Method for extracting indigo blue and indirubin from baphicacanthus cusia stems and leaves

Abstract

The invention discloses a method for extracting indigo blue and indirubin through baphicacanthus cusia stems and leaves, and belongs to the technical field of system.The method comprises the steps that a baphicacanthus cusia stem and leaf extracting solution is subjected to adsorption treatment, elution is conducted after saturation is achieved, and vacuum concentration is conducted on eluent; diluting the concentrated solution with pure water, regulating the temperature, and adding hydrolase for constant-temperature reaction to obtain a target precipitate; and carrying out vacuum filtration and collection on the sedimentation product, and carrying out fractional separation by adopting a multi-layer filter membrane to respectively obtain indigo blue and indirubin. According to the scheme, the precipitation and separation efficiency of indigo and indirubin can be improved, and the product quality is improved.

Description

Technical Field

[0001] The present application relates to the field of system control, and in particular to a method for extracting indigo and indigotin through the roots, stems and leaves of Isatis indigotica. Background Art

[0002] Indigo and indirubin are a class of natural indole compounds with important application value, and have broad application prospects in the fields of medicine, dyes and functional materials. Among them, indigo, as a natural dye, has excellent dyeing properties and environmental protection characteristics, and occupies an important position in the textile industry; while indirubin has attracted much attention in the field of medicine due to its significant anti-tumor activity and immunomodulatory function. The research on the extraction methods of these two compounds has important practical significance and application value for the development of related industries. Traditional extraction methods mainly include solvent extraction, ultrasonic assisted extraction and microwave assisted extraction. Among them, solvent extraction is widely used in industrial production due to its advantages such as simple operation and low equipment requirements. However, this method has problems such as low extraction efficiency, high solvent consumption and serious environmental pollution. Although ultrasonic assisted extraction and microwave assisted extraction have improved the extraction efficiency to a certain extent, the equipment investment cost is high, and there are technical bottlenecks such as high energy consumption and difficulty in equipment maintenance in large-scale production. In addition, there are generally problems such as poor extraction selectivity and low product purity in the existing technology, which seriously restricts the high-quality large-scale production of indigo and indirubin.

[0003] As a traditional Chinese medicinal material, the stems and leaves of Isatis indigotica Fort. are rich in effective ingredients such as indigo and indigotin, and are one of the important sources of raw materials for obtaining these two compounds. At present, the methods for extracting indigo and indigotin from the stems and leaves of Isatis indigotica Fort. mainly include the following aspects: First, the traditional water extraction method, which is to soak the stems and leaves of Isatis indigotica Fort. in water and obtain the target product through fermentation, oxidation and other processes. Although the process of this method is simple, it has significant defects such as long extraction cycle, low yield and high energy consumption. Secondly, the organic solvent extraction method, commonly used solvents include ethanol, methanol, ethyl acetate, etc., although this method improves the extraction efficiency, there are problems such as solvent residue, environmental pollution and safety hazards. The third is supercritical CO2 extraction technology, which is environmentally friendly and efficient, but the equipment investment is large, the operating conditions are harsh, and the extraction selectivity is not ideal. In addition, the actual production process also faces the following technical difficulties: improper pretreatment of raw materials leads to serious loss of effective ingredients; improper temperature control during extraction causes product degradation; it is difficult to clarify the extract, affecting product quality; it is difficult to optimize the extraction process parameters, making it difficult to achieve industrial production; oxidation side reactions are prone to occur during the extraction process, reducing product purity; the selection and recycling of extractants need to be solved urgently. These technical problems seriously restrict the efficient extraction and industrial application of indigo and indirubin in southern isatis root.

[0004] Therefore, there is an urgent need for a technical solution to improve the precipitation and separation efficiency of indigo and indirubin and improve product quality. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention discloses a method for extracting indigo and indirubin from the roots, stems and leaves of Isatis indigotica. The present invention solves the technical problems of product degradation caused by improper temperature control during the prior art extraction process, difficulty in clarifying the extract, and affecting product quality.

[0006] The embodiment of the present application discloses a method for extracting indigo and indirubin through the roots, stems and leaves of Isatis indigotica, comprising: subjecting the extract of the roots, stems and leaves of Isatis indigotica to adsorption treatment, eluting after reaching saturation, and vacuum concentrating the eluate; diluting the concentrate with pure water and adjusting the temperature, adding a hydrolase to react at a constant temperature to obtain a target precipitate; collecting the precipitated product by vacuum filtration, and performing graded separation using a multi-layer filtration membrane to respectively obtain indigo and indirubin.

[0007] In one possible implementation, the extract of the roots, stems and leaves of Isatis indigotica is subjected to adsorption treatment, eluted after reaching saturation, and the eluate is vacuum concentrated, including: treating the room temperature extract through a resin column filled with macroporous adsorption resin; eluting with methanol solution when the resin column reaches a saturated adsorption state; vacuum concentrating the eluate and recovering the methanol solution.

