CN106967622B

CN106967622B - Aspergillus flavus BP6T2 for producing paclitaxel and application thereof

Info

Publication number: CN106967622B
Application number: CN201710354729.2A
Authority: CN
Inventors: 曹军卫; 涂毅; 金卫华
Original assignee: Wuhan Donghu University
Current assignee: Wuhan Donghu University
Priority date: 2017-05-17
Filing date: 2017-05-17
Publication date: 2019-12-13
Anticipated expiration: 2037-05-17
Also published as: CN106967622A

Abstract

The invention discloses an endophyte strain which is separated and screened from tissues of torreya grandis of Taxaceae and is used for producing paclitaxel, and the endophyte strain is classified, identified and named as aspergillus flavus BP6T2, and has a preservation number of CCTCC NO: M2016318. The strain has the activity of resisting staphylococcus aureus and escherichia coli, the content of an extract in a fermentation liquid can reach 93mg/L, wherein the relative percentage content of paclitaxel is 8.4%, the yield is 7.81mg/L, and is higher than the yield disclosed in the prior art; the CCK-8 reagent method is adopted to prove that the extract of the fermentation liquor of the aspergillus flavus BP6T2 has the inhibition effect on Hela tumor cells, and the inhibition rate can reach 66.20%.

Description

Aspergillus flavus BP6T2 for producing paclitaxel and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to aspergillus flavus BP6T2 separated from torreya grandis, a strain has the characteristic of high taxol yield, and application of the aspergillus flavus BP6T2 in treating tumor cells.

Background

Paclitaxel (Paclitaxel) named Paclitaxel, viologen, tertin, molecular weight 853.92, molecular formula C₄₇H₅₁NO₁₄(FIG. 1), inhibitors of mitosis in cells, are used in cancer therapy (Wani MC, Taylor HL, Wall ME, et al, plant inhibitor agents, VI, the isolation and Structure of taxol, a novel anti-inflammatory and anti-oxidant agent from Taxus brevifolia [ J].J Am Chem Soc,1971,93:2325～2327.)。

paclitaxel and its analogues are considered as high-efficiency, broad-spectrum, low-toxicity anticancer drugs, especially the best drugs for treating ovarian cancer, uterine cancer and breast cancer at present, and have certain curative effects on lung cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma and cerebroma. It is a highly potent cytotoxin and has a unique anti-cancer mechanism compared to other chemically synthesized anti-tumor or plant anti-cancer drugs already on the market. It binds specifically to the vascular bundle proteins, especially beta-tubulin, of dividing cells such as cancer cells, promotes tubulin polymerization, inhibits depolymerization, maintains tubulin stability, stops tumor cells at G2 and M, inhibits cell mitosis, and thus prevents proliferation of cancer cells, thereby gradually reducing tumor volume, rather than directly killing cancer cells. In tumor cells, paclitaxel induces the expression of pro-apoptotic genes, such as Bax, p53 and p21, C-Mos, TNF- α, etc., to induce apoptosis. Paclitaxel has also been found to enhance tyrosine kinase phosphorylation, activate apoptotic signal transduction pathways, and promote apoptosis. Paclitaxel can also regulate immunity, promote secretion and release of IL-1, IFN-alpha, IFN-beta, INF-alpha, etc., and kill or inhibit tumor cells.

Paclitaxel was first discovered in 1967 at the national cancer institute, and by the end of the fifties of the last century, a vast program was developed by the national cancer institute for screening anticancer actives from whole american plants. With the help of this project, Monroe e.wall and Mansukh c.wani extracted crude taxol extract from the bark of Taxus pacifica (Taxus breveifolia) in 1963. In 1966, paclitaxel was purified and its structure was determined. It was subsequently demonstrated to have higher activity against B16 melanoma. In 1979, Schiff and Horwitz demonstrated that paclitaxel specifically binds to dividing cells such as cancer cells. By 29.12.1992, the United states Food and Drug Administration (FDA) approved paclitaxel, marketed under the trade name Taxol, by Bristol-Myers-Squibb (BMS), for the treatment of ovarian and breast cancer.

however, the taxol content in the taxus and other plants is very low, and is only 0.003-0.069%. The amount of paclitaxel required for treating 1 cancer patient is obtained by cutting off 3 yew trees growing for 60 years or bark of 1 thousand yew. For each 1kg of paclitaxel extracted, approximately 2000 centuries of big trees were felled. And the natural resources of the yew and the east African Japanese pine grow very slowly, the number is limited, and the requirements of human beings are difficult to meet, so the price is very expensive. Even if all natural resources are harvested, the short-term requirements can only be met, and irreparable losses are caused to forests, human living environments and biodiversity. Therefore, the guarantee of the paclitaxel raw material becomes a key factor for the success of the medicine to market.

