CN111534553A - Method for biotransformation of dihydrodeoxyartemisinin B by artemisia annua cells and application of dihydrodeoxyartemisinin B - Google Patents

Abstract

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.

Description

Method for biotransformation of dihydrodeoxyartemisinin B by artemisia annua cells and application of dihydrodeoxyartemisinin B

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

The first embodiment is as follows: induction, solid culture and liquid suspension culture of artemisia annua dedifferentiated cells

Induction of dedifferentiated cells: cutting the tender branch of herba Artemisiae Annuae aseptic seedling into 1cm long sections with scissors, and cutting off the leaf edge of the leaf into 1cm²And (4) clamping the MS culture medium by using forceps to ensure that the wound is fully contacted with the culture medium. The culture medium is placed in a thermostat at 25 ℃ and is divided into 12h illumination culture and continuous dark culture.

Solid culture of dedifferentiated cells: subculturing the two dedifferentiated cells to an MS culture medium containing 30g/L of sucrose, 7.5g/L of agar, 50.0mg/L of vitamin C, 0.5mg/L of 6-BA and 0.5mg/L of NAA, wherein the pH value is 5.70-5.72. Subculturing is carried out once every 14d,

culturing suspension cells: adding 4.43g/L MS minimal medium, 30g/L sucrose, 0.5mg/L NAA and 0.5 mg/L6-BA into a beaker, transferring the solution into a measuring cylinder, adding distilled water to a constant volume, adjusting the pH to 5.70-5.72, and sterilizing at 121 ℃ for 20 min. The subculture cells with the amount of 50g/L were suspended and cultured on a shaker at a rotation speed of 120rpm and a culture temperature of 25 ℃ under continuous light and dark conditions, respectively.

Example two: optimization of Dihydrodeoxyartemisinin B bioconversion conditions

After the suspension culture cells of the artemisia annua are pre-cultured for 10 days, DHEDB is added, the substrate concentration is respectively 0, 10, 30, 50, 70 and 90mg/L, 3 bottles of each group are respectively cultured for 12, 24, 36, 48, 60, 72 and 84 hours, and then the transformation is stopped. And (3) carrying out suction filtration on the suspended cells to separate the cells from the culture medium, and placing the cells in a vacuum freeze dryer to remove water. Pulverizing the dried cells in a mortar, ultrasonically extracting with appropriate amount of ethyl acetate for 3 times, mixing the extractive solutions, and concentrating at 45 deg.C to dry; the medium was extracted 3 times with an equal volume of ethyl acetate, the organic layers were combined and concentrated to dryness at 45 ℃ under reduced pressure. With chromatographic grade methanol: chloroform (1: 1 by volume) 2mL of the dissolved sample was filtered through a 0.45 μm organic microfiltration membrane and stored in a liquid phase vial for HPLC-ELSD and GC-MS detection.

As shown in fig. 1 and 2: HPLC-ELSD detection shows that three product peaks 1, 2 and 3 appear in the light culture and the dark culture of the experimental group (I) and the control group (II) within 9.5-10.5 min.

As shown in fig. 3 and 4: GC-MS detection shows that three product peaks 1, 2 and 3 appear in the experimental group (I) and the control group (II) under the conditions of light culture and dark culture.

As shown in fig. 5: under the condition of illumination culture, the optimal transformation time is determined to be 60 h.

As shown in fig. 6: under dark culture conditions, the optimal transformation time was determined to be 72 h.

As shown in fig. 7: under the condition of light culture, the optimal transformation concentration is determined to be 90 mg/L.

As shown in fig. 8: under dark culture conditions, the optimal transformation concentration was determined to be 90 mg/L.

Example three: accumulation, separation and purification and structural identification of transformation products

In order to isolate the product of the biological transformation of the artemisia annua suspension cells into the DHEDB and identify the structure of the artemisia annua suspension cells through an analytical method, the culture amount of the artemisia annua cells needs to be increased. Subculturing 100mL herba Artemisiae Annuae suspension cells in 100 bottles, culturing under illumination for 10 days, adding 90mg/L DHEDB, culturing under illumination for 60 hr, stopping transformation, filtering, extracting with culture medium for 3 times, mixing ethyl acetate layers, and concentrating under reduced pressure at 45 deg.C to dry.

Separating the sample by 200-mesh silica gel with 300 meshes, performing gradient elution by using petroleum ether and ethyl acetate as eluent, continuously detecting by TLC, merging the same fractions, and developing the color by using vanillin-concentrated sulfuric acid. And further carrying out Sephadex LH-20 and high performance liquid chromatography separation to obtain three compounds. And carrying out structural identification on the converted product by using methods such as HR-ESI-MS, NMR and the like.

FIGS. 9-11, FIGS. 12-14, and FIGS. 15-22 show mass spectra and NMR data for compounds 1, 2, and 3, respectively. As shown in fig. 23, the transformation products were the same in both the light and dark culture conditions, and three compounds were obtained: 3 alpha-hydroxydihydroep-epi-deoxyranuin B (1), 9 beta-hydroxydihydroep-epi-deoxyranuin B (2) and 14-hydroxydihydroep-epi-deoxyranuin B (3), wherein the compound 3 is a novel sesquiterpene compound.