[0008] In one possible implementation, the concentrated solution is diluted with pure water and the temperature is adjusted, and then a hydrolase is added to carry out a constant temperature reaction to obtain a target precipitate, which includes: diluting the concentrated solution with pure water and adjusting the temperature to a predetermined temperature; adding an exoglycoside hydrolase to carry out a constant temperature reaction; and after the reaction is completed, standing to allow the conversion product to naturally settle.

[0009] In a possible implementation, the precipitated product is collected by vacuum filtration and then graded and separated using a multi-layer filter membrane to obtain indigo and indirubin respectively, including: vacuum filtration of the reaction solution after standing and collecting the precipitate; separation of the mixed product using a temperature gradient field to grade and separate indigo and indirubin through multi-layer filtration, and washing the separated filter cakes separately; and drying the precipitate using a pulse vacuum drying method.

[0010] In a possible implementation, a temperature gradient field is used to separate the mixed product, so as to grade and separate indigo and indigo carmine through multi-layer filtration, and the separated filter cakes are washed separately, including: transferring the reaction liquid to a separation device with a double-layer jacket structure; passing circulating water of a first preset temperature into the inner jacket; passing heat transfer oil of a second preset temperature into the outer jacket; forming a first temperature gradient field through the temperature difference between the inner and outer layers; adjusting the vacuum degree according to a first preset control equation to allow indigo to precipitate preferentially; adjusting the temperatures of the inner and outer layers to form a second temperature gradient field; adjusting the vacuum degree according to a second preset control equation to promote the selective precipitation of indigo carmine; between two separation stages, performing transition adjustment of the temperature gradient and vacuum degree according to a third preset control equation; setting three filter layers with different pore sizes at the bottom of the separation device; preventing filter cake from compacting through tangential flow; and washing the filter cakes on different filter layers at different temperatures.

[0011] In one possible implementation, the precipitate is dried by pulse vacuum drying, including: applying a preset vacuum degree to the precipitate at a preset temperature for drying; performing pulse drying on the precipitate according to a set pulse time; performing intermittent drying on the precipitate according to a set interval time; and stopping the drying operation when the moisture content of the precipitate drops below a preset value.

[0012] In a possible implementation, before the extract of the roots, stems and leaves of Isatis indigotica is subjected to adsorption treatment and eluted after reaching saturation, and before the eluate is vacuum concentrated, it also includes: washing, draining and cutting fresh roots, stems and leaves of Isatis indigotica into sections; performing two hot water extractions in sequence, and temporarily storing the extract in a liquid storage tank after the first extraction; and combining the two extracts and letting them stand to room temperature.

[0013] In a possible implementation, after the precipitated product is collected by vacuum filtration, it is graded and separated using a multi-layer filtration membrane to obtain indigo and indirubin respectively, and the process also includes: collecting and squeezing the plant residue after extraction; recovering the supernatant of the squeezed liquid for the next batch of extraction; and regenerating the resin using an ethanol solution.

[0014] In a method for extracting indigo and indigotin from the roots, stems and leaves of Isatis indigotica as disclosed above, the embodiments of the present application can selectively destroy different types of intermolecular interactions by setting a temperature gradient, ensure the orderly progress of the dissociation process, and prevent product agglomeration. Further, in some embodiments, the introduction of vacuum pulses can disclose moderate mechanical disturbances, which help to break the eutectic structure and hydrophobic interactions without causing mechanical damage to the product itself. Further, in some embodiments, by setting a multi-layer filtration structure, the product can be separated step by step under the condition of the size characteristics of various complexes brought by the previous process. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 A schematic diagram of a process for extracting indigo and indirubin from the roots, stems and leaves of Isatis indigotica disclosed in an embodiment of the present application;

[0017] Figure 2 A schematic diagram of a process for fractionating and separating indigo and indirubin disclosed in an embodiment of the present application;

[0018] Figure 3 A separation efficiency surface diagram disclosed in an embodiment of the present application;

[0019] Figure 4 A schematic diagram of a temperature field simulation of a multi-layer filtration cross section disclosed in an embodiment of the present application. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present disclosure unless otherwise specifically stated.

[0021] Those skilled in the art can understand that the terms "first", "second" and the like in the embodiments of the present disclosure are only used to distinguish different steps, devices or modules, etc., and neither represent any specific technical meaning nor represent the necessary logical order between them. It should also be understood that in the embodiments of the present disclosure, "multiple" can refer to two or more, and "at least one" can refer to one, two or more. It should also be understood that for any component, data or structure mentioned in the embodiments of the present disclosure, in the absence of explicit limitation or contrary revelation given in the context, it can generally be understood as one or more. In addition, the term "and / or" in the present disclosure is only a description of the association relationship of the associated objects, indicating that there can be three relationships, for example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in the present disclosure generally indicates that the associated objects before and after are an "or" relationship. It should also be understood that the description of each embodiment in the present disclosure emphasizes the differences between the embodiments, and the same or similar parts can refer to each other. For the sake of brevity, they will not be repeated one by one.

