阅读说明：本技术 黄花蒿细胞生物转化二氢去氧青蒿素b的方法和用途 (Method for biotransformation of dihydrodeoxyartemisinin B by artemisia annua cells and application of dihydrodeoxyartemisinin B ) 是由 朱建华 于荣敏 陈昌 于 2020-05-13 设计创作，主要内容包括：本发明是黄花蒿细胞生物转化二氢去氧青蒿素B的方法和用途。涉及到黄花蒿脱分化细胞的诱导、悬浮培养,利用TLC、HPLC-ELSD和GC-MS法考察并优化二氢去氧青蒿素B最佳转化时间和最佳转化浓度。光照培养条件下,最佳转化条件为60h和90mg/L；暗培养条件下,最佳转化条件为72h和90mg/L。进一步积累转化产物,利用硅胶柱、Sephadex LH-20及半制备液相进行分离纯化后得到三个化合物,通过HR-ESI-MS、NMR进行结构鉴定,确定为3α-hydroxy dihydro-epi-deoxyarteannuin B,9β-hydroxy dihydro-epi-deoxyarteannuin B和14-hydroxy dihydro-epi-deoxyarteannuin B；细胞冻干、乙酸乙酯超声提取以及GC-MS检测青蒿素的含量,光照培养和暗培养条件下,实验组的青蒿素含量分别增加了3.6％和23.9％。利用qRT-PCR法检测最佳转化条件下黄花蒿细胞中HMGR、FPS、ADS、CYP71AV1、CPR、DBR2和ALDH1的表达水平。(The invention relates to a method for biotransformation of dihydrodeoxyartemisinin B by artemisia annua cells and application thereof. Relates to the induction and suspension culture of the artemisia annua dedifferentiated cells, and utilizes TLC, HPLC-ELSD and GC-MS methods to investigate and optimize the optimal conversion time and the optimal conversion concentration of dihydrodeoxyartemisinin B. Under the illumination culture condition, the optimal transformation condition is 60h and 90 mg/L; under dark culture conditions, the optimal transformation conditions were 72h and 90 mg/L. Further accumulating the transformation products, separating and purifying by utilizing a silica gel column, Sephadex LH-20 and a semi-prepared liquid phase to obtain three compounds, and carrying out structure identification through HR-ESI-MS and NMR to determine the three compounds as 3 alpha-hydroxy dihydro-epi-deoxy-anhydronin B, 9 beta-hydroxy-epi-deoxy-anhydronin B and 14-hydroxy dihydro-epi-anhydronin B; cell freeze-drying, ethyl acetate ultrasonic extraction and GC-MS detection of the artemisinin content, the artemisinin content of the experimental groups was increased by 3.6% and 23.9% respectively under the conditions of light culture and dark culture. And detecting the expression levels of HMGR, FPS, ADS, CYP71AV1, CPR, DBR2 and ALDH1 in the artemisia annua cells under the optimal transformation condition by using a qRT-PCR method.)

1. A method and use of the artemisia annua cells to biologically convert dihydrodeoxyartemisinin B as claimed in claim 1. Through cell culture, the optimal transformation condition of dihydrodeoxyartemisinin B is inspected and optimized, the product is accumulated, separated and purified, the structure is identified, the content of artemisinin in cells is detected, and the expression level of genes in artemisia annua cells under the optimal biotransformation condition is detected by a qRT-PCR method. The method comprises the following steps:

(1) inducing the dedifferentiated cells of the artemisia annua, carrying out solid culture and carrying out liquid suspension culture;

(2) optimizing the biotransformation condition of dihydrodeoxyartemisinin B under the conditions of illumination culture and dark culture;

(3) accumulation, separation and purification of the conversion product and utilization of HR-ESI-MS,¹H-NMR、¹³C-NMR is carried out for structural identification;

(4) freeze-drying artemisia annua cells, carrying out ultrasonic extraction by ethyl acetate and detecting the content of artemisinin by GC-MS.

(5) And detecting the expression levels of HMGR, FPS, ADS, CYP71AV1, CPR, DBR2 and ALDH1 in the artemisia annua cells by using a qRT-PCR method.

