CN115572748A - Method for producing antitumor deoxy harringtonine by using suspension culture cells - Google Patents

Method for producing antitumor deoxy harringtonine by using suspension culture cells Download PDF

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CN115572748A
CN115572748A CN202211479304.1A CN202211479304A CN115572748A CN 115572748 A CN115572748 A CN 115572748A CN 202211479304 A CN202211479304 A CN 202211479304A CN 115572748 A CN115572748 A CN 115572748A
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cephalotaxus
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江雪飞
王亮
乔飞
彼得·尼克
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Sanya Nanfan Research Institute Of Hainan University
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Abstract

The invention discloses a method for producing antitumor deoxy harringtonine by using suspension culture cells, which comprises the following steps: the method for producing antitumor deoxy cephalotaxus fortunei ester alkaloids by using precursor feeding (cephalotaxine, L-homoleucine) + inducing (stimulating agent) in cephalotaxus hainanensis axillary cell suspension culture solves the problem of low biomass by establishing a cell line with high growth speed, has the core content of converting cephalotaxus fortunei into deoxy cephalotaxus fortunei ester alkaloids by using a cell bioreactor, has the characteristics of short and stable production period, no cultivated land occupation, stable yield, high content of effective components and no natural resource damage, and has great development and utilization values.

Description

Method for producing antitumor deoxy harringtonine by using suspension culture cells
Technical Field
The invention belongs to the technical field of harringtonine, and mainly relates to a method for producing antitumor deoxy harringtonine by using suspension culture cells.
Background
The plant of the family Cephalotaxaceae is evergreen, a heterosexual plant, a coniferous tree or a shrub. Wherein Cephalotaxus is the only genus of the family, and has only 7 varieties and 2 varieties, including cephalotaxus pectinatus, cephalotaxus japonica, cephalotaxus latifolia, cephalotaxus sinensis, cephalotaxus hainanensis, cephalotaxus fortunei, cephalotaxus alpina, cephalotaxus sinensis, cephalotaxus chinensis, and Cephalotaxus gongyasana. In addition, most of the plants in this genus contain cephalotaxins and homoerythrines.
At present, cephalotaxus ester alkaloids in cephalotaxaceae plants have proved to have medicinal value for treating various diseases, including non-lymphoid leukemia such as acute (chronic) myelocytic leukemia, monocytic leukemia, promyelocytic leukemia and the like; meanwhile, the medicine also has obvious curative effect on malignant lymphoma, breast cancer, chorioadenoma, cervical cancer, polycythemia vera and the like.
In cephalotaxus plants, cephalotaxus hainanensis is a local characteristic variety in the south of the hainanensis, and is recorded in Xinhua Bencao compendium and Guangdong common Chinese herbal medicines, and the root, stem bark, branches and leaves, flowers and seeds of the cephalotaxus hainanensis have pharmacological activity and are main raw materials of various Chinese patent medicines; it is recorded in the book of Chinese materia medica that the root and stem bark of the Chinese materia medica can dispel wind and remove dampness and mainly treat rheumatic arthralgia and the like when taken orally, the branches and leaves can also dispel wind and remove dampness, reduce swelling and eliminate stagnation, relieve cough and moisten lung and the like and mainly treat rheumatic swelling and pain, and the Chinese torreya flower can promote diuresis, kill parasites and mainly treat hydrosphere swelling and ascariasis; torreya grandis seeds (Chinese medicine name: torreya grandis seeds) are recorded in medical works such as Bie Lu, ben Cao Jing Ji Zhu, ben Cao Jing Ji Yi, ben Cao Yan Yi, and Ben Cao Dian, and can be used for treating intestinal parasitic diseases, infantile malnutrition, cough due to lung dryness, constipation due to intestinal dryness, hemorrhoid, etc.
In 1963, cephalotaxine and harringtonine were first isolated from cephalotaxus and cephalotaxus plants by Paudler et al, and their structures were later established by Powll et al in 1969. Studies have shown that cephalotaxine itself is not biologically active (discovered by Powell in 1972), but its very many polyester alkaloid derivatives such as cephalotaxine (HRT), isocephalotaverine (IHT), deoxycephalotaxine (DHT) and homoharringtonine (HHT) 4 alkaloid compounds were found to have inhibitory effect on growth of leukemia cells in mice. In the early 70 s to the end of the 70 s in the 20 th century, china respectively carries out researches on separation and extraction of anticancer substances, structural analysis, pharmacological action and the like on 8 cephalotaxus plants in China, and more than 30 alkaloids are separated and identified, wherein only the 4 ester alkaloids with anticancer activity are mainly used.
