Method for preparing shikonin on large scale by utilizing lithospermum cells
By adsorbing and transferring cypress cells with magnetic microsphere carriers, the problems of high cost and low yield in the two-step culture method of cypress cells were solved, and efficient and stable cypress cells were achieved.
Patent Information
- Application Number
- CN202510170289.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-06-24
AI Technical Summary
The existing two-step culture method for comfreycein preparation is too costly and has low yield, making it difficult to meet market demand.
Magnetic microsphere carriers are used to adsorb suspended cypress cells to improve cell proliferation speed and quickly transfer it to pigment production medium through magnetic rods to reduce the damage to cells by the filter and improve production efficiency.
It reduces production costs, improves the yield and quality of cyperin, is suitable for large-scale industrial production, and is superior.
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Figure CN120193028A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological tissue culture, in particular to a preparation technology of shikonin, and specifically to a method for preparing shikonin in large quantities using Lithospermum erythrorhizon cells. Background Art
[0002] Lithospermum erythrorhizon is a representative source of shikonin. In recent years, it has faced the risk of extinction and is very difficult to cultivate. Its germination rate is low, the seedlings are very vulnerable to virus infection, and the plants are sensitive to disinfectants. It takes three years for Lithospermum erythrorhizon to grow enough roots to have commercial application value, from which shikonin can be prepared. However, it is usually very difficult to grow without pests and diseases for three years. However, with the application of shikonin in anti-tumor, anti-inflammatory, antibacterial and other products, the demand for shikonin is continuously increasing. Therefore, the price of shikonin on the market is constantly rising, and the traditional planting method is difficult to meet the market demand. The current research direction mainly focuses on cell culture and chemical synthesis.
[0003] Currently, in the technical route of Lithospermum erythrorhizon cell culture, Tabata et al. proposed a two-step culture method for Lithospermum erythrorhizon cells, that is, first, MG-5 medium is used for proliferation culture. At this time, Lithospermum erythrorhizon cells rapidly multiply in a bioreactor. After culturing for about 9 days, the obtained Lithospermum erythrorhizon cell culture is transferred to the M-9 pigment production medium proposed by Fujita et al. for light-free culture. At this time, the proliferation of Lithospermum erythrorhizon cells is inhibited, and Lithospermum erythrorhizon cells begin to produce shikonin derivatives and secrete the shikonin derivatives outside the cells, attaching to the cell wall surface.
[0004] The above two-step culture method for Lithospermum erythrorhizon cells is suitable for preparing shikonin in large quantities, but the cost of preparing shikonin by this method is too high and the yield of shikonin is low. Summary of the Invention
[0005] The purpose of the present invention is to address the problems that the process of preparing shikonin by the current two-step culture method for Lithospermum erythrorhizon cells has too high preparation cost, low shikonin yield, and no commercial application value. The invention provides a method for preparing shikonin in large quantities using Lithospermum erythrorhizon cells. It uses magnetic microsphere carriers to adsorb suspended Lithospermum erythrorhizon cells, provides support for the growth of Lithospermum erythrorhizon cells, improves the proliferation rate of Lithospermum erythrorhizon cells, and then uses the magnetic attraction of the magnetic microsphere carriers. Through a magnetic rod, the magnetic microsphere carriers adsorbed with Lithospermum erythrorhizon cells can be quickly transferred to the pigment production medium for culture, so as to reduce the damage to Lithospermum erythrorhizon cells by the filter screen and improve production efficiency.
[0006] The technical solution of the present invention is as follows:
[0007] A method for preparing shikonin in large quantities using Lithospermum erythrorhizon cells, characterized in that the method comprises the following steps:
[0008] Step 1 Cell preparation: Add Lithospermum erythrorhizon cells to PBS buffer, and then add magnetic microsphere carriers, stir evenly to prepare a high-yield cell mixture;
[0009] Step 2 Proliferation culture: Add Lithospermum erythrorhizon cells and magnetic microsphere carriers together to a cell growth medium, perform light culture, stir during the culture, after the culture is completed, drain the cell growth medium to obtain a tissue culture;
[0010] Step 3 Transformation culture: Transfer the tissue culture to a pigment production medium, perform dark culture, after the culture is completed, obtain Lithospermum erythrorhizon cell liquid;
[0011] Step 4 Separation and purification: Separate the Lithospermum erythrorhizon cell liquid from the pigment production medium, and extract and purify shikonin.