In order to solve the problems of medicine source shortage and ecological destruction caused by slow growth and resource shortage of some medicinal plants, people have been trying to produce taxol by other methods, such as chemical synthesis, semi-chemical synthesis, cell tissue culture, etc., which are mainly characterized as follows:

(1) The method for chemically synthesizing taxol completely succeeds, but the whole process is very expensive and has low yield, so that the method is not an ideal way.

(2) Semi-synthesis, while effective, is also difficult to isolate and purify semi-synthetic precursors from plant tissues.

(3) Plant tissue culture requires simpler steps, but paclitaxel yields are lower and yield is unstable.

The isolation of endophytic fungi producing paclitaxel or analogues is a new way to effectively solve the problem of paclitaxel resources developed in recent years. The method for producing paclitaxel in large amount by microbial fermentation is expected to improve the current situations of high price and short supply and demand of paclitaxel. Even if the yield is equal to or slightly lower than the content of the taxol in the plant, the fermentation scale can be artificially controlled due to the short fermentation period (10-15 days) of the fungi, and the production capacity of the plant material is greatly superior to that of the plant material. Therefore, the plant endophytic fungi capable of producing the paclitaxel is searched, the paclitaxel is produced in large quantity by a microbial fermentation method, and the current situations that the paclitaxel is high in price and is short in supply and demand are expected to be improved.

Endophytes are widely present in almost every plant and, during long-term evolution, form a harmonious ecological relationship with the plant host. The host provides a relatively stable environment for the bacteria, and the endophytes endow the host with antibacterial, antiviral, drought-resistant and insect-resistant properties. Through long-term co-evolution, it is possible that the same or similar pharmaceutically active substances as the host can be produced also in endophytes of certain medicinal plants. Thus, many medicinal plant endophytic fungi studies have received much attention, including isolation studies of plant endophytic fungi producing taxol or the like.

In 1993, Stierle et al isolated a new endosymbiotic fungus, Taxus andersonii (Taxomyces andreanae), from the phloem of Taxus brevifolia for the first time, could produce paclitaxel and taxane compounds in semi-synthetic culture solution, indicating that some endophytic fungi have the ability to synthesize the same or similar active ingredients as the host plant. Thereafter, various researchers in various countries have developed the work of separating taxol-producing endophytic fungi. Li et al isolated taxol-producing endophytes (Pestalotiopsismicrospora) from both the sub-bark phloem and xylem of Calipea balifera (Taxodiumdistichum). Strobel et al isolated a taxol-producing endophytic fungus Pestalotiopsis microspora (Pestalotiopsis microspora) from Taxus cuspidata (T. wallachiana), the culture broth of which can produce taxol in an amount of 50. mu.g/L; thereafter, the strain of Deerhorn (Seimatolerum nepalense) was isolated, and the amount of taxol produced in the cultured fungal solution was 62-80 ng/L. This indicates that the taxol-producing endophytic fungi exist not only in Taxus plants but also in some non-Taxus plants, further expanding the screening range of the taxol-producing endophytic fungi.

Chinese scholars also obtain certain achievements in the research of separating taxol-producing endophytic fungi. In 1994, qiudou et al isolated from the bark of taxus yunnanensis an endophytic fungus YFI with a morphology similar to that of taxus andersoni found by stiierle, and by TLC analysis, it was shown that paclitaxel was also produced, but with a very low biosynthetic yield. An endophytic fungus, namely nodulisporium arborescens (nodulisporium sp.) is found from taxus cuspidata (T. cuspidata) in Zhoutongpo and the like, can generate paclitaxel, is a new record genus in China, and has the yield of 51.06-125.7 mu g/L. The plant is 9 endophytes separated from the trunk, lateral branch bark and subcutaneous part of taxus chinensis (T.chinensis isvar mairei), 6 of which can secrete taxane compounds and are respectively identified to belong to Cephalosporium sp, streptomyces sp and aseptic (Mycelia sterila). The substituent culture is also separated from Chinese taxus chinensis var mairei to obtain a taxol-producing endophyte which is identified as Fusarium (Fusarium mairei) and has the yield of about 20 mu g/L.