The alkaloids are distributed in bark, root, stem, leaf and fruit of Cephalotaxus fortunei, but the content is low, and 100-150kg of Cephalotaxus fortunei branches and leaves are required for extracting 1g of Cephalotaxus fortunei ester alkaloids. In addition, the cephalotaxus plants have extremely slow growth speed, narrow ecological distribution and sporadic distribution and are not easy to obtain. At present, cephalotaxus hainanensis is listed as a national second-level key protection endangered plant, so that the large-scale acquisition of medicine raw materials by cutting down plants is difficult to realize. The artificial synthesis mode becomes the key for crossing the obstacle, and promotes the comprehensive development of the cephalotaxine total synthesis path research. In 1972, weinreb et al successfully completed total synthesis of cephalotaxine for the first time by constructing C-rings by intramolecular Friedel-Crafts reaction. In 2002, the Nagasaka group has completed the total synthesis of cephalotaxine by substituting the benzazepine heptatomic ring. In addition, china has a breakthrough in the synthesis of harringtonine alkaloids, for example, huang Wen Queen, which is 1975, firstly synthesizes deoxy harringtonine through Reformatsky chemical reaction, and then is widely applied to the synthesis of harringtonine, homoharringtonine and iso-harringtonine. However, the chemical synthesis of these compounds has many highly complex chiral center structures and is susceptible to steric hindrance, resulting in loss of activity.
Plant cell culture has the advantages of homogeneity, rapid proliferation, short and stable period, and the like, and is considered to be the most promising alternative method for continuously producing the compounds which are difficult to extract from plants and synthesize by chemical methods. The production of plant functional secondary metabolites using suspension cell lines has been studied over 1000 plants and has been exemplified successfully. For example, paclitaxel, shikonin, rosmarinic acid, ginsenoside, etc. have been industrially produced. The cephalotaxus callus culture and the cephalotaxus cell suspension culture have been reported, but the cell growth is slow, and the alkaloid yield is very low. Therefore, how to obtain a stable, rapidly growing, high-yielding cell line becomes a major challenge for commercial production of the compound.
The cephalotaxus alkaloids extracted from plant of family Cephalotaxaceae have the highest cephalotaxus content. However, as a precursor of deoxyharringtonine, harringtonine itself has no biological activity. Therefore, how to efficiently convert the cephalotaxine without bioactivity into the deoxycephalotaxine with antitumor activity becomes a key link for solving the production problem of the medicine. Thus, the subject matter of the present invention includes methods for creating high-yielding, stable cell lines and methods for enhancing the conversion of cell lines to cephalotaxine to deoxyharringtonine.
Disclosure of Invention
The invention aims to provide a method for producing antitumor deoxy harringtonine by using suspension culture cells, which solves the problem of low biomass by establishing a cell line with high growth speed, and converts the cephalotaxine without bioactivity into the deoxy harringtonine with antitumor activity by using a cell bioreactor.
The technical problem to be solved by the invention can be realized by the following technical scheme: a method for producing antitumor deoxy harringtonine by using suspension culture cells comprises the following steps:
(1) Selecting cephalotaxus hainanensis explants to inoculate to a callus culture medium, firstly carrying out dark culture to obtain cephalotaxus hainanensis leaf axillary callus, and then carrying out subculture;
(2) Selecting axillary callus of cephalotaxus hainanensis leaves obtained in the step (1), inoculating the axillary callus into a liquid proliferation culture medium, and carrying out dark culture under a shaking condition to obtain a suspension cell line of liquid culture;
(3) Subculturing the suspension cell line obtained in the step (2), feeding cephalotaxine, L-homoleucine and a stimulant with the final concentration of 8-12mg/L into the suspension cell line culture in 8-12 days of the subculturing of the suspension cell line, and continuously incubating for 7-12 days;
(4) Filtering the product incubated in the step (3), separating the cells from the culture medium, homogenizing the cells, respectively adding methanol into the homogenized cells and the culture medium for extraction, then performing ultrasonic treatment and centrifugation, combining the supernatants, and performing rotary evaporation treatment to obtain the deoxyharringtonine.