[0012] Preferably, the magnetic microsphere carrier includes a magnetic core, an intermediate layer, and an outer coating layer, and the diameter of the magnetic microsphere carrier is 50 - 250 μm; wherein,
[0013] The magnetic core is any one of cobalt chloride, nickel chloride, and cobalt-nickel alloy;
[0014] The intermediate layer is SiO2, and the intermediate layer wraps the magnetic core;
[0015] The outer coating layer is any one of agar, collagen, cellulose, chitin and its derivatives, and alginate.
[0016] Preferably, the cell growth medium in Step 2 uses MG-5 cell growth medium, the culture time is 9 days, and the culture temperature is 21 - 30 °C;
[0017] The pigment production medium in Step 3 uses M-9 pigment production medium, the culture time is 14 days, and the culture temperature is 21 - 30 °C.
[0018] Preferably, in the transformation culture of Step 3, after the pigment production medium turns red, add petroleum ether to the pigment production medium, stir and culture for 1 - 10 h, suck away the upper layer of petroleum ether, and then start stirring and culturing.
[0019] Preferably, the number of times of adding petroleum ether is 2 times, which are added on the 5th day and the 10th day of culture respectively, and the addition amount of petroleum ether is 0.2 - 0.5 of the amount of M-9 pigment production medium used.
[0020] Preferably, the separation and purification in Step 4 includes the following steps:
[0021] S41: Separate the Lithospermum erythrorhizon cell liquid from the pigment production medium, dry and crush the filtered cells;
[0022] S42: Add 2 mol / L KOH solution for hydrolysis to hydrolyze the shikonin derivative into shikonin.
[0023] S43: After hydrolysis, add 2 mol / L hydrochloric acid solution to the hydrolysate for acidification, adjust the pH to 2 - 3, and shikonin precipitates out. Centrifuge and filter to remove impurities such as potassium ions to obtain the finished product.
[0024] Preferably, in the cell preparation of step 1, the mass - volume ratio is: Lithospermum erythrorhizon cells: PBS buffer: magnetic microsphere carrier = 1 g: 20 ml: 10 ml.
[0025] Preferably, in the proliferation culture of step 2, the volume ratio of the added cell growth medium to the magnetic microsphere carrier is 100:1 - 5.
[0026] Preferably, in the proliferation culture of step 2 and the transformation culture of step 3, after the proliferation culture is completed, the magnetic microsphere carrier is adsorbed by an electromagnet, and then the tissue culture is obtained by filtering through a filter screen; the magnetic microsphere carrier and the tissue culture are all transferred to a bioreactor containing M - 9 pigment production medium.
[0027] Preferably, in step S41, the method for separating the Lithospermum erythrorhizon cell liquid from the pigment production medium is: adsorb the magnetic microsphere carrier by an electromagnet, and then filter through a filter screen to remove the M - 9 pigment production medium.
[0028] Advantages of the present invention:
[0029] The present invention uses magnetic microsphere carriers to adsorb suspended Lithospermum erythrorhizon cells, providing support for the growth of Lithospermum erythrorhizon cells and increasing the proliferation rate of Lithospermum erythrorhizon cells. By adding petroleum ether in the transformation culture stage, the shikonin derivative is eluted by the petroleum ether. On the one hand, the conversion rate can be increased. On the other hand, after stopping stirring, the petroleum ether floats on the medium and forms a layer. The petroleum ether can be directly sucked out and then transferred to the purification workshop for subsequent purification processes, accelerating the acquisition of shikonin. By using the magnetic attraction of the magnetic microsphere carrier, the magnetic microsphere carrier adsorbed with Lithospermum erythrorhizon cells can be quickly transferred to the pigment production medium by a magnetic rod, reducing the production time. The shikonin produced has stable quality, is suitable for large - scale industrial production, and has great superiority.