Because the content of taxol or analogues produced by wild-type fungi is generally low, the current research mainly focuses on the following aspects in order to improve the yield: for example, the conventional mutagenesis was used to improve the strain (Zhoudong slope, Pingxiangxiang, Sunweiqiu, etc.. Studies on isolation of taxol-producing bacteria [ J ]. J. Microbiol., 2001, 21 (1): 18-21.); utilizing protoplast to mutate and improve strains (Zhao Kai, Pingxiang, Mali, etc., protoplast mutagenesis breeding of paclitaxel high-producing strains and initial detection of genetic variation thereof, microbiological report, 2005,45(3), 355-358.); constructing an engineering strain improved strain by using a genetic engineering technology; optimizing culture medium and fermentation conditions, and the like; the key issue for applying these taxol or analog producing fungi to production is how much it is produced. However, the yield of the producing bacteria found at present does not meet the requirement of industrial production.

To date, taxol-producing endophytic fungal strains have been isolated mainly from plants of the genus Taxus, an endangered species, while other plants of the genus Taxus, the family Taxaceae, have been ignored. Research shows that taxanes are found in plants in the genus Torreya of the family Taxaceae, but the content of the taxanes is extremely low, the content of the taxol is lower than 0.003%, and the medicinal development value is not large (Yi guan Mei, Qiu Ying spring Torreya tree research status and development [ J ] resources and environment, 2013, 29 (8): 844-848). The strain which is separated from Cephalotaxus hainanensis Li by Liuyan and the like and produces secondary metabolites, and Cephalotaxus hainanensis endophytic fungi CH1307 identification and antitumor activity research of the compounds (Liuyan and the like) which are mainly produced and possibly have antitumor effect [ D ]. Hainan university, 2012.Jianchujie.Hainan endophytic fungi antitumor activity strain screening [ D ]. Hainan university, 2014.)

The invention separates endophytic fungi from Torreya grandis (Taxaceae, Torreya fargesii) tissues in geological forest park of Shennongjia, screens out the endophytic fungi of plants with antibacterial activity through an antibacterial test; then, screening out strains which produce secondary metabolites of paclitaxel or analogues through high performance liquid chromatography analysis and anti-tumor activity analysis; and qualitatively detecting by using a liquid chromatograph-mass spectrometer, and presuming that the taxane compound generated by the strain is paclitaxel.

The method for producing paclitaxel in large amount by microbial fermentation is expected to improve the current situations of high price and short supply and demand of paclitaxel. Even if the yield is equal to or slightly lower than the content of the taxol in the plant, the fermentation scale can be artificially controlled due to the short fermentation period (10-15 days) of the fungi, and the production capacity of the plant material is greatly superior to that of the plant material.

Disclosure of Invention

The invention aims to provide an endophyte Aspergillus flavus BP6T2 separated from torreya grandis, which has the function of resisting staphylococcus aureus (G)⁺) And Escherichia coli (G)^-) Has high activity and high paclitaxel yield.

The invention also aims to provide the application of Aspergillus flavus BP6T2 in preparing the medicine for treating tumor cells, and experiments prove that the Aspergillus flavus BP6T2 fermentation broth extract has obvious inhibition effect on Hela tumor cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

The method for separating and screening Aspergillus flavus BP6T2 comprises the following steps:

1. Respectively washing roots, stems, leaves and barks of torreya grandis with sterile water, drying by suction, sequentially disinfecting with 75% (v/v) alcohol for 5min and 2% (v/v) hypochlorous acid for 8min, then washing with sterile water, cutting the collected roots, stems and barks into small segments with a sterile knife, taking materials of each part, placing the materials in a PDA solid culture medium plate, placing the plate in a thermostat at 28 ℃ for culture, and when fine hyphae grow from the edges of the section of an inoculum in the plate, picking the hyphae and inoculating the hyphae to a new PDA solid plate for purification until the hyphae are a pure culture;