In the method for producing antitumor deoxy harringtonine by using suspension culture cells, the method comprises the following steps:
preferably, the cephalotaxus hainanensis explants in the step (1) are cephalotaxus hainanensis young stems.
Preferably, the cephalotaxus hainanensis juvenile stem in the step (1) is 1-2 cm in length and contains 1-2 axillary buds.
Preferably, the cephalotaxus hainanensis explants are subjected to cutting, leaf removing and disinfection treatment before culture in the step (1).
Preferably, the callus culture medium in the step (1) is a basal culture medium containing vitamin DKW, 5 to 6 g/L, 20 to 40 g/L of cane sugar, 100 to 200 mg/L of hydrolyzed retinas, 5 to 10mg/L of Picloram and 7 to 10 g/L of agar, and the pH value is 5.8 to 5.9; in dark culture, the culture temperature is 25 to 27 ℃, the culture time is 40 to 45 days, and then the grown callus is subcultured once every 1.5 to 2 months.
More preferably, the callus culture medium in the step (1) is a basal medium containing vitamin DKW 5g/L + sucrose 30g/L + hydrolyzed retin 100 mg/L + Picloram 5mg/L + agar 7 g/L, the pH is 5.8 to 5.9, the culture temperature is 25 ℃ during dark culture, the culture time is 40 to 45 days, and then the grown callus is subcultured once every 2 months.
The callus culture medium formula and the culture method adopted in the step (1) can meet the dual requirements of cephalotaxus hainanensis axillary cell elongation growth and cell division, the cell division and the cell expansion and elongation have obvious cycles, the cell types are consistent, and the callus culture medium formula and the culture method have the characteristics of non-embryonic cells.
Preferably, the liquid proliferation medium in the step (2) is a basal medium containing vitamin DKW, the basal medium contains 5-6 g/L of sucrose, 20-40 g/L of sucrose, 100-200 mg/L of hydrolyzed retin and 5-10 mg/L of Picloram, and the pH value is 5.8-5.9; in dark culture, the culture temperature is 25 to 27 ℃, and the shaking culture is carried out in an orbital shaker under the shaking condition that the rotation speed is adjusted to be 120 to 150 rpm.
More preferably, the liquid multiplication culture medium in the step (2) is a vitamin-containing DKW basic culture medium of 5g/L, sucrose of 30g/L, hydrolyzed retinin of 100 mg/L and Picloram of 5mg/L, and the pH is 5.8 to 5.9; in the dark culture, the culture temperature is 25 ℃, and the shaking condition is that the shaking culture is carried out in an orbital shaker at the rotation speed of 150 rpm.
In the step (2) of the invention, the liquid culture is carried out by the liquid culture medium, and the cells with consistent cell types, rapid growth and high proliferation coefficient can be rapidly obtained.
Preferably, in the step (3), the subculture period is 8 to 12 days, and the volume ratio of the suspension cell line to the liquid proliferation medium is 1:5 to 1: and 6, adjusting the rotating speed to be 120 to 150 rpm in an orbital oscillator to carry out shake culture.
More preferably, in the case of the subculture in the step (3), the subculture cycle is 10 days, the inoculation volume in 30mL of the liquid growth medium is 5mL, and the orbital shaker (Φ =25 mm) is used for shaking culture at 150 rpm.
The fresh weight of the cells is increased by about 3 times when the subculture is carried out for 10 days in the step (3) of the invention, and sufficient raw materials can be provided for the deoxyharringtonine.
Preferably, the stimulant in the step (3) is one or more of abscisic acid (ABA), methyl jasmonate (MeJA), salicylic Acid (SA), silver Nitrate (SN), ibuprofen (IBU), ethephon (ETH), chitosan Glutamate (CG), hydrolyzed collateral protein (CH) and beta-Cyclodextrin (CD).
Preferably, the final concentration of the abscisic acid is 80-100 μ M, the final concentration of the methyl jasmonate is 100-200 μ M, the final concentration of the salicylic acid is 50-100 μ M, the final concentration of the ethephon is 50-100 μ M, the final concentration of the ibuprofen is 50-100 μ M, the final concentration of the silver nitrate is 10-50 μ M, the final concentration of the chitosan glutamate is 20-40 mg/L, the final concentration of the aquaporin is 200-500mg/L, and the final concentration of the beta-cyclodextrin is 10-50 μ M.