[0022] At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present disclosure and its application or use. The techniques, methods and devices known to ordinary technicians in the relevant fields may not be discussed in detail, but where appropriate, the techniques, methods and devices should be considered part of the specification. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0023] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0024] Figure 1A schematic flow chart of a method for extracting indigo and indirubin from the roots, stems and leaves of southern isatis disclosed in the embodiments of the present application. It should be understood that in the existing southern isatis root extraction process, the mixed product of indigo and indirubin obtained after the enzymatic reaction is usually treated by a simple vacuum filtration or centrifugal separation method. This conventional separation method has the following specific technical problems: when separated under a constant vacuum degree, due to the overlapping particle size distribution range of indigo and indirubin in the mixed product (indigo particles 75-168 microns, indigo particles 62-145 microns), and the surface charge properties of the two substances are similar (indigo isoelectric point 6.8 ± 0.3, indigo isoelectric point 6.5 ± 0.4), it is very easy to form a co-deposition layer in the traditional separation process. This co-deposition causes the indigo content in the separated indigo to fluctuate greatly, usually between 4.8% and 12.5%. In addition, it is easy to form a dense layer on the surface of the filter cake under constant vacuum conditions, resulting in a significant reduction in filtration rate and a decrease in separation efficiency of 42% to 58%. When centrifugal separation is used, the density difference between the two substances is small (indigo density 1.01g / cm 3 , density of indigo carmine is 1.417 g / cm 3 ), even under the condition of high-speed centrifugation at 12000r / min, the separation degree can only reach 68%-75%, and the shear force generated during high-speed centrifugation will cause some particles to break, affecting the product quality.

[0025] In response to the above technical problems, this technology uses temperature gradient coupled vacuum pulse separation technology to separate indigo and indirubin by using the difference in solubility changes at different temperatures (when the temperature rises from 32.5°C to 42.8°C, the solubility of indigo increases by 0.82 mg / L, while the solubility of indirubin increases by 2.35 mg / L). By constructing a temperature gradient field in the separation device and combining the control of vacuum pulses, the two substances present different movement trajectories and deposition positions during the sedimentation process. Specifically, when the temperature is distributed in a gradient from the inner layer to the outer layer, the solubility of indirubin in the high temperature area is significantly higher than that of indigo. Under the action of vacuum pulses, the solubility difference causes the sedimentation rates of the two substances to be significantly stratified. At the same time, the periodic changes of vacuum pulses can effectively prevent the formation of a dense layer on the surface of the filter cake and maintain stable permeability during the separation process. The design of the multi-layer filtration structure further enhances the selectivity of the separation, and filter layers of different pore sizes can further physically separate the two substances that have been preliminarily stratified. This separation method increases the separation degree of indigo and indirubin in the final product to 92%-96%, which is significantly better than the existing technical level. Moreover, the entire separation process is carried out under mild vacuum conditions (maximum vacuum degree 0.068MPa), avoiding mechanical damage to product particles caused by high-speed centrifugation. The particle size distribution of the product is more concentrated, which is conducive to subsequent refining and application.

[0026] During the implementation of the process, the synergistic effect of temperature gradient and vacuum pulse is the key to achieving efficient separation. By dynamically adjusting the rate of change of the temperature gradient (0.82°C / minute) and the frequency characteristics of the vacuum pulse (fluctuation period between 218-435 seconds), the separation process of the two substances can be precisely controlled. The introduction of tangential flow optimizes the separation process from the perspective of fluid mechanics. The existence of the shear flow field not only prevents the filter cake from compacting, but also controls the residence time of the particles on the filter layer by adjusting the tangential flow rate (base flow rate 0.32 m / s), thereby further improving the selectivity of the separation. This multi-parameter coordinated control separation technology not only ensures the separation effect, but also has the advantages of low energy consumption (about 35% lower than traditional centrifugation) and simple equipment requirements (no high-speed centrifuge is required).

[0027] like Figure 1 As shown, in step S101, the extract of the roots, stems and leaves of Isatis indigotica is subjected to adsorption treatment, eluted after reaching saturation, and the eluate is vacuum concentrated. This includes: treating the room temperature extract through a resin column filled with macroporous adsorption resin; eluting with methanol solution when the resin column reaches a saturated adsorption state; vacuum concentrating the eluate and recovering the methanol solution.

[0028] In one implementation scenario, the extract at room temperature is treated with a macroporous adsorption resin column, and the resin column is filled with DM130 or D101 macroporous adsorption resin, and the column height is controlled at 55-65 cm. The extract passes through the resin column at a flow rate of 2.8 column volumes / hour, and the effluent is collected and recovered for the next batch of extraction. When the resin column reaches the saturated adsorption state, a methanol solution with a mass fraction of 82% is used for elution, the amount of eluent is 3 times the column volume, and the elution flow rate is controlled at 2.2 column volumes / hour. After the elution is completed, the eluent is collected and vacuum concentrated, the temperature is controlled at 48°C, the vacuum degree is maintained at 0.085MPa, and the methanol solution is recovered for reuse.

[0029] In step S102, the concentrated solution is diluted with pure water and the temperature is adjusted, and then a hydrolase is added to react at a constant temperature to obtain a target precipitate. This includes: the concentrated solution is diluted with pure water and adjusted to a predetermined temperature; an exoglycoside hydrolase is added to react at a constant temperature; and after the reaction is completed, the concentrated solution is allowed to stand to allow the conversion product to settle naturally.

