. The deposit number is CGMCC No. 29125. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is: Bacillus subtilis.

[0050] Bacillus megaterium (phosphate-solubilizing bacterium), purchased from Yancheng Shenwei Microbial Strain Technology Co., Ltd., brand: Shuiguxin, product number: Bacillus megaterium 10 billion viable bacteria)

[0051] Probiotic natto fermentation powder, purchased from Funa Technology Co., Ltd.

[0052] Enzyme solution or enzyme powder of Bacillus megaterium or Bacillus subtilis YR08: Bacillus megaterium or Bacillus subtilis YR08 was cultured in TSB medium at 35°C and 220 rpm until the bacterial concentration reached 108 - 109 CFU / ML. After centrifugation at 3000 r / min, the supernatant was removed. The precipitate was washed with 0.01 mol / L phosphate buffer (pH = 7.0) to obtain a bacterial suspension with a certain concentration. The cells were broken by ultrasonic disruption method, and the supernatant was collected by centrifugation to obtain the enzyme solution. The enzyme solution was freeze-dried to obtain the enzyme powder.

[0053] Comparative Example 1

[0054] In this example, the stems and leaves of Strobilanthes cusia were pretreated, and the specific steps are as follows:

[0055] (1) Pretreatment of fresh leaves of Baphicacanthus cusia: After removing impurities such as stems and dust and mud, the intact leaves of fresh Baphicacanthus cusia stems and leaves had a moisture content of 83%. They were laid flat on a tray with a laying thickness of 0.5 cm and left to dry in the shade for 30 days with a moisture content of 5% to obtain dried Baphicacanthus cusia leaves.

[0056] (2) Grind the dried Baphicacanthus cusia leaves with a pulverizer or a high-throughput grinder. The frequency of the high-throughput grinder was set to 50 Hz, the running time was 60 s, the interval time was 5 s, and the number of running times was 5 times. After pulverization or grinding, Baphicacanthus cusia leaf powder was obtained.

[0057] (3) Weigh 1 g of Baphicacanthus cusia leaf powder and add distilled water according to a solid-liquid ratio of 1:50, and ultrasonicate for 1.0 h to obtain an extract. Inoculate Bacillus megaterium into TSB medium and shake-culture at 35 °C and 220 rpm until the bacterial concentration reaches 10 8 -10 9 CFU / ML. After centrifugation at 3000 r / min, the supernatant was removed, and the bacterial sludge at the bottom was inoculated into the conversion solution and shaken and converted at 35 °C and 220 rpm for 60 h. After filtration and drying of the product, it was ground into powder. Using high-performance liquid chromatography analysis, the indigo yield of the dried Baphicacanthus cusia leaf powder prepared from 1 g of fresh Baphicacanthus cusia leaves was 4.79 mg / g, the indirubin yield was 1.80 mg / g, and the tryptanthrin yield was 0.43 mg / g.

[0058] Comparative Example 2

[0059] The preparation process was the same as that of Comparative Example 1, except that the bacteria in Comparative Example 1 were replaced with Bacillus subtilis YR08, and other conditions remained unchanged. Using high-performance liquid chromatography analysis, the indigo yield of the dried Baphicacanthus cusia leaf powder prepared from 1 g of fresh Baphicacanthus cusia leaves was 3.55 mg / g, the indirubin yield was 1.32 mg / g, and the tryptanthrin yield was 0.51 mg / g.

[0060] Comparative Example 3

[0061] The preparation process was the same as that of Comparative Example 1, except that the bacteria in Comparative Example 1 were replaced with probiotic natto fermentation powder, and other conditions remained unchanged. And using high-performance liquid chromatography analysis, the indigo yield of the dried Baphicacanthus cusia leaf powder prepared from 1 g of fresh Baphicacanthus cusia leaves was 3.87 mg / g, the indirubin yield was 1.32 mg / g, and the tryptanthrin yield was 0.32 mg / g.