In the invention, after the cell culture is fed with precursors (cephalotaxine and L-homoleucine) in 8 to 12 days of cell subculture, a stimulant is immediately added into a culture solution, the functions of regulating and controlling cell aging, starting the synthesis of secondary metabolites, promoting the metabolic flow direction and reducing the self toxicity of the metabolites are realized through the stimulant, and the total culture time is 7 to 12 days.
Preferably, in step (4), the supernatant is detected by UPLC-MS to confirm the deoxyharringtonine.
Preferably, the UPLC-MS assay uses an UPLC system with a mass spectrometer on a C18 column using a 0.1% v/v aqueous formic acid-acetonitrile gradient elution conditions of 0.3 mL/min to separate the cell extract (2 μ L), the gradient elution procedure comprising: and (3) 0 minute: 98:2,2 minutes: 98:2,8 minutes: 65:35 And 10 minutes: 15:85 And 12 minutes: 15:85 12.8 minutes: 55:45 15 minutes: 98:2, 16 minutes: 98:2, all are volume ratios.
As a preferred embodiment of the present invention, in the step (4), the cells are separated from the culture medium, the cells are homogenized, methanol is added into the homogenized cells and the culture medium respectively for extraction, and then the ultrasonic and centrifugal processes comprise: separating the culture medium from the cells in the cell suspension culture fed with the precursor in the step (3) by using a Mie funnel, recording the sample collection result, and taking 0.3 g of the cells to be filled into a 2mL EP tube, wherein the culture medium is taken 0.5mL to be filled into a 2mL EP tube, and freezing and storing at-80 ℃ for later use; adding 1.5 mL of methanol into 2mL EP centrifuge tubes containing 0.3 g of cells and 0.5mL of culture medium respectively, placing the cells and the culture medium homogenized by a tissue grinder (preferably 60 Hz,60 s) in an ultrasonic cleaning instrument (preferably 600W, 40 kHz) for 20 min by ultrasonic treatment at 25 ℃, centrifuging at 12000 rmp for 10 min, combining the supernatants, passing the combined supernatants through a 0.22 mu M membrane, detecting by UPLC-MS (ultra performance liquid chromatography-mass spectrometry) or combining the supernatants, and performing rotary evaporation treatment to obtain the deoxy-harringtonine with the purity of more than 97%.
The invention has the following advantages:
(1) The method of the invention is a method for producing antitumor deoxy cephalotaxus fortunei ester alkaloid in cephalotaxus hainanensis leaf axillary cell suspension culture by using a mode of 'precursor feeding (cephalotaxus fortunei, L-homoleucine) + inducing (stimulant)', the core content of the method is that cephalotaxus fortunei ester alkaloid is converted into deoxy cephalotaxus fortunei ester alkaloid by using a cell bioreactor, and the method has the characteristics of short and stable production cycle, no occupation of cultivated land, stable yield, high content of effective components and no damage to natural resources, and has great development and utilization values;
(2) The cells obtained by the method are non-embryonic cells, maintain the obvious characteristic of the elongation growth of explants (leaf axils), are fully dispersed, and have extremely high proliferation efficiency;
(3) The cell type obtained by the method is single, the growth cycle is consistent, and the yield is easy to regulate and control;
(4) The method can be used for industrially producing the deoxyharringtonine, and gets rid of the dependence on natural conditions;
(5) Compared with the cephalotaxus hainanensis planting method, the production period is short (15 to 20 days), the production scale is convenient to control, and the investment risk is small;
(6) The method of the invention takes the cephalotaxine and the L-homoleucine which are relatively cheap and easy to obtain as precursors, and finally realizes the biosynthesis of the deoxycephalotaxine with the anti-tumor activity through the induction of different stimulators.
Drawings
FIG. 1 is the morphology of Cephalotaxus hainanensis C.Li axillary cells and the growth of cell suspension culture in example 1 of the present invention, wherein A is callus induced by axillary, bar =2 mm, B is the morphology of suspension culture cells, bar = 20 μm, C is the growth of cell suspension culture cells, FW is fresh weight;
FIG. 2 is an identification chart of the present invention, in example 1, the administration of cephalotaxine and the conversion of homoleucine to generate deoxyharringtonine, wherein A is a total ion flow chart and B is an MS spectrogram;
FIG. 3 is a control of the yield of deoxyharringtonine obtained in example 3 with incubation times of 5 days and 7 days at the time of induction of the feeding precursors and stimulants.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The starting materials used in the following examples were all commercially available, unless otherwise specified.