2. Three compounds obtained by the artemisia annua suspension cell bioconversion method as claimed in claim 2: 3 alpha-hydroxydihydroep-epi-deoxyranuin B (1), 9 beta-hydroxydihydroep-epi-deoxyranuin B (2), and 14-hydroxydihydroep-epi-deoxyranuin B (3).

Technical Field

The invention belongs to the field of biotransformation of natural active products in medicinal chemistry, and particularly relates to induction, solid culture and liquid suspension culture of artemisia annua dedifferentiated cells, and investigation and optimization of optimal transformation time and optimal transformation concentration of dihydrodeoxyartemisinin B; accumulation, separation and purification of the conversion product and the purification of the product by HR-ESI-MS,¹H-NMR、¹³C-NMR is carried out for structural identification; freeze drying of artemisia annua cells and acetic acid BPerforming ester ultrasonic extraction and GC-MS (gas chromatography-Mass spectrometer) detection on the artemisinin content; and detecting the expression level of the genes in the artemisia annua cells under the optimal transformation condition by using a qRT-PCR method.

Background

Artemisinin is a sesquiterpene compound with a peroxide bridge separated from the traditional Chinese medicine Artemisia annua L in 1971 by Chinese scientists, and has multiple biological activities such as malaria resistance and the like. At present, the combined therapy mainly based on artemisinin drugs becomes the standard anti-malaria therapy recommended by the world health organization. Because the content of the artemisinin in the plants is low (0.02-1.07 percent) and the artemisinin has regional difference, the yield of the raw material medicine can not meet the requirements of patients. At present, the artemisinin acquisition pathway includes direct extraction from artemisia annua, chemical synthesis and biosynthesis.

The domestic medicinal artemisinin is mainly extracted from artemisia annua, and the defects of complicated planting, harvesting and processing steps, time and labor waste, large occupied area and the like of the artemisia annua bring challenges to the commercial production of the artemisia annua. The chemical synthesis of artemisinin has been carried out in the 80 s of the 20 th century. The chemical synthesis has low cost, the reaction can be amplified to dozens of grams, but the synthesis route is complex, the yield is low, the production process causes great pollution to the environment, and the waste liquid treatment cost is high. The semi-synthesis of the artemisinin mainly adopts biosynthesis of arteannuic acid, and then the arteannuic acid is used as a raw material to chemically synthesize the artemisinin. The method has high yield and simple operation, and is expected to realize large-scale industrial production. However, semisynthesis relies on the availability of artemisinin derivatives, which is costly to produce industrially. Heretofore, the method for producing arteannuic acid by fermentation of yeast engineering bacteria and synthesizing artemisinin by using arteannuic acid as a raw material is widely applied. The biological total synthesis is favored because of simple operation and low cost, thereby having attractive prospect. The biological total synthesis of artemisinin has great application value, but the biological synthesis terminal reaction mechanism is not clear, so that the biological total synthesis of artemisinin cannot be realized at present.

Exogenous organic compounds are added into a biological system or an enzyme system in a growth state, and a substrate is catalyzed by the enzyme in the biological system to generate a chemical structure change, and the process is called biotransformation. The biotransformation reaction has the advantages of strong selectivity, mild condition, environmental protection and the like. The biological systems generally used for transformation research mainly include bacteria, fungi, algae, plant suspension cells and tissues, and the most widely used are plant suspension cells. These conversion products are generally difficult to achieve by chemical reactions.

The biotransformation of suspension cells is generally used for the structural modification of natural products and organic synthetic products, thereby obtaining lead compounds. The derivatives obtained by the biotransformation method are further researched, and the problems are expected to be solved. The method has obvious difference and advantages by using the plant suspension culture cells to carry out biotransformation reaction, chemical reaction and the like. The method is simple to operate, environment-friendly and suitable for large-scale production. However, plant cells grow slowly during the culture process, are easily affected by the environment such as temperature and the like, and need to be operated in a sterile environment, and the disadvantages limit the utilization of plant suspension cells.

Previous studies have shown that dihydrodeoxyartemisinin B is a possible precursor of artemisinin and thus can promote the increase of the content of artemisinin and its conversion products. The study was carried out by feeding dihydrodeoxyartemisinin B to artemisia annua suspension cells.