[0062] Comparative Examples 1 - 3 are traditional indigo-making processes (such as Figure 1As shown in the figure, the yields of three alkaloids of Comparative Examples 1-3 were compared. It can be seen from Comparative Examples 1 to 3 that when the precursor substance was not used, the indirubin yield prepared by Bacillus megaterium was the highest, while the tryptanthrin yield prepared by Bacillus subtilis YR08 was the highest. More products during the conversion process were indigo, accounting for more than 65% of the total alkaloids.

[0063] Example 1

[0064] The experimental procedure was the same as that of Comparative Example 1, except that the precursor substance indoxyl was added to the conversion solution.

[0065] (1) The preparation of Strobilanthes cusia leaves powder was the same as steps (1)-(2) in Comparative Example 1.

[0066] (2) Take Strobilanthes cusia powder and add distilled water according to a solid-liquid ratio of 1:50, add indoxyl accounting for 0.5% of the mass of Strobilanthes cusia leaves and ultrasonicate for 1.0 h to obtain an extract. Take Bacillus megaterium and inoculate it into TSB medium, shake culture at 35 °C and 220 rpm until the bacterial concentration reaches 108-109 CFU / ML. After centrifugation at 3000 r / min, remove the supernatant, inoculate the bacterial sludge into the extract, and carry out conversion at 35 °C and 220 rpm for 120 h. Filter to obtain a product rich in indirubin and tryptanthrin. The high-performance liquid chromatography analysis is as Figure 4 shown, compared with Figure 2 and Figure 3 by comparative analysis, the indigo yield of the dry Strobilanthes cusia leaves powder prepared from each gram of fresh Strobilanthes cusia leaves was 1.55 mg / g, the indirubin yield was 5.64 mg / g, and the tryptanthrin yield was 1.86 mg / g. The obtained product rich in indirubin and tryptanthrin was purified by column chromatography to obtain indirubin with a purity of 88% and a recovery rate of 83%.

[0067] Among them, the column chromatography separation method: add the obtained product rich in indirubin and tryptanthrin to ethyl acetate according to a solid-liquid ratio of 1:50, ultrasonicate for 0.5 h, filter and discard the precipitate. After rotary evaporation and concentration of the solution, add silica gel powder for sample mixing, and carry out silica gel column chromatography purification to obtain indirubin with a purity of 88% and a recovery rate of 83%.

[0068] Example 2

[0069] The preparation process was the same as that of Example 1, except that the addition of the precursor substance indoxyl in Example 1 was replaced with cysteine, and other conditions remained unchanged. The high-performance liquid chromatography analysis method was used to determine that the indigo yield of the dry Strobilanthes cusia leaves powder prepared from fresh Strobilanthes cusia leaves was 2.89 mg / g, the indirubin yield was 2.37 mg / g, and the tryptanthrin yield was 0.47 mg / g. After purification by column chromatography, the purity of indirubin was 75% and the recovery rate was 61%.

[0070] Example 3

[0071] The preparation process was the same as that of Example 1, except that the precursor substance indoxyl used in Example 1 was replaced with indole, and other conditions remained unchanged. The indigo yield of the dry Strobilanthes cusia leaves prepared from fresh Strobilanthes cusia leaves was measured by high performance liquid chromatography to be 3.11 mg / g, the indirubin yield was 1.06 mg / g, and the tryptanthrin yield was 0.92 mg / g.

[0072] Example 4

[0073] The preparation process was the same as that of Example 1, except that the precursor substance indoxyl used in Example 1 was replaced with indoxyl and cysteine (the mass of indoxyl and cysteine was 0.5% of the mass of Strobilanthes cusia leaves respectively), and other conditions remained unchanged. The indigo yield of the dry Strobilanthes cusia leaves prepared from 1 g of fresh Strobilanthes cusia leaves was measured by high performance liquid chromatography to be 1.37 mg / g, the indirubin yield was 4.32 mg / g, and the tryptanthrin yield was 1.45 mg / g. After purification by column chromatography, indirubin with a purity of 80% was obtained, and the yield was 72%.