[0030] Specifically, for enzymatic conversion, the concentrated solution can be diluted with 5 times the volume of pure water and the temperature can be adjusted to 37°C. Add 0.42% of exoglycoside hydrolase by mass to the dilution, control the stirring rate at 150 rpm, and the reaction time is 23 hours. During the reaction, the jacket heating method is used to maintain a constant temperature, and the temperature fluctuation range is controlled within ±1.5°C by the circulating water system. After the enzymatic reaction is completed, the reaction solution is allowed to stand for 2 hours to allow the insoluble product generated by the conversion to settle naturally.

[0031] In step S103, the precipitated product is collected by vacuum filtration and then graded and separated using a multi-layer filter membrane to obtain indigo and indirubin, respectively. Figure 2 As shown): the reaction solution after standing is vacuum filtered and the precipitate is collected; the mixed product is separated by using a temperature gradient field, so as to grade indigo and indirubin through multi-layer filtration, and the separated filter cakes are washed separately; the precipitate is dried by pulse vacuum drying.

[0032] In addition, before the extract of the roots, stems and leaves of Isatis indigotica is adsorbed and eluted after reaching saturation, and the eluate is vacuum concentrated, it also includes: washing, draining and cutting the fresh roots, stems and leaves of Isatis indigotica into sections; performing two hot water extractions in sequence, and temporarily storing the extract in a liquid storage tank after the first extraction; and combining the two extracts and letting them stand to room temperature.

[0033] During the raw material processing and initial extraction process, the freshly picked southern isatis root stems and leaves are washed to remove soil impurities, drain the surface water and cut into sections of 2.5-3.5 cm in length. Weigh the treated stems and leaves, add pure water with a volume-to-mass ratio of 8.5:1, heat to 82°C and maintain constant temperature for extraction. During the extraction process, the stirring rate is maintained at 180 rpm, and the extraction time is controlled at 58 minutes. After the extraction is completed, the extract is transferred to a liquid storage tank for temporary storage, and the filter residue is re-added with pure water with a volume-to-mass ratio of 9:1 for a second extraction. The second extraction temperature is also controlled at 82°C, the time is 55 minutes, and the stirring rate is maintained at 180 rpm. Combine the two extracts and let stand to cool to room temperature. Through repeated experiments, it was found that under this solid-liquid ratio and temperature-time conditions, the indole glycosides and indole phenols in the southern isatis root raw material were fully extracted, and the extraction efficiency was more than 90%.

[0034] Furthermore, after the precipitated product is collected by vacuum filtration, it is graded and separated using a multi-layer filter membrane to obtain indigo and indirubin respectively, and it also includes: collecting and squeezing the plant residue after extraction; recovering the supernatant of the squeezed liquid for the next batch of extraction; and regenerating the resin using an ethanol solution.

[0035] Specifically, in the by-product treatment, the plant residues after extraction can be collected and squeezed, the squeezing pressure is controlled at 2.8MPa, and the squeezing time is 15 minutes. The moisture content of the residue after squeezing is controlled below 65%, and it can be directly used as animal feed processing and edible fungus culture medium. After the squeezing liquid is settled, the supernatant is recovered for the next batch of extraction, and the precipitate is treated together with the squeezing residue. The resin is regenerated by backwashing with 95% ethanol solution, and the amount of flushing liquid is 4 times the column volume. After the flushing is completed, it is replaced with pure water to neutral. The regenerated resin has been tested to reach more than 92% of the initial adsorption capacity and can be reused.

[0036] Figure 2 The present invention is a schematic flow chart of a method for fractionating and separating indigo and indirubin disclosed in an embodiment of the present application.

[0037] like Figure 2 As shown, in step S201, the reaction solution after standing is vacuum filtered and the precipitate is collected. For product separation and purification, the reaction solution after standing can be vacuum filtered, and the filtrate can be recovered and reused.

[0038] At step S202, the mixed product is separated by a temperature gradient field, so as to grade and separate indigo and indirubin through multi-layer filtration, and the separated filter cakes are washed separately. The process includes: transferring the reaction liquid to a separation device with a double-layer jacket structure; introducing circulating water of a first preset temperature into the inner jacket; introducing heat transfer oil of a second preset temperature into the outer jacket; forming a first temperature gradient field through the temperature difference between the inner and outer layers; adjusting the vacuum degree according to the first preset control equation to make indigo settle preferentially; adjusting the temperature of the inner and outer layers to form a second temperature gradient field; adjusting the vacuum degree according to the second preset control equation to promote the selective sedimentation of indirubin; between the two separation stages, performing transition adjustment of the temperature gradient and vacuum degree according to the third preset control equation; setting three filter layers with different pore sizes at the bottom of the separation device; preventing the filter cake from hardening by tangential flow; and washing the filter cakes on different filter layers at different temperatures.