[0030] The present invention uses magnetic microsphere carriers to adsorb suspended Lithospermum erythrorhizon cells, providing support for the growth of Lithospermum erythrorhizon cells and increasing the proliferation rate of Lithospermum erythrorhizon cells. Then, by using the magnetic attraction of the magnetic microsphere carrier, the magnetic microsphere carrier adsorbed with Lithospermum erythrorhizon cells can be quickly transferred to the pigment production medium by a magnetic rod for cultivation, so as to reduce the damage of the filter screen to Lithospermum erythrorhizon cells and improve the production efficiency.
[0031] The present invention creatively adds 0.1-0.5% agar to the M-9 pigment production medium, which can improve the secretion of shikonin derivatives by Lithospermum erythrorhizon cells. The reason is that the Lithospermum erythrorhizon cells will adsorb on the agar and grow, enabling the enrichment of Lithospermum erythrorhizon cells.
[0032] The present invention uses magnetic microspheres as carriers. The outside of the magnetic microspheres is wrapped with agar. The Lithospermum erythrorhizon cells will adhere to the agar and grow, enabling the enrichment of Lithospermum erythrorhizon cells and obtaining a good yield of shikonin.
[0033] The present invention also makes targeted improvements to the shortcomings of the existing process. That is, after the proliferation culture is completed, it is necessary to completely filter out the MG-5 medium with a filter screen, leaving the Lithospermum erythrorhizon cells. However, during filtration, the Lithospermum erythrorhizon cells will be retained on the filter screen, blocking the mesh holes, resulting in slow filtration. And during filtration, when the Lithospermum erythrorhizon cells come into contact with the filter screen and are squeezed, they will inevitably be damaged, causing the death of a part of the Lithospermum erythrorhizon cells. Based on this, the present invention improves the two-step culture method of Lithospermum erythrorhizon cells. Description of the Drawings
[0034] Figure 1 It is the HPLC graph of the reference substance of 98% shikonin reference substance.
[0035] Figure 2 It is the HPLC graph of the shikonin finished product prepared in Example 1. Detailed Description of the Invention
[0036] The following further describes the present invention in detail with reference to the drawings and specific embodiments. According to the following description, the advantages and features of the present invention will be clearer. It should be noted that the drawings are all in a very simplified form and use non-precise scales, only for the purpose of conveniently and clearly assisting in explaining the purpose of the embodiments of the present invention.
[0037] Description of experimental materials:
[0038] Lithospermum erythrorhizon cells: After obtaining callus from the root tip tissue culture of Lithospermum erythrorhizon in Xinjiang, they are dispersed by enzymatic method or mechanical method; the selection and breeding of Lithospermum erythrorhizon cells have been publicly disclosed in many journals, and the present invention will not elaborate here.
[0039] Magnetic microsphere carrier: In the following embodiments, cobalt chloride is used as the core, the outside of the core is wrapped with silica, and the outermost layer is wrapped with agar, with a diameter of 50-250 μm; the magnetic microsphere carrier can also use any one of nickel chloride and cobalt-nickel alloy as the core, the outside of the core is wrapped with SiO2, and the outermost layer is wrapped with any one of collagen, cellulose, chitin and its derivatives, and alginate.
[0040] 10L bioreactor: BS10L model plant cell incubator of Henan Ospbo Machinery Co., Ltd.
[0041] Example 1.
[0042] 1) Cell preparation: Take 5 g of Lithospermum erythrorhizon cells and add them to 100 ml of PBS buffer, then add 50 ml of magnetic microsphere carriers, and stir evenly to prepare a high-yield cell mixture.
[0043] 2) Proliferation culture: Add 5 L of MG-5 cell growth medium and 150 ml of magnetic microsphere carriers (cell growth medium: magnetic microsphere carriers = 100:3) to a 10 L bioreactor. Add the high-yield cell mixture to the 10 L bioreactor and perform light culture for 9 days. Control the culture temperature at 21 - 30°C and stir during the culture to avoid sedimentation of the magnetic microsphere carriers. Obtain the tissue culture by filtration through a filter screen.