[0074] Example 5

[0075] The preparation process was the same as that of Example 1, except that the precursor substance indoxyl used in Example 1 was replaced with indole and cysteine (the mass of indole and cysteine was 0.5% of the mass of Strobilanthes cusia leaves respectively), and other conditions remained unchanged. The indigo yield of the dry Strobilanthes cusia leaves prepared from fresh Strobilanthes cusia leaves was measured by high performance liquid chromatography to be 3.89 mg / g, the indirubin yield was 0.48 mg / g, and the tryptanthrin yield was 0.39 mg / g.

[0076] Example 6

[0077] The preparation process was the same as that of Example 1, except that the precursor substance indoxyl used in Example 1 was replaced with indigoquinone, and other conditions remained unchanged. The indigo yield of the dry Strobilanthes cusia leaves prepared from fresh Strobilanthes cusia leaves was measured by high performance liquid chromatography to be 2.50 mg / g, the indirubin yield was 4.15 mg / g, and the tryptanthrin yield was 1.90 mg / g. The obtained product rich in indirubin and tryptanthrin was purified by column chromatography. Indirubin with a purity of 80% was obtained, and the yield was 87%.

[0078] Example 7

[0079] The preparation process was the same as that of Example 1, except that the addition amount of indolones in Example 1 was replaced by 0.1% of the mass of Strobilanthes cusia leaves, and other conditions remained unchanged. Using high performance liquid chromatography analysis, the indigo yield of 1 g of dry Strobilanthes cusia leaf powder was measured to be 2.95 mg / g, the indirubin yield was 4.14 mg / g, and the tryptanthrin yield was 1.53 mg / g. The obtained product rich in indirubin and tryptanthrin was purified by column chromatography. Indirubin with a purity of 68% was obtained, and the yield was 75%.

[0080] Example 8

[0081] The preparation process was the same as that of Example 1, except that the addition amount of indolones in Example 1 was replaced by 0.3% of the mass of Strobilanthes cusia leaves, and other conditions remained unchanged. Using high performance liquid chromatography analysis, the indigo yield of Strobilanthes cusia leaf powder was measured to be 1.90 mg / g, the indirubin yield was 4.99 mg / g, and the tryptanthrin yield was 1.78 mg / g. The obtained product rich in indirubin and tryptanthrin was purified by column chromatography. Indirubin with a purity of 75% was obtained, and the yield was 76%.

[0082] Combined with the analysis of Example 1 and Examples 7 to 8, it can be seen that with the increase of the addition amount of indolones, the yields of indirubin and tryptanthrin both increase steadily.

[0083] Comparative Example 4

[0084] (1) Pretreatment of fresh Strobilanthes cusia leaves: Use the leaves of fresh Strobilanthes cusia stems and leaves picked within 12 hours on the day, which are intact after removing impurities such as stems and dust and mud, with a moisture content of 83%. Place the fresh leaves in a metal can and freeze them in liquid nitrogen for 5 min.

[0085] (2) Grind the frozen fresh Strobilanthes cusia leaves with a high-throughput grinder. The frequency of the high-throughput grinder is set to 50 Hz, the running time is 60 s, the interval time is 5 s, and the number of running times is 5 times to obtain Strobilanthes cusia leaf powder.

[0086] (3) Weigh 1 g of Strobilanthes cusia leaf powder, add distilled water according to the solid-liquid ratio of 1:10, place it under 100 W ultrasonic for 1.0 h to obtain a conversion solution. Then place it for static conversion at 35 °C for 48 h, filter to obtain the filter residue, dry it, and grind it into powder to obtain a product containing tryptanthrin and indirubin. Using high performance liquid chromatography analysis, the indigo yield of 1 g of fresh Strobilanthes cusia leaf was measured to be 2.36 mg / g, the indirubin yield was 0.03 mg / g, and the tryptanthrin yield was 0.66 mg / g.

[0087] Example 9

[0088] The experimental process was the same as that of Comparative Example 4, except that the precursor substance anthranilic acid was added to the conversion solution.

[0089] (1) The preparation of Strobilanthes cusia leaves powder is the same as steps (1)-(2) in Comparative Example 4.