[0039] In one embodiment, when separating indigo and indirubin from the mixed product after the enzymatic hydrolysis reaction, firstly, the solubility difference between the two under different temperature and vacuum conditions is used for graded separation. The reaction solution is transferred to a separation device, which is equipped with a double-layer jacket structure, and the inner jacket is passed through circulating water with a temperature of 32.5°C, and the outer jacket is passed through heat transfer oil with a temperature of 42.8°C, forming a temperature gradient field.

[0040] The separation process uses temperature gradient coupled vacuum pulse technology to achieve graded sedimentation of indigo and indirubin by adjusting the ratio of temperature gradient and vacuum intensity. In the first stage, the inner layer temperature is maintained at 32.5°C and the outer layer temperature is maintained at 42.8°C, forming a temperature gradient of 10.3°C. Under this temperature condition, an initial vacuum degree of 0.042MPa is applied, and then the vacuum degree is adjusted according to the following equation: P(t) = 0.042 + 0.018sin(πt / 435) + 0.012cos(πt / 282) × (1-e^(-0.175t)), where t represents time (seconds). The vacuum fluctuations generated by this equation cause indigo to precipitate preferentially.

[0041] In the second stage, the temperature of the inner and outer layers is adjusted to 36.8℃ and 45.2℃, and the temperature gradient is increased to 8.4℃, while the vacuum pulse characteristics are changed. At this time, the vacuum degree changes according to the following equation: P(t)=0.068+0.015sin(πt / 325)×(1-e^(-0.223t))+0.009cos(πt / 218). This stage mainly promotes the selective precipitation of indigo carmine. The vacuum fluctuation period is shortened, the fluctuation amplitude is reduced, and the change in temperature gradient is combined to enrich indigo carmine in a specific area.

[0042] To further improve the separation effect, a transition link is set between the two main separation stages. In the transition link, the temperature gradient changes at a rate of 0.82°C / minute, and the vacuum degree is adjusted according to: P(t) = Pm + (Ph-Pm) (1-e^(-0.168t)) × cos(πt / 375), where Pm is 0.042MPa and Ph is 0.068MPa. This gradual change process helps prevent the aliasing and sedimentation of the two components.

[0043] Three filter layers with different pore sizes are set at the bottom of the separation device, from top to bottom: polytetrafluoroethylene filter membrane with a pore size of 0.82 microns, modified cellulose filter membrane with a pore size of 0.58 microns, and composite ceramic filter plate with a pore size of 0.35 microns. The spacing between the three layers of filter media is 4.2 mm and 3.8 mm respectively. When the mixed liquid passes through multiple layers of filtration, indigo is mainly retained on the first layer of filter membrane, indirubin is enriched on the second layer of filter membrane, and the last layer is used to ensure the integrity of the separation.

[0044] In order to prevent the filter cake from hardening, tangential flow is introduced during the vacuum pulse process. The tangential flow velocity satisfies: v(r) = v0(1-(r / R) 2 )(1+0.15sin(2πt / T)), where v0 is the reference flow velocity of 0.32 m / s, r is the distance from the central axis, R is the separator radius, and T is the pulse period. This flow pattern can maintain the looseness of the filter cake structure without destroying the formed stratification.

[0045] At the end of the separation process, the filter cakes on the first and second filter membranes are treated separately. The indigo filter cake enriched on the first filter membrane is gently rinsed with 42°C pure water, with the amount of rinse liquid being 2 to 3 times the mass of the filter cake; the indirubin enriched on the second filter membrane is rinsed with 35°C pure water, with the amount being 1 to 2 times the mass of the filter cake. The two products are collected separately and then dried.

[0046] In step S203, the precipitate is dried by pulse vacuum drying, which includes: applying a preset vacuum degree to the precipitate at a preset temperature for drying; performing pulse drying on the precipitate according to a preset pulse time; performing intermittent drying on the precipitate according to a set interval time; and stopping the drying operation when the moisture content of the precipitate drops below a preset value.

[0047] Specifically, the precipitate is collected and dried by pulse vacuum drying. The drying temperature is controlled at 42°C, the vacuum degree is set to 0.092MPa, each pulse time is 8 minutes, and the interval time is 3 minutes. Drying is stopped when the moisture content of the product drops below 8.5%.

[0048] In summary, in the process of extracting indigo and indirubin from the roots, stems and leaves of Isatis indigo, cutting the raw materials into 2.5-3.5 cm length segments and extracting at 82°C will lead to partial degradation of tissue fibers, and these degradation products will have intermolecular interactions with indigo and indirubin in the subsequent steps. Especially under the extraction temperature of 82°C, the oligosaccharide chains generated by partial hydrolysis of cellulose will form hydrogen bond networks with indigo and indirubin molecules (each oligosaccharide chain can form hydrogen bonds with 2-4 indigo or indirubin molecules on average), and these complexes will interfere with the separation process of the two target products in the final separation stage. When DM130 or D101 macroporous adsorption resins are used for treatment, due to the large difference between the pore size distribution range of the two resins (DM130 is 28-32nm, D101 is 25-28nm) and the kinetic diameters of indigo and indirubin molecules (indigo is 1.85nm, indigo is 1.72nm), some small molecules aggregate in the resin pores. These aggregates will not completely dissociate in the subsequent elution process, thus affecting the dispersion state of the final product.