Example 1
Obtaining a cephalotaxus hainanensis leaf axillary non-embryonic cell suspension cell line and drawing a growth curve of the cephalotaxus hainanensis leaf axillary cell suspension culture line.
(I) obtaining cephalotaxus hainanensis axillary non-embryonic cell suspension cell line
(1) Taking a tender stem of cephalotaxus hainanensis as an explant after leaf removal, wherein the length of the tender stem is 1-2 cm and contains 1-2 axillary buds, sterilizing the surface of the cephalotaxus hainanensis, cutting the cephalotaxus hainanensis into 1cm small sections, inoculating the small sections to a callus culture medium, culturing at the temperature of 25 ℃, performing dark culture in a culture mode for 40-45 days, and then subculturing the grown callus every 2 months, wherein the subculture is shown in a picture A in figure 1;
wherein the callus culture medium comprises 5g/L of vitamin DKW basic culture medium, 30g/L of cane sugar, 100 mg/L of hydrolyzed retinin, 5mg/L of Picloram and 7 g/L of agar, and the pH value is 5.8-5.9.
(2) Taking 2 to 3 cm 3 Transferring the cell mass with rapid growth into a 100 mL triangular flask with a baffle plate containing 30mL cell suspension culture liquid proliferation culture medium, and carrying out shake dark culture at 25 ℃ and 150 rpm for 10 days to obtain a liquid culture non-embryonic suspension cell line with non-axial division characteristics, as shown in a diagram B in figure 1;
wherein the proliferation culture medium comprises vitamin DKW basic culture medium 5g/L, sucrose 30g/L, hydrolyzed retinin 100 mg/L, picloram 5mg/L, and pH 5.8-5.9.
Drawing growth curve of cephalotaxus hainanensis leaf axillary cell suspension culture line
(3) The cells obtained by the method grow rapidly, are subcultured once every 10 days, are shake-cultured in an orbital shaker (phi =25 mm) at the rotation speed of 150 rpm, the inoculation amount of the non-embryonic suspension cell line is 1/6 of the volume of the liquid proliferation medium, the cells and the medium of the cell suspension culture are separated by using a buchner funnel, the cells and the medium are collected once every 2 days from the beginning of the subculture to the 14 th day after the subculture, and the sample collection result is recorded, and the cell growth process presents an S-shaped growth curve, as shown in a C diagram in fig. 1. It can be seen that the subculture cycle of the cells is 10 days, the fresh weight of the cells reaches about 10 g, the fresh weight of the cells is increased by more than three times after one subculture cycle, and sufficient raw materials can be provided for the deoxyharringtonine.
(4) Feeding cephalotaxine and L-homoleucine with final concentration of 10mg/L to cephalotaxus sinensis leaf axillary cell suspension culture on the 10 th day of cell subculture, incubating for 5 days, and extracting deoxycephalotaxine from cells and culture medium.
The method specifically comprises the following steps:
(4.1) precursor feeding treatment: after feeding cephalotaxine and L-homoleucine to cephalotaxus sinensis leaf axillary cell suspension culture at the final concentration of 10mg/L on the 10 th day of cell subculture, incubating for 5 days, separating the cells from the culture medium by filtration through Whitman No.1 filter paper, and recording the sample collection result.
Fresh cells were dispensed into sterilized 2mL EP centrifuge tubes, 0.3 g per tube, 3 aliquots of 0.5mL of media were placed into sterilized 2mL EP centrifuge tubes, and the remaining aliquot was placed into 50 mL centrifuge tubes. All samples were flash frozen in liquid nitrogen and stored at-80 ℃ until use.
(4.2) preparation of sample solvent: a2 mL EP centrifuge tube containing 0.3 g of cells and 0.5mL of medium was taken in one portion, and 1.5 mL of methanol was added thereto as an extraction solvent. The cells were homogenized by a tissue disruptor (60 Hz, 60S), then two EP centrifuge tubes containing the homogenized cells and the medium, respectively, were placed together in an ultrasonic cleaner (600W, 40 kHz) with ultrasound at 25 ℃ for 20 min,12000 rmp for 10 min, and the supernatant was removed and passed through a 0.22 μ M pore membrane for UPLC-MS detection.
(4.3) qualitative and quantitative analysis of cephalotaxus alkaloids: the qualitative and quantitative determination of cephalotaxus alkaloids was carried out in a mass spectrometer in combination with a UPLC system.