Disclosure of Invention

The invention aims to research a method for feeding dihydrodeoxyartemisinin B to artemisia annua suspension cells to improve the content of conversion products and artemisinin.

Based on cell culture, the invention inspects and optimizes the optimal transformation time and the optimal transformation concentration of dihydrodeoxyartemisinin B, the accumulation, separation and purification and structure identification of products, the detection of the content of artemisinin in cells, and the qRT-PCR method detects the expression level of genes in artemisia annua cells under the optimal biotransformation condition. The method comprises the following steps:

(1) inducing the dedifferentiated cells of the artemisia annua, carrying out solid culture and carrying out liquid suspension culture;

(2) optimizing the biotransformation condition of dihydrodeoxyartemisinin B under the conditions of illumination culture and dark culture;

(3) accumulation, separation and purification of conversion products and utilization of HR-ESI-MS、¹H-NMR、¹³C-NMR is carried out for structural identification;

(4) freeze-drying artemisia annua cells, performing ultrasonic extraction by ethyl acetate and detecting the content of artemisinin by GC-MS;

(5) and detecting the expression levels of HMGR, FPS, ADS, CYP71AV1, CPR, DBR2 and ALDH1 in the artemisia annua cells by using a qRT-PCR method.

The invention utilizes artemisia annua suspension cell biotransformation method to obtain three compounds: 3 alpha-hydroxy di-epi-deoxyarteannin B, 9 beta-hydroxy di-epi-deoxyarteannin B, 14-hydroxy di-epi-deoxyarteannin B.

Drawings

FIG. 1 is a HPLC-ELSD detection spectrum of the product in the culture medium under the illumination condition.

FIG. 2 is a HPLC-ELSD detection profile of the product in the medium under dark culture conditions.

FIG. 3 is a GC-MS detection spectrum of the product in the medium under light conditions.

FIG. 4 is a GC-MS detection spectrum of a product in a medium under dark culture conditions.

FIG. 5 is a graph showing the content of DHEDB conversion products in light culture systems as a function of conversion time.

FIG. 6 is a graph of DHEDB conversion product content versus conversion time in dark culture systems.

FIG. 7 is a graph of DHEDB conversion product content as a function of substrate concentration in light culture systems.

FIG. 8 is a graph of DHEDB conversion product content as a function of substrate concentration in dark culture systems.

FIG. 9 is a HR-ESI-MS spectrum of Compound 1.

FIG. 10 is a drawing of Compound 1¹H-NMR spectrum.

FIG. 11 is a drawing of Compound 1¹³C-NMR spectrum.

FIG. 12 is a HR-ESI-MS spectrum of Compound 2.

FIG. 13 is a drawing of Compound 2¹H-NMR spectrum.

FIG. 14 is a drawing of Compound 2¹³C-NMR spectrum.

FIG. 15 is a HR-ESI-MS spectrum of Compound 3.

FIG. 16 is a drawing of Compound 3¹H-NMR spectrum.

FIG. 17 is a drawing of Compound 3¹³C-NMR spectrum.

Figure 18 is a DEPT 135 map of compound 3.

FIG. 19 is a drawing of Compound 3¹H-¹H COSY map.

Figure 20 is an HSQC spectrum of compound 3.

Figure 21 is an HMBC map of compound 3.

FIG. 22 is a NOESY spectrum of Compound 3.

FIG. 23 is a schematic of the DHEDB roadmap for suspension cell biotransformation of Artemisia annua.

FIG. 24 is a GC-MS spectrum of the product in cells cultured under light.

FIG. 25 is a GC-MS spectrum of the product in cells in dark culture.

FIG. 26 shows the content of artemisinin in cells.

FIG. 27 shows a reaction system of reverse transcription.

FIG. 28 shows the primer sequences of the key enzyme genes of Artemisia annua.

FIG. 29 shows the reaction system of qRT-PCR.

FIG. 30 shows the reaction sequence of qRT-PCR.

FIG. 31 shows the effect of substrate on key enzyme genes under light culture conditions.

FIG. 32 shows the effect of substrate on key enzyme genes under dark culture conditions.

Detailed Description