[0049] The enzymatic conversion process was carried out at 37°C, using 0.42% of exoglycoside hydrolase, and the reaction time was 23 hours. At this stage, due to the inhomogeneity of the enzymatic reaction (the diffusion coefficient of the enzyme molecule in the reaction system is 2.8×10 -6 cm 2 / s), resulting in the formation of products with temporal and spatial differences. Some areas may form eutectics of indigo and indirubin (lattice constant ), this co-crystal is difficult to be effectively separated by traditional separation methods. In addition, the long enzymatic hydrolysis process will cause some product molecules to form hydrophobic interactions with residual protein fragments. The presence of these complexes makes simple vacuum filtration or centrifugal separation methods ineffective. Although the sedimentation process of standing for 2.5 hours can allow some insoluble products to precipitate naturally, due to various intermolecular interactions in the system, the distribution of indigo and indirubin in the precipitate is not uniform, and the composition of local areas may show significant deviations (the indigo content fluctuates from 32% to 58%, and the indirubin content fluctuates from 2.2% to 8.5%).

[0050] In the context of this complex system, the introduction of temperature gradient coupled vacuum pulse separation technology can effectively solve the above problems. This technology can selectively destroy different types of intermolecular interactions by setting a temperature gradient of 32-43°C. Within this temperature range, the hydrogen bond network formed with the oligosaccharide chain will gradually dissociate (the hydrogen bond dissociation energy decreases by about 0.15 kJ / mol with every 1°C increase in temperature), and the existence of the temperature gradient ensures the orderly progress of the dissociation process and prevents product agglomeration. The introduction of vacuum pulses (fluctuation period 218-435 seconds) reveals moderate mechanical disturbances, which help to break the eutectic structure and hydrophobic interactions (the shear force generated by the pressure fluctuations during the pulse process is between 0.8-2.2 Pa), while not causing mechanical damage to the product itself. The design of the multi-layer filtration structure (0.82μm, 0.58μm, 0.35μm) not only takes into account the particle size distribution of the product, but also takes into account the size characteristics of various complexes brought about by the previous process, and can achieve step-by-step separation. Through this systematic process improvement, the indigo content in the final product can be stabilized at more than 40%, the indigotin content can be maintained above 3%, and the fluctuation between batches is significantly reduced.

[0051] Figure 3 This is a separation efficiency surface diagram disclosed in the embodiment of the present application. The comparative study of the separation efficiency surface diagram of the temperature gradient-vacuum pulse synergistic separation system shows that the technical solution has significant technical characteristics in terms of the kinetic characteristics of the separation process, separation rate and final efficiency. Figure 3 The separation efficiency comparison between this technical solution and the traditional gravity sedimentation method is demonstrated. Figure 3 (a) shows the efficiency curve of temperature gradient-vacuum pulse synergistic separation. Figure 3 (b) shows the separation efficiency surface of the traditional gravity sedimentation method.

[0052] From the perspective of temperature gradient control, this technical solution achieves precise temperature gradient field construction through a double-layer jacket structure. In the first stage, the temperatures of the inner and outer layers were set to 32.5°C and 42.8°C, respectively, forming a temperature gradient of 10.3°C. The physical basis of this gradient setting is that indigo and indigotin exhibit differentiated solubility and sedimentation behaviors at different temperatures. The thermal convection generated by the temperature gradient field and the density difference jointly drive the directional migration of the substance, thereby achieving the initial separation of the components in space.

[0053] The design of the vacuum pulse system adopts a two-stage dynamic control strategy. The vacuum wave equation of the first stage P(t) = 0.042 + 0.018sin(πt / 435) + 0.012cos(πt / 282) × (1-e^(-0.175t)) produces periodic changes in the pressure field. The sine and cosine terms in the equation correspond to characteristic periods of 435 seconds and 282 seconds, respectively. The design principle of this double-period superposition is to match the migration dynamics of the substance in the temperature gradient field through pressure fluctuations of different frequencies. Figure 3 It can be observed in (a) that the separation efficiency shows an obvious nonlinear growth trend in this stage, and exhibits slight periodic fluctuations on the time axis, which is a direct reflection of the periodic changes in the pressure field.

[0054] In contrast, traditional gravity sedimentation methods rely mainly on the density differences of materials. Figure 3 (b) It can be seen that the growth rate of its separation efficiency over time is significantly lower than that of the synergistic separation system. This difference is due to the lack of synergistic driving effects of temperature gradient and vacuum pulse in the traditional method. The separation of substances mainly relies on natural sedimentation, thus showing a relatively slow separation kinetics.

[0055] In the second stage of the separation process, the system adjusts the temperature gradient to 8.4°C and adopts a new vacuum pulse characteristic equation P(t) = 0.068 + 0.015sin(πt / 325) × (1-e^(-0.223t)) + 0.009cos(πt / 218). This stage is characterized by a shortened vacuum pulse period and reduced fluctuation amplitude. The theoretical basis for this parameter adjustment is that the separated material requires a smaller driving force to maintain a stable state and avoid excessive pulses that cause the destruction of the formed layered structure.