[0044] 3) Transformation culture: Add 5 L of M-9 pigment production medium to a 10 L bioreactor. Transfer the tissue culture to the 10 L bioreactor and perform dark culture for 14 days. Control the culture temperature at 21 - 30°C to obtain Lithospermum erythrorhizon cell liquid.
[0045] 4) Separation and purification: Filter through a filter screen to remove the M-9 pigment production medium. Dry the magnetic microsphere carriers and the filtered cells and break them by ultrasonic wave. Add 2 mol / L KOH solution for hydrolysis to hydrolyze the shikonin derivatives into shikonin. After hydrolysis, add 2 mol / L hydrochloric acid solution to the hydrolysis solution for acidification, adjust the pH to 2 - 3, and the shikonin precipitates out. Centrifuge and filter to remove impurities such as potassium ions to obtain the finished product.
[0046] Example 2.
[0047] 1) Cell preparation: Take 5 g of Lithospermum erythrorhizon cells and add them to 100 ml of PBS buffer, then add 50 ml of magnetic microsphere carriers, and stir evenly to prepare a high-yield cell mixture.
[0048] 2) Proliferation culture: Add 5 L of MG-5 cell growth medium and 150 ml of magnetic microsphere carriers to a 10 L bioreactor. Add the high-yield cell mixture to the 10 L bioreactor and perform light culture for 9 days. Stir during the culture to avoid sedimentation of the magnetic microsphere carriers. Obtain the tissue culture by filtration through a filter screen.
[0049] 3) Transformation culture: Add 5 L of M-9 pigment production medium to a 10 L bioreactor. Transfer the tissue culture to the 10 L bioreactor and perform dark culture for 14 days. On the 10th day, add 2 L of petroleum ether to the 10 L bioreactor, stir and mix for 5 h and then let it stand. Suck out the upper-layer petroleum ether, and then start stirring and culturing. Obtain the Lithospermum erythrorhizon cell liquid after the culture is completed.
[0050] 4) Separation and purification: Filter through a filter screen to remove the M-9 pigment production medium. Dry the magnetic microsphere carrier and the filtered cells, and then break them by ultrasonic waves. Add 2 mol / L KOH solution for hydrolysis to hydrolyze the shikonin derivatives into shikonin. After hydrolysis, add 2 mol / L hydrochloric acid solution to the hydrolyzate for acidification, adjust the pH to 2-3, and shikonin will precipitate out. Centrifuge and filter to remove impurities such as potassium ions to obtain the finished product.
[0051] Example 3.
[0052] Compared with Example 2, the difference lies in: 3) Transformation culture: Add 5 L of M-9 pigment production medium to a 10 L bioreactor, transfer the tissue culture to the 10 L bioreactor, and perform dark culture for 14 days. On the 5th day, add 2 L of petroleum ether (equal to 0.4 times of the M-9 pigment production medium. Specifically, during implementation, the addition amount of petroleum ether can also be adjusted to any value between 0.2 and 0.5 times of the M-9 pigment production medium according to the situation) to the 10 L bioreactor, stir and mix for 5 h and then let it stand. After sucking off the upper-layer petroleum ether, start stirring culture. On the 10th day, add 2 L of petroleum ether (equal to 0.4 times of the M-9 pigment production medium. Specifically, during implementation, the addition amount of petroleum ether can also be adjusted to any value between 0.2 and 0.5 times of the M-9 pigment production medium according to the situation) to the 10 L bioreactor, stir and mix for 5 h and then let it stand (specifically, during implementation, the stirring and mixing time can be controlled between 1 and 10 h and then let it stand according to the specific situation). After sucking off the upper-layer petroleum ether, start stirring culture, and obtain the shikonin cell liquid after the culture is completed. Specifically, during implementation, the number of times of adding petroleum ether can also be one time.
[0053] Example 4.