Cell extracts (2 μ L) were separated on a C18 column (2.1 × 100 mm, 2.7 μm particle size) using 0.1% v/v aqueous formic acid-acetonitrile under 0.3 mL/min gradient elution conditions, 0 min: 98:2 (v/v), 2 min: 98:2 (v/v), 8 min: 65:35 (v/v), 10 min: 15:85 (v/v), 12 min: 15:85 (v/v), 12.8 min: 55:45 (v/v), 15 min: 98:2 (v/v), 16 min: 98:2 (v/v). The UV1 channel was set at 291 nm. The DAD detection range is 200 to 800 nm, and the data collection rate is 5 Hz.
The detection results are shown in fig. 2, and the results show that: 5.6 mg of harringtonine alkaloid can be obtained per liter of culture system, wherein the proportion of the deoxyharringtonine in the four kinds of harringtonine is more than 97%, and the characterization of the deoxyharringtonine is realized by comparing with a standard substance and molecular weight. However, it is difficult to continue to improve the yield of the deoxyharringtonine greatly by feeding the precursor alone, and the yield is limited by the feeding amount of the harringtonine. In the latter work, therefore, the present application attempts to increase the production of deoxyharringtonine in cell culture by induction of pro-feeding in combination with a stimulant.
Example 2
The steps (1) to (3) are the same as in example 1.
Step (4) 9 elicitors, 100. Mu.M abscisic acid (ABA), 100. Mu.M methyl jasmonate (MeJA), 100. Mu.M Salicylic Acid (SA), 10. Mu.M Silver Nitrate (SN), 100. Mu.M Ibuprofen (IBU), 100. Mu.M Ethephon (ETH), 20 mg/L Chitosan Glutamate (CG), 200 mg/L hydrolin (CH) and 35. Mu.M beta-Cyclodextrin (CD), were added to the cell suspension culture while feeding the precursor of example 1, respectively, with an incubation time of 5 days, and the sample extraction and detection method was identical to that of example 1. The yield of the deoxyharringtonine obtained by the method is slightly improved compared with that after the feeding of the precursor, but the yield is not greatly different.
Example 3
The steps (1) to (3) are the same as in example 1.
Step (4) the incubation time was extended to 7 days based on the feeding precursor in example 2 in combination with stimulator induction. The sample extraction and detection methods were in accordance with example 1. The yield of the deoxyharringtonine obtained by the method is obviously improved compared with that obtained by incubation for 5 days (figure 3), and the yield of the deoxyharringtonine is related to the incubation time after the induction of the precursor feeding combined stimulant. Wherein the yield of the deoxyharringtonine induced by stimulants abscisic acid (ABA), ethephon (ETH), ibuprofen (IBU) and Silver Nitrate (SN) is more than 2 times of that induced by feeding a precursor (8.325 mg/L), and the yield of the deoxyharringtonine in the 4 stimulants induced cell cultures is as follows:
18.620 mg/L of deoxyharringtonine available from abscisic acid (ABA) as an irritant;
18.743mg/L of deoxyharringtonine available from Ethephon (ETH), a stimulant;
17.135 mg/L of deoxyharringtonine available from the stimulant Ibuprofen (IBU);
stimulant Silver Nitrate (SN) available as 18.995mg/L of deoxyharringtonine.
In addition, the production of deoxyharringtonine in the stimulator, methyl jasmonate (MeJA), salicylic Acid (SA), chitosan Glutamate (CG), hydrolyzed collateral protein (CH) and β -Cyclodextrin (CD) induced cell cultures was also more than 1.2 times higher than that of the control alone (fed precursor). The nine stimulators are probably realized by regulating the related enzyme gene activity in the biosynthesis of the deoxyharringtonine, and meanwhile, the incubation time is also an important factor influencing the yield of the deoxyharringtonine.
The above description is only for the purpose of illustrating the embodiments of the present invention, and the scope of the present invention is not limited thereto, and any modifications, equivalents and improvements made by those skilled in the art within the technical scope of the present invention as disclosed herein are all covered by the scope of the present invention.