[0090] (2) Weigh 1 g of Strobilanthes cusia leaves powder, add distilled water according to a solid-liquid ratio of 1:10, add 2.0% of anthranilic acid based on the mass of Strobilanthes cusia leaves, and ultrasonicate for 1.0 h to obtain an extract. Then, it is left to statically transform at 35 °C for 48 h, and the filtrate is obtained to get the tryptanthrin product. Through high-performance liquid chromatography analysis, the indigo yield of the Strobilanthes cusia leaves powder made from 1 g of fresh Strobilanthes cusia leaves is 1.35 mg / g, the indirubin yield is 0.49 mg / g, and the tryptanthrin yield is 4.32 mg / g. The obtained product rich in tryptanthrin is purified by column chromatography. Tryptanthrin with a purity of 92% is obtained, and the yield is 77%.

[0091] Example 10

[0092] The preparation process is the same as that in Example 9, except that the precursor substance added in Example 9 is replaced with anthranilic acid and cysteine (the mass of both anthranilic acid and cysteine is 0.5% of the mass of Strobilanthes cusia leaves), and other conditions remain unchanged. Through high-performance liquid chromatography analysis, the indigo yield of the Strobilanthes cusia leaves powder made from 1 g of fresh Strobilanthes cusia leaves is 1.57 mg / g, the indirubin yield is 0.95 mg / g, and the tryptanthrin yield is 2.52 mg / g.

[0093] Example 11

[0094] The preparation process is the same as that in Example 9, except that the precursor substance added in Example 9 is replaced with anthranilic acid and indigoquinone (the mass of both anthranilic acid and indigoquinone is 0.5% of the mass of Strobilanthes cusia leaves), and other conditions remain unchanged. Through high-performance liquid chromatography analysis, the indigo yield of the Strobilanthes cusia leaves powder made from 1 g of fresh Strobilanthes cusia leaves is 1.22 mg / g, the indirubin yield is 0.76 mg / g, and the tryptanthrin yield is 1.50 mg / g.

[0095] Example 12

[0096] The preparation process is the same as that in Example 9, except that the precursor substance added in Example 9 is replaced with anthranilic acid and indolyl-cysteine (the mass of both anthranilic acid and indolyl-cysteine is 0.5% of the mass of Strobilanthes cusia leaves), and other conditions remain unchanged. Through high-performance liquid chromatography analysis, the indigo yield of the Strobilanthes cusia leaves powder made from 1 g of fresh Strobilanthes cusia leaves is 1.25 mg / g, the indirubin yield is 0.81 mg / g, and the tryptanthrin yield is 5.28 mg / g. After purification by column chromatography, tryptanthrin with a purity of 85% is obtained, and the yield is 81%.

[0097] Among them, the synthesis method of indolinone-cysteine is as follows: Take 1.47 g of indolinone and 1.21 g of cysteine and add them to a round-bottom flask, add 100 mL of water, and reflux with condensation at 100 °C for 1.0 h. After the reaction is completed, indolinone-cysteine is obtained.

[0098] Combined with Examples 9 to 12 and Comparative Example 4 of the traditional indigo-making process, after adding the precursor anthranilic acid, the yield of tryptanthrin increased significantly, and the proportion of tryptanthrin in the alkaloid products increased from 9.6% to 72.3%.

[0099] Example 13

[0100] The preparation process is the same as that of Example 12, except that the addition amounts of the precursor anthranilic acid and indolinone-cysteine in Example 12 are both replaced by 1.0% of the mass of Strobilanthes cusia leaves, and other conditions remain unchanged. Using high-performance liquid chromatography analysis, the indigo yield of the dry Strobilanthes cusia leaf powder made from 1 g of fresh Strobilanthes cusia leaves is 1.41 mg / g, the indirubin yield is 0.98 mg / g, and the tryptanthrin yield is 2.99 mg / g. After purification by column chromatography, tryptanthrin with a purity of 81% is obtained, and the yield is 85%

[0101] Example 14

[0102] The preparation process is the same as that of Example 12, except that the addition amount of the precursor is replaced by 3.0% of the mass of Strobilanthes cusia leaves, and other conditions remain unchanged. And using high-performance liquid chromatography analysis, as Figure 5 shown. The indigo yield of the dry Strobilanthes cusia leaf powder made from 1 g of fresh Strobilanthes cusia leaves is 1.30 mg / g, the indirubin yield is 1.17 mg / g, and the tryptanthrin yield is 7.68 mg / g. After purification by column chromatography, tryptanthrin with a purity of 89% is obtained, and the yield is 80%

[0103] Combined with the analysis of Examples 12 to 14, it can be seen that with the increase of the addition amounts of anthranilic acid and indolinone-cysteine, the yield of tryptanthrin increases steadily.