Example four: freeze-drying artemisia annua cells, performing ethyl acetate ultrasonic extraction and detecting artemisinin content by GC-MS

After the suspension cells of the artemisia annua are pre-cultured for 10 days, DHEDB (90mg/L) is added, and 3 bottles are put in each group. And the culture is stopped after the illumination culture and transformation are carried out for 60 hours, and the culture is stopped after the dark culture and transformation are carried out for 72 hours. And (3) carrying out suction filtration on the suspended cells to separate the cells from the culture medium, and placing the cells in a vacuum freeze dryer to remove water. Pulverizing the dried cells in a mortar, ultrasonically extracting with ethyl acetate for 3 times, combining the extractive solutions, concentrating at 45 deg.C to dryness, and purifying with chromatographic pure methanol: 2mL of chloroform (volume ratio of 1:1) and 1mL of chromatographically pure ethyl acetate are used for dissolving samples, and the samples are filtered by a 0.22 mu m organic microporous filter membrane and stored in a liquid phase bottle for testing. The samples were subjected to HPLC-ELSD and GC-MS detection, respectively.

As shown in fig. 24: GC-MS detection shows that the artemisinin peak appears at 18.10min in the experimental group (I) and the control group (II) under the illumination culture condition, the artemisinin content in the control group is 167.74 mug/L, the artemisinin content in the experimental group is 173.85 mug/L, and the artemisinin content in the experimental group is increased by 3.6% compared with the control group.

As shown in fig. 25: GC-MS detection shows that compared with a control group (II), the artemisinin content of the control group is 133.96 mu g/L, the artemisinin content of the experimental group is 165.99 mu g/L, and the artemisinin content of the experimental group is increased by 23.9 percent compared with that of the control group (I) under the dark culture condition.

FIG. 26 shows the artemisinin content of experimental and control groups under light and dark culture conditions.

Example five: qRT-PCR method for detecting expression level of gene in artemisia annua cells

After the suspension cells of the artemisia annua are pre-cultured for 10 days, 3 bottles of experiment groups with the concentration of 90mg/L DHEDB are arranged and are put into the experiment groups, and Light-culturing is carried out for 60 hours (LB); 3 bottles of Light culture control group added with 100 mul DMSO, and culturing for 60h (Light-cut DMSO group; LD); 3 bottles were put into the experiment group at 90mg/L DHEDB, and Dark-cultured for 72 hours (Dark-cultured DHEDB group; DB)3 bottles were added with 100. mu.L DMSO of the Dark-culture control group, and cultured for 72 hours (Dark-cultured control group; DD). After the cells were separated by suction filtration, the cells were packed in tinfoil paper and placed in a-80 ℃ freezer for further use.

The total RNA extraction procedure was as follows:

(1) placing the reagent and the articles in a superclean bench, opening an ultraviolet lamp for sterilization for 30min, and ventilating; meanwhile, the medicine spoon, the forceps and the cell sample prepared in advance are put into a liquid nitrogen bottle for cooling.

(2) 1mL of Trizol, 200mL of chloroform, 500mL of isopropanol, and 1mL of 75% ethanol were added to a 1.5mL EP tube using a pipette in this order, and the EP tube cap was closed.

(3) Burning the mortar with absolute ethyl alcohol, and pouring liquid nitrogen for cooling. Liquid nitrogen was poured into the mortar, and the cells to be ground were added and ground to a uniform powder. Liquid nitrogen is continuously added in the grinding process to ensure that the liquid nitrogen completely submerges the sample.

(4) About 2 spoons of the ground sample were taken, added to 1mL Trizol, vortexed for 15s, and then centrifuged at 12000rpm for 10min at 4 ℃.

(5) The centrifuged sample was removed, and about 900mL of the supernatant was pipetted into an EP tube containing 200mL of chloroform, vortexed for 15s, and centrifuged at 12000rpm for 15min at 4 ℃. .

(6) The centrifuged sample was taken out, and about 500mL of the supernatant was aspirated by a pipette gun, added to an EP tube containing 500mL of isopropyl alcohol, left at room temperature for 10min, centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was discarded.

(7) 1mL of 75% ethanol was added to the EP tube and gently tapped with a pipette until the pellet was completely dissolved.

(8) Centrifuge at 7500rpm for 5min at 4 deg.C, carefully aspirate off the supernatant, place the EP tube in a clean bench to air dry.

(9) 20mL RNase-Free H was used₂O solubilized RNA samples in EP tubes.

(10) 2mL of RNA sample was subjected to electrophoresis.

(11) And detecting the concentration of the sample and the OD260/OD280 value by using an ultramicro ultraviolet spectrophotometer.

Use of Goldenstar^TMRT6 cDNA Synthesis Mix kit for reverse transcription. A reaction system was prepared in an RNase free PCR tube according to FIG. 27, gently mixed with a pipette tip, centrifuged for a short time, and reacted at 50 ℃ for 15 min. And after the reaction is finished, obtaining a DNA template, cooling to 4 ℃, and carrying out subsequent qRT-PCR experiments.

The primers used are shown in FIG. 28, the reference gene is UBC, the target genes are ADS, CPR, CYP71AV1, FPS, HMGR, ALDH1 and DBR 2. qRT-PCR detection was performed using the 2 × T5 Fast qPCR Mix (SYBRGreenI) kit, the reaction system was prepared in a qPCR tube according to FIG. 29, and the settings are shown in FIG. 30

480, reaction program of the real-time fluorescence quantitative PCR system, and recording Ct value of each gene.

The expression levels of the target genes are shown in FIGS. 31 and 32. Under the condition of light culture, the expression levels of HMGR, FPS, CYP71AV1, DBR2 and ALDH1 in the LB group are 1.76, 3.16, 1.65, 2.03 and 2.06 times of those in the LD group respectively. The expression levels of ADS and ALDH1 in the DB group were 4.17-fold and 2.30-fold respectively for the DD group.

Claims (2)

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).

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