Claims (9)

1. A method for producing antitumor deoxy harringtonine by using suspension culture cells is characterized by comprising the following steps:
(1) Selecting cephalotaxus hainanensis explants to inoculate to a callus culture medium, firstly carrying out dark culture to obtain cephalotaxus hainanensis leaf axillary callus, and then carrying out subculture;
(2) Selecting axillary callus of cephalotaxus hainanensis leaves obtained in the step (1), inoculating the axillary callus into a liquid proliferation culture medium, and carrying out dark culture under a shaking condition to obtain a suspension cell line of liquid culture;
(3) Subculturing the suspension cell line obtained in the step (2), feeding cephalotaxine, L-homoleucine and a stimulant with the final concentration of 8-12mg/L into the suspension cell line culture in 8-12 days of the subculturing of the suspension cell line, and continuously incubating for 7-12 days;
(4) And (4) filtering the product incubated in the step (3), separating the cells from the culture medium, homogenizing the cells, respectively adding methanol into the homogenized cells and the culture medium for extraction, then performing ultrasonic treatment and centrifugation, combining the supernatants, and performing rotary evaporation treatment to obtain the deoxy harringtonine.
2. The method for producing antitumor deoxy harringtonine using suspension culture cells according to claim 1, wherein: the cephalotaxus hainanensis explants in the step (1) are cephalotaxus hainanensis young stems, the cephalotaxus hainanensis young stems are 1-2 cm in length and contain 1-2 axillary buds, and the cephalotaxus hainanensis explants are subjected to cutting, leaf removing and disinfection treatment before culture.
3. The method for producing antitumor deoxy harringtonine using suspension culture cells according to claim 1, wherein: the callus culture medium in the step (1) is a basic culture medium containing vitamin DKW, 5-6 g/L, 20-40 g/L sucrose, 100-200 mg/L hydrolyzed collaterals protein, 5-10 mg/L Picloram and 7-10 g/L agar, and the pH value is 5.8-5.9; in dark culture, the culture temperature is 25 to 27 ℃, the culture time is 40 to 45 days, and then the grown callus is subcultured once every 1.5 to 2 months.
4. The method for producing antitumor deoxy harringtonine using suspension culture cells according to claim 1, wherein: the liquid propagation medium in the step (2) is a basal medium of DKW containing vitamins 5-6 g/L, sucrose 20-40 g/L, hydrolyzed netoglobin 100-200 mg/L, picloram 5-10 mg/L, and the pH is 5.8-5.9; in the dark culture, the culture temperature is 25 to 27 ℃, and the shaking culture is carried out by adjusting the rotation speed in an orbital shaker to 120 to 150 rpm.
5. The method for producing antitumor docetaxel using suspension culture cells as set forth in claim 1, wherein: and (3) in the step (3), the subculture period is 8 to 12 days, and the volume ratio of the suspension cell line to the liquid proliferation culture medium is 1:5 to 1: and 6, adjusting the rotation speed to be 120 to 150 rpm in an orbital oscillator, and carrying out oscillation culture.
6. The method for producing antitumor deoxy harringtonine using suspension culture cells according to claim 1, wherein: the stimulant in the step (3) is one or more of abscisic acid, methyl jasmonate, salicylic acid, silver nitrate, ibuprofen, ethephon, chitosan glutamate, hydrolyzed complexing protein and beta-cyclodextrin.
7. The method for producing antitumor deoxy harringtonine using suspension culture cells according to claim 6, wherein: the final concentration of the abscisic acid is 80 to 100 mu M, the final concentration of methyl jasmonate is 100 to 200 mu M, the final concentration of salicylic acid is 50 to 100 mu M, the final concentration of ethephon is 50 to 100 mu M, the final concentration of ibuprofen is 50 to 100 mu M, the final concentration of silver nitrate is 10 to 50 mu M, the final concentration of chitosan glutamate is 20 to 40 mg/L, the final concentration of aquaxosin is 200 to 500mg/L, and the final concentration of beta-cyclodextrin is 10 to 50 mu M.
8. The method for producing antitumor docetaxel using suspension culture cells as set forth in claim 1, wherein: in the step (4), the supernatant is detected by UPLC-MS to confirm the deoxyharringtonine.
9. The method for producing antitumor docetaxel using suspension culture of cells as claimed in claim 8, wherein: for UPLC-MS detection, the cell extract was separated using a UPLC system and mass spectrometer on a C18 column using 0.1% v/v aqueous formic acid-acetonitrile under a 0.3 mL/min gradient elution, the gradient elution procedure comprising: and (3) 0 minute: 98:2,2 minutes: 98:2,8 minutes: 65:35 And 10 minutes: 15:85 And 12 minutes: 15:85 12.8 minutes: 55:45 15 minutes: 98:2, 16 minutes: 98:2, are volume ratios.
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