[0104] Example 15

[0105] The preparation process is the same as that of Example 14, except that the raw materials are replaced with fresh leaves of Indigofera tinctoria, and other conditions remain unchanged. Using high-performance liquid chromatography analysis, the indigo yield of 1 g of fresh leaves of Indigofera tinctoria is 1.66 mg / g, the indirubin yield is 1.70 mg / g, and the tryptanthrin yield is 5.15 mg / g.

[0106] Comparative Example 5

[0107] The preparation process was the same as that of Example 15, except that no precursor substances were added, the raw material was replaced with fresh leaves of Polygonum tinctorium, and other conditions remained unchanged. The indigo yield of 1 g of fresh leaves of Polygonum tinctorium was measured to be 2.57 mg / g, the indirubin yield was 0.11 mg / g, and the tryptanthrin yield was 0.74 mg / g by high performance liquid chromatography analysis.

[0108] Example 16

[0109] The preparation process was the same as that of Example 12, except that the raw material was replaced with fresh leaves of Isatis indigotica, and other conditions remained unchanged. The indigo yield of 1 g of fresh leaves of Isatis indigotica was measured to be 0.76 mg / g, the indirubin yield was 1.05 mg / g, and the tryptanthrin yield was 2.10 mg / g by high performance liquid chromatography analysis.

[0110] Comparative Example 6

[0111] The preparation process was the same as that of Example 16, except that no precursor substances were added and other conditions remained unchanged. The indigo yield of 1 g of fresh leaves of Isatis indigotica was measured to be 1.20 mg / g, the indirubin yield was 0.69 mg / g, and the tryptanthrin yield was 0.50 mg / g by high performance liquid chromatography analysis.

[0112] Example 17

[0113] The preparation process was the same as that of Example 12, except that the raw material was replaced with fresh leaves of Clerodendrum cyrtophyllum, and other conditions remained unchanged. The indigo yield of the fresh leaves was measured to be 1.24 mg / g, the indirubin yield was 0.80 mg / g, and the tryptanthrin yield was 1.98 mg / g by high performance liquid chromatography analysis.

[0114] Comparative Example 7

[0115] The preparation process was the same as that of Example 17, except that no precursor substances were added and other conditions remained unchanged. The indigo yield of 1 g of fresh leaves of Clerodendrum cyrtophyllum was measured to be 2.58 mg / g, and neither indirubin nor tryptanthrin was detected by high performance liquid chromatography analysis.

[0116] Example 18

[0117] The preparation process was the same as that of Example 12, except that the raw material was replaced with fresh leaves of Isatis tinctoria, and other conditions remained unchanged. The indigo yield of the fresh leaves was measured to be 1.58 mg / g, the indirubin yield was 2.04 mg / g, and the tryptanthrin yield was 3.24 mg / g by high performance liquid chromatography analysis.

[0118] Comparative Example 8

[0119] The preparation process was the same as that of Example 18, except that no precursor substances were added and other conditions remained unchanged. The indigo yield of 1 g of fresh Isatis indigotica leaves was measured to be 3.41 mg / g, the indirubin yield was 0.04 mg / g, and the tryptanthrin yield was 0.25 mg / g by high performance liquid chromatography analysis.

[0120] Comparative analysis of Examples 15 to 18 and Comparative Examples 5 to 8 shows that the addition of precursor substances (anthranilic acid and indolyl-cysteine) to various bluegrass plants can significantly increase the yield of tryptanthrin.