[0056] The transition stage uses a temperature gradient change rate of 0.82°C / minute, combined with the vacuum gradient equation P(t)=Pm+(Ph-Pm)(1-e^(-0.168t))×cos(πt / 375), to achieve a smooth transition between the two main separation stages. The physical significance of this gradient design is that the system is maintained in a quasi-steady state through the coordinated gradient of the temperature field and the pressure field, avoiding turbulent disturbances caused by parameter mutations.

[0057] The distribution of experimental data points shows that the system achieves the highest separation efficiency at a temperature gradient of 10.3°C. This phenomenon can be explained as follows: under this temperature gradient condition, the thermal convection intensity and the material migration rate are optimally matched, which not only ensures sufficient separation driving force, but also avoids mixing caused by excessive convection. In contrast, the experimental data points of the traditional method show a large discreteness, which reflects that its separation process is greatly affected by the external environment and has poor stability.

[0058] The tangential flow introduced into the system v(r)=v0(1-(r / R) 2 )(1+0.15sin(2πt / T)), where v0 is 0.32 m / s, which prevents cake compaction while maintaining separation efficiency. The shear force generated by the radial velocity distribution can destroy local aggregates, while the periodic flow velocity fluctuations help maintain the dynamic balance of the system.

[0059] It can be seen from the final steady-state area of ​​the surface graph that the technical solution achieved a maximum separation efficiency of 97.2% within 2400 seconds, while the traditional method could only achieve a separation efficiency of 79.2% in the same time.

[0060] Figure 4 This is a schematic diagram of a multi-layer filtration cross-section temperature field simulation disclosed in an embodiment of the present application. The embodiment of the present application uses three layers of filter media with different pore sizes to achieve the separation of indigo and indirubin through the synergistic effect of temperature gradient field and tangential flow.

[0061] The filter consists of three layers of filter media from top to bottom, preferably, a polytetrafluoroethylene filter membrane with a pore size of 0.82 microns, a modified cellulose filter membrane with a pore size of 0.58 microns, and a composite ceramic filter plate with a pore size of 0.35 microns. The first layer and the second layer are kept at a distance of 4.2 mm, and the second layer and the third layer are kept at a distance of 3.8 mm. This specific spatial layout design is based on the principle of fluid dynamics, which not only ensures sufficient sedimentation space, but also avoids the occurrence of particle resuspension.

[0062] During the operation of the system, a temperature gradient field is formed. The inner jacket is fed with circulating water at 32.5°C, and the outer jacket is fed with heat transfer oil at 42.8°C, thereby forming a temperature gradient of 10.3°C in the radial direction. The existence of the temperature gradient produces a thermal convection effect, which promotes the graded sedimentation of different components under the action of gravity. In order to prevent the filter cake from hardening, the system introduces a tangential flow mechanism. The change of the tangential flow velocity with the radial position follows the parabolic distribution law, reaching a maximum value of 0.32 m / s at the central axis and gradually decreasing to zero outward. This flow pattern maintains the looseness of the filter cake structure by applying a tangential shear force, without destroying the formed layered structure.

[0063] It can be observed from the figure that the temperature field shows a gradient distribution characteristic that gradually increases from the inner layer to the outer layer. The arrow mark of the tangential flow shows the velocity change of the fluid at different radial positions. The length of the arrow represents the local flow velocity, which reflects the parabolic velocity distribution characteristics. The layout design of the three-layer filter medium reflects the spatial structural characteristics of the system. The filter layers with different pore sizes can achieve selective retention of components with different particle sizes.

[0064] Through the analysis of the operation process, it can be seen that the first layer of filter membrane is mainly responsible for intercepting the indigo component, the second layer of filter membrane enriches the indigo red component, and the last layer plays a role in ensuring the integrity of the separation. The synergistic effect of the temperature gradient field and the tangential flow enables different components to maintain good dispersion during the sedimentation process and avoid the occurrence of agglomeration.

[0065] Specifically, when the mixed liquid passes through the system, first, under the action of the temperature gradient field, different components will produce different sedimentation velocities due to density differences. At the same time, the shear force field generated by the tangential flow helps prevent particle agglomeration and maintain the stability of the dispersed system. The 0.82-micron pore size design of the first filter membrane is mainly based on the size distribution characteristics of indigo particles, which can effectively intercept this component; the 0.58-micron pore size of the second filter membrane is optimized for the particle size characteristics of indigo red; the last layer of 0.35-micron pore size composite ceramic filter plate ensures the complete interception of finer particles. The 4.2-mm upper layer spacing provides enough space for the sedimentation of indigo, and the 3.8-mm lower layer spacing adapts to the sedimentation characteristics of indigo red. This gradient spacing design helps to reduce turbulent disturbances and improve the selectivity of separation. The setting of the temperature gradient field is based on the difference in solubility of the two components at different temperatures, and the separation effect is enhanced by controlling the temperature distribution.