[0054] 1) Cell preparation: Take 5 g of shikonin cells and add them to 100 ml of PBS buffer solution, and then add 50 ml of magnetic microsphere carrier, stir evenly to prepare a high-yield cell mixture;
[0055] 2) Proliferation culture: Add 5 L of MG-5 cell growth medium and 150 ml of magnetic microsphere carrier to a 10 L bioreactor, add the high-yield cell mixture to the 10 L bioreactor, and perform light culture for 9 days. Stir during the culture to avoid deposition of the magnetic microsphere carrier. After the culture is completed, adsorb the magnetic microsphere carrier by an electromagnet, and then filter through a filter screen to obtain the tissue culture;
[0056] 3) Transformation and culture: Add 5 L of M-9 pigment production medium to a 10 L bioreactor. Directly transfer the magnetic microsphere carriers adsorbed on the electromagnet to the 10 L bioreactor containing the M-9 pigment production medium. Then transfer the filtered tissue culture to the 10 L bioreactor and perform light-free culture for 14 days to obtain Lithospermum erythrorhizon cell liquid.
[0057] 4) Separation and purification: Adsorb the magnetic microsphere carriers through an electromagnet, then filter through a filter screen to remove the M-9 pigment production medium. Dry and ultrasonically crush the magnetic microsphere carriers and the filtered cells, add 2 mol / L KOH solution for hydrolysis to hydrolyze the shikonin derivatives into shikonin. After hydrolysis, add 2 mol / L hydrochloric acid solution to the hydrolysis solution for acidification, adjust the pH to 2 - 3, and shikonin precipitates out. Centrifuge and filter to remove impurities such as potassium ions to obtain the finished product.
[0058] Specifically in implementation, the volume ratio of the cell growth medium to the magnetic microsphere carriers in Examples 1 - 4 can also be adjusted to any value between 100:1 and 100:5.
[0059] Comparative Example 1.
[0060] 1) Cell preparation: Take 5 g of Lithospermum erythrorhizon cells and add them to 100 ml of PBS buffer to prepare a high-yield cell mixture.
[0061] 2) Proliferation culture: Add 5 L of MG-5 cell growth medium to a 10 L bioreactor. Add the high-yield cell mixture to the 10 L bioreactor and perform light culture for 9 days. Stir during the culture and obtain the tissue culture through filtration with a filter screen.
[0062] 3) Transformation and culture: Add 5 L of M-9 pigment production medium to a 10 L bioreactor. Transfer the tissue culture to the 10 L bioreactor and perform light-free culture for 14 days to obtain Lithospermum erythrorhizon cell liquid.
[0063] 4) Separation and purification: Filter through a filter screen to remove the M-9 pigment production medium. Dry and ultrasonically crush the filtered cells, add 2 mol / L KOH solution for hydrolysis to hydrolyze the shikonin derivatives into shikonin. After hydrolysis, evaporate to recover the shikonin dry powder. Add the shikonin dry powder to hydrochloric acid solution with pH 2 for acidification, and shikonin precipitates out. Remove impurities such as potassium ions to obtain the finished product.
[0064] Experimental results:
[0065] Analyze the finished products prepared in Examples 1 - 4 and Comparative Example 1 and measure their quality. The results are shown in Table 1:
[0066] Table 1 Quality table of the finished products prepared in Examples 1 - 4 and Comparative Example 1
[0067] Finished product quality (g) Shikonin production level (g / L) Example 1 4.250 0.850 Example 2 5.760 1.152 Example 3 6.215 1.243 Example 4 4.817 0.963 Comparative Example 1 2.887 0.577
[0068] As shown in Table 1, from the comparison of the finished product quality between Example 1 and Comparative Example 1, it can be seen that by adding magnetic microsphere carriers to adsorb Lithospermum erythrorhizon cells and provide a proliferation bed for the cells, the cells are ensured to be in an organized state, and their microenvironment is closer to the state in the plant. Therefore, compared with the conventional suspension culture method used in Comparative Example 1, the production of shikonin can be increased by 1.47 times.