[0121] Example 19

[0122] The preparation process was the same as that of Example 1, except that the bacterial sludge in Example 1 was replaced with the enzyme solution prepared from Bacillus megaterium and other conditions remained unchanged. The indigo yield of the dry Strobilanthes cusia leaves made from fresh Strobilanthes cusia leaves was measured to be 2.80 mg / g, the indirubin yield was 4.55 mg / g, and the tryptanthrin yield was 1.64 mg / g by high performance liquid chromatography analysis. The obtained product rich in indirubin and tryptamine was purified by column chromatography. Indirubin with a purity of 80% was obtained, and the recovery rate was 78%.

[0123] Example 20

[0124] The preparation process was the same as that of Example 1, except that the bacterial sludge in Example 1 was replaced with the enzyme powder prepared from Bacillus megaterium and other conditions remained unchanged. The indigo yield of the dry Strobilanthes cusia leaves made from fresh Strobilanthes cusia leaves was measured to be 1.40 mg / g, the indirubin yield was 3.28 mg / g, and the tryptanthrin yield was 1.17 mg / g by high performance liquid chromatography analysis. The obtained product rich in indirubin and tryptamine was purified by column chromatography. Indirubin with a purity of 70% was obtained, and the recovery rate was 75%.

Claims

1. A method for synchronously and efficiently preparing indirubin and tryptanthrin from indigofera tinctoria raw materials, characterized in that, It includes the following steps: (1) Pretreat the indigo plant: Air-dry the fresh leaves of the indigo plant in the shade or blanch them with microwave to obtain dry leaves of the indigo plant, and then grind them after grinding or freezing, or make a slurry by pulping the fresh leaves of the indigo plant; (2) Add water to the indigo plant powder or slurry, and then add the precursor substance, and perform ultrasonic or microwave treatment; (3) Perform static conversion or dynamic conversion, or inoculate microorganisms producing cellulase and oxygenase and then perform static conversion or dynamic conversion, dry, and grind.

2. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, characterized in that, In step (1), the indigo plant is one or more of Baphicacanthus cusia, Isatis indigotica, Indigofera tinctoria, Polygonum tinctorium, Clerodendrum cyrtophyllum or Wrightia tinctoria.

3. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, characterized in that, In step (1), when the target product is tryptanthrin, grind after freezing the dry leaves. When the target product is indirubin, use dry leaf grinding or fresh leaf pulping.

4. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, characterized in that, In step (2), add 1 to 10 times the amount of water to the indigo plant powder or slurry.

5. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, characterized in that, In step (2), when the target product is indirubin, the precursor substance is one or several of indol-3-one, indigoquinone, cysteine or derivatives of the foregoing substances.

6. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, characterized in that, In step (2), when the target product is tryptanthrin, the precursor substance is one or several of anthranilic acid, indol-3-one, indigoquinone, cysteine, indol-3-one-cysteine or derivatives of the foregoing substances.

7. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, wherein In step (2), the addition amount of the precursor substance is 0.1% to 3.0% of the mass of the indigo plant leaves. The ultrasonic treatment frequency is 100 - 200W, the ultrasonic treatment time is 0.1 to 3h, the microwave treatment power is 500 - 800W, and the treatment time is 0.1 to 3h.

8. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, wherein, In step (3), when the target product is indirubin, inoculate microorganisms producing cellulase and oxygenase and then perform static conversion or dynamic conversion. When the target product is tryptanthrin, directly perform static conversion or dynamic conversion.

9. The method for simultaneously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, wherein In step (3), the microorganisms producing cellulase and oxygenase are one or several of Bacillus subtilis YR08, Bacillus megaterium or Bacillus natto. The inoculation form of the microorganisms producing cellulase and oxygenase can adopt strains, bacterial solutions, bacterial powders, enzyme solutions and / or enzyme powders.

10. The method for synchronously and efficiently preparing indirubin and tryptanthrin from indigo plant raw materials according to claim 1, wherein In step (3), the static conversion is to stand still at 15 - 60°C for 0.5 - 120h, and the dynamic conversion is to oscillate at 15 - 60°C and 180 - 220r / min for 0.5 - 120h.

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