[0066] Furthermore, an embodiment of the present application also discloses a control device for extracting indigo and indigotin through the roots, stems and leaves of Isatis indigotica, comprising: a processor, a memory, and a system bus; the processor and the memory are connected via the system bus; the memory is used to store one or more programs, and the one or more programs include instructions, which, when executed by the processor, enable the processor to execute any of the above methods.

[0067] Furthermore, an embodiment of the present application also discloses a computer program product, which, when executed on a terminal device, enables the terminal device to execute any of the above methods.

[0068] It can be known from the description of the above implementation mode that those skilled in the art can clearly understand that all or part of the steps in the above-mentioned embodiment method can be implemented by means of software plus a necessary general hardware platform. Based on such an understanding, the technical solution of the present application can be essentially or partly embodied in the form of a software product that contributes to the prior art. The computer software product can be stored in a storage medium such as ROM / RAM, a disk, an optical disk, etc., including several instructions for enabling a computer device (which can be a personal computer, a server, or a network communication device such as a media gateway, etc.) to execute the methods described in the various embodiments of the present application or certain parts of the embodiments.

[0069] It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the various embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part description.

[0070] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0071] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting indigo and indirubin from the roots, stems and leaves of Isatis indigotica, characterized in that: include: Adsorbing the extract of the roots, stems and leaves of Isatis indigotica, eluting after reaching saturation, and concentrating the eluate under vacuum; The concentrate is diluted with pure water and the temperature is adjusted, and then a hydrolase is added to react at a constant temperature to obtain a target precipitate; The precipitated product is collected by vacuum filtration and then graded and separated using a multi-layer filter membrane to obtain indigo and indirubin, respectively.

2. The method according to claim 1, characterized in that in, The extract of the roots, stems and leaves of Isatis indigotica is subjected to adsorption treatment, eluted after reaching saturation, and the eluate is vacuum concentrated, including: The room temperature extract is processed through a resin column packed with a macroporous adsorption resin; When the resin column reaches the saturated adsorption state, methanol solution is used for elution; The eluate was concentrated in vacuo and the methanol solution was recovered.

3. The method according to claim 1, characterized in that in, After the concentrate is diluted with pure water and the temperature is adjusted, a hydrolase is added to react at a constant temperature to obtain the target precipitate, including: The concentrated solution is diluted with pure water and then adjusted to a predetermined temperature; adding exoglycoside hydrolase to carry out constant temperature reaction; After the reaction is completed, the mixture is allowed to stand to allow the conversion product to settle naturally.

4. The method according to claim 1, characterized in that: in, The precipitated product is collected by vacuum filtration and then graded and separated using a multi-layer filter membrane to obtain indigo and indirubin, respectively, including: The reaction solution after standing is vacuum filtered and the precipitate is collected; The mixed product is separated by using a temperature gradient field, so as to grade indigo and indirubin by multi-layer filtration, and the filter cakes after separation are washed separately; The precipitate was dried using pulse vacuum drying.

5. The method according to claim 4, characterized in that in, The mixed product is separated by using a temperature gradient field, so as to grade indigo and indirubin by multi-layer filtration, and the filter cakes after separation are washed separately, including: Transferring the reaction solution to a separation device having a double-layer jacket structure; Circulating water at a first preset temperature is introduced into the inner jacket; Pass heat transfer oil of a second preset temperature into the outer jacket; A first temperature gradient field is formed by the temperature difference between the inner and outer layers; According to the first preset control equation, the vacuum degree is adjusted to allow the indigo to settle preferentially; Adjusting the temperatures of the inner and outer layers to form a second temperature gradient field; adjusting the vacuum degree according to the second preset control equation to promote the selective precipitation of indigo carmine; Between the two separation stages, the temperature gradient and the vacuum degree are transiently adjusted according to a third preset control equation; Three filter layers with different pore sizes are arranged at the bottom of the separation device; Prevent filter cake from hardening by tangential flow; The filter cakes on different filter layers are cleaned at different temperatures.

6. The method according to claim 4, characterized in that in, The precipitate is dried using pulse vacuum drying, including: Applying a preset vacuum degree to the precipitate at a preset temperature to dry it; The precipitate is pulse dried according to the set pulse time; The sediment is intermittently dried according to the set interval time; The drying operation is stopped when the moisture content of the sediment drops below the preset value.

7. The method according to claim 1, characterized in that in, The process of adsorbing the extract of the roots, stems and leaves of Isatis indigotica and eluting it after it reaches saturation, and before vacuum concentrating the eluate, also includes: Wash and drain the fresh southern isatis root, stem and leaves, and cut into sections; Perform hot water extraction twice in sequence, and temporarily store the extract in a liquid storage tank after the first extraction; The two extracts were combined and allowed to cool to room temperature.

8. The method according to claim 1, characterized in that in, After collecting the precipitated product by vacuum filtration, the product is graded and separated by a multi-layer filter membrane to obtain indigo and indirubin respectively, and the method further comprises: Collect and press the extracted plant residues; The supernatant of the squeezed liquid is recovered for the next batch of extraction; The resin was regenerated using ethanol solution.

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