[0069] From the comparison of the finished product quality between Example 1 and Example 2, it can be seen that by adding petroleum ether in the transformation culture stage, the production of shikonin can be increased by 1.36 times. This is because the shikonin derivatives generated by Lithospermum erythrorhizon cells during the transformation stage will be transferred to the cell outer wall, and the attached shikonin derivatives will hinder the absorption of the culture medium and oxygen, slowing down the transformation process. By eluting the shikonin derivatives with petroleum ether, on the one hand, the conversion rate can be increased. On the other hand, after stopping stirring, the petroleum ether floats on the culture medium and forms a layer. After directly sucking out the petroleum ether, it can be transferred to the purification workshop for subsequent purification processes, accelerating the acquisition of shikonin.
[0070] From the comparison of the finished product quality between Example 2 and Example 3, it can be seen that by increasing the addition times of petroleum ether, the production of shikonin can be further increased.
[0071] From the comparison of the finished product quality between Example 1 and Example 4, it can be seen that by adopting the dual methods of electromagnet adsorption and filter screen filtration, the production of shikonin can be increased by 1.13 times. The reason is that only using filter screen filtration will cause the magnetic microsphere carriers and Lithospermum erythrorhizon cells to attach and block the filter screen holes, resulting in slow filtration efficiency. At the same time, the filtration pressure will squeeze the Lithospermum erythrorhizon cells, leading to the death of the cells and reducing the survival rate of the Lithospermum erythrorhizon cells. By adopting the dual methods of electromagnet adsorption and filter screen filtration, the electromagnet quickly transfers the magnetic microsphere carriers and the Lithospermum erythrorhizon cells attached to the magnetic microsphere carriers, and the remaining small amount of suspended Lithospermum erythrorhizon cells are filtered by the filter screen, avoiding the blockage of the filter screen, reducing the death of the Lithospermum erythrorhizon cells and the time required for filtration, thus reducing the production time.
[0072] The 98% shikonin reference substance purchased and Example 1 were analyzed by high performance liquid chromatography:
[0073] Liquid phase conditions: mobile phase acetonitrile: 0.1% glacial acetic acid water = 80:20, wavelength 516 nm, flow rate 1 ml / min, column temperature 30 °C; the solvent is methanol;
[0074] The prepared concentration of the 98% shikonin reference substance is 102 ug / ml; a total of 2 repeated experiments were carried out. The HPLC results are shown in Table 2, and the HPLC graph is as Figure 1 shown.Figure 1 For repeated experimental results;
[0075] Table 2 HPLC results of 98% shikonin reference substance
[0076] Title Component 1 -> Time Component 1 -> Concentration Component 1 -> Area [1] - 98% shikonin reference substance 6.1069 0.0000 2122014.5000 [2] - 98% shikonin reference substance 6.0986 0.0000 2128804.7500 RSD 0.0010 0.0000 0.0023 Average value 6.1027 0.0000 2125409.5000 Maximum value 6.1069 0.0000 2128804.7500 Minimum value 6.0986 0.0000 2122014.5000 Range 0.0083 0.0000 6790.2500
[0077] In Example 1, the prepared concentration of shikonin finished product is 104 ug / ml; a total of 3 repeated experiments were carried out, and the HPLC results are shown in Table 3, and the HPLC graph is as Figure 2 shown, Figure 2 For repeated experimental results;
[0078] Table 3 HPLC results of shikonin finished product prepared in Example 1
[0079] Title Component 1 -> Time Component 1 -> Concentration Component 1 -> Area [1] - Shikonin finished product prepared in Example 1 6.3764 0.0000 2141432.2500 [2] - Shikonin finished product prepared in Example 1 6.3569 0.0000 2070235.8750 [3] - Shikonin finished product prepared in Example 1 6.3028 0.0000 2133855.2500 RSD 0.0060 0.0000 0.0185 Average value 6.3454 0.0000 2115174.5000 Maximum value 6.3764 0.0000 2141432.2500 Minimum value 6.3028 0.0000 2070235.8750 Range 0.0736 0.0000 71196.3750
[0080] Combined with Table 2 - 3 and Figure 1 - 2 It can be seen that calculated by liquid phase normalization method for purity, the purity of the shikonin finished product prepared in Example 1 can reach 96.57%, which has medical application value.
[0081] In summary, the present invention is an improvement based on the two - step culture method of Lithospermum erythrorhizon cells proposed by Tabata et al. It uses magnetic microsphere carriers as culture carriers, elutes with petroleum ether, and uses an electromagnet for rapid transfer of magnetic microsphere carriers, which improves the yield of shikonin, reduces the production time, the produced shikonin has stable quality, and the purity can reach 96.57%, which is suitable for large - scale industrial production and has great superiority.
[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.
[0083] The parts not involved in the present invention are the same as the prior art or can be realized by the prior art.
Claims
1. A method for preparing shikonin by cultivating lithospermum erythrorhizon cells, characterized in that: It includes the following steps: Step 1: Cell preparation: Add the lithospermum officinale cells into PBS buffer, then add the magnetic microsphere carrier, stir evenly, and prepare a high-yield cell mixture; Step 2 proliferation culture: adding the comfrey cells and the magnetic microsphere carriers to a cell growth medium, performing light culture, stirring during the culture, and after the culture is completed, draining the cell growth medium to obtain a tissue culture; Step 3: transformation culture: transferring the tissue culture to a pigment production medium and culturing in the dark, and obtaining lithospermum cell fluid after the culturing is completed; Step 4: separation and purification: separating the lithospermum cell fluid from the pigment production medium, and extracting and purifying shikonin.
2. The method according to claim 1, characterized in that: The magnetic microsphere carrier comprises a magnetic core, an intermediate layer and an outer coating layer, and the diameter of the magnetic microsphere carrier is 50-250 μm; wherein the magnetic core is made of any one of cobalt chloride, nickel chloride, and cobalt-nickel alloy; the intermediate layer is SiO2, and the intermediate layer wraps the magnetic core; the outer coating layer is made of any one of agar, collagen, cellulose, chitin and its derivatives and seaweed salt.
3. The method according to claim 1, characterized in that: The cell growth medium in step 2 adopts MG-5 cell growth medium, the culture time is 9 days, and the culture temperature is 21-30°C; the pigment production medium in step 3 adopts M-9 pigment production medium, the culture time is 14 days, and the culture temperature is 21-30°C.
4. The method according to claim 1, characterized in that: In the transformation culture of step 3, after the pigment production medium turns red, petroleum ether is added to the pigment production medium, and the culture is stirred for 1-10 hours, the petroleum ether on the upper layer is sucked off, and the stirring culture is started again.
5. The method according to claim 4, characterized in that: The petroleum ether is added twice, on the 5th day and the 10th day of culture respectively. The amount of petroleum ether added is 0.2-0.5 times the amount of the M-9 pigment production culture medium.
6. The method according to claim 1, characterized in that: The separation and purification step 4 comprises the following steps: S41: separating the lithospermum cell fluid from the pigment production medium, and drying and crushing the filtered cells; S42: adding 2 mol / L KOH solution for hydrolysis to hydrolyze the shikonin derivative into shikonin; S43: After the hydrolysis, 2 mol / L hydrochloric acid solution is added to the hydrolyzate for acidification, and the pH is adjusted to 2-3, so that shikonin is precipitated and centrifuged to remove impurities to obtain a finished product.
7. The method according to claim 6, characterized in that: In the cell preparation of step 1, the mass volume ratio is: Lithospermum officinale cells: PBS buffer: magnetic microsphere carrier = 1g: 20ml: 10ml.
8. The method according to claim 7, characterized in that: In the proliferation culture of step 2, the volume ratio of the added cell growth medium and the magnetic microsphere carrier is 100:1-5.
9. The method according to claim 8, characterized in that; In step 2, proliferation culture and step 3, transformation culture, after the proliferation culture is completed, the magnetic microsphere carrier is adsorbed by an electromagnet and then filtered through a filter to obtain a tissue culture; the magnetic microsphere carrier and the tissue culture are all transferred to a bioreactor filled with M-9 pigment production culture medium.
10. The method according to claim 6, characterized in that; In step S41, the method for separating the lithospermum cell fluid from the pigment production medium is: adsorbing the magnetic microsphere carrier by an electromagnet, and then filtering through a filter to remove the M-9 pigment production medium.