2. inoculating the purified fungi to a PDA (personal digital assistant) plate, culturing at 28 ℃, and recording colony size, color, hypha compactness, colony edge uniformity, colony growth speed, pigment production and other colony morphologies;

3. The antibacterial activity of the endophytic fungi is measured by an agar block method: activating test bacteria (Staphylococcus aureus and Escherichia coli) with LB culture medium, respectively coating on the surface of LB solid culture medium, inoculating endophyte with agar culture medium in each plate, culturing at 37 deg.C for 1-2 days, observing the inhibition zone around the agar block to obtain a strain with anti-Staphylococcus aureus (G)⁺) And Escherichia coli (G)^-) Active strain, denoted BP6T 2;

4. observing the colony color of the strain in grass green through the plate culture characteristic, wherein the colony texture is in a velvet shape, observing that the head of a conidium of the strain is loose and radial under a microscope, the top capsule is approximately spherical, and a single-layer peduncle and a double-layer peduncle exist at the same time, so that the strain is identified as Aspergillus flavus (BP 6T 2);

5. Activating the strain BP6T2 in a PDA liquid culture medium at 180rpm and 28 ℃ for 3 days to serve as a seed solution, inoculating the seed solution into the PDA liquid culture medium at an inoculation amount of 5% (v/v), and culturing at 180rpm and 28 ℃ for 7-10 days to obtain a fermentation liquid;

6. After fermentation, collecting filtrate, adding 1/3 volume of ethyl acetate into the filtrate, performing countercurrent extraction for 3 times, collecting upper layer organic phase, and removing organic solvent in a rotary evaporator to obtain fat-soluble extract with average content of 93 mg/L;

7. High Performance Liquid Chromatography (HPLC) finds that the extract contains components with retention time substantially consistent with that of a paclitaxel standard product, the retention time is 13.208min, and is similar to that of the standard product, which indicates that the extract contains paclitaxel or analogues;

8. The structure of taxol analogs in the metabolic extract is determined by a liquid chromatography-mass spectrometer (HPLC-MS), and the parent ion Da is 876.300, the daughter ions Da are 307.900, 591.200 and 531.200 respectively, the source voltage is 150, the collision energy is 36, 36 and 40 respectively, and the structure is completely consistent with a taxol standard product of a taxane compound, so that the metabolic extract generated by Aspergillus flavus BP6T2 is judged to be taxol.

the endophyte Aspergillus flavus BP6T2 separated from the torreya grandis tree tissue is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address: wuhan university, preservation date: 2016, 6/8/month, accession number: CCTCC NO. M2016318, which is characterized in that it has activity against staphylococcus aureus and Escherichia coli, and the content of paclitaxel in fermentation liquid reaches 7.81 mg/L.

the application of the aspergillus flavus BP6T2 in preparing the medicine for treating the tumor cells comprises the following steps: the CCK-8 method is used for determining the inhibition effect of the fermentation liquor extract of the aspergillus flavus BP6T2 on Hela cells, and the result shows that the inhibition effect on cell proliferation is enhanced along with the increase of the concentration of the extract, wherein the inhibition effect is strongest when the extract is treated at the concentration of 3.104 mu g/mL, and the inhibition rate is 66.20%, so that the anti-Hela cell substance generated by the aspergillus flavus BP6T2 is presumed to be paclitaxel.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. Heretofore, taxol-producing endophytic fungal strains have been isolated mainly from plants of the genus Taxus, which is an endangered species, and from plants of Japanese cypress, and it has been studied to isolate taxol-producing endophytes also from Japanese cypress. The invention also separates the taxol-producing endophytic fungi from the tissues of plants except taxus, such as torreya grandis (taxaceae, torreya), further expands the resource range for obtaining the taxol or analogue-producing strains, and is beneficial to more widely utilizing microbial resources.

2. The research is to obtain Aspergillus flavus for producing taxol compounds for the first time, the endophyte Aspergillus flavus BP6T2 can produce taxol analogs very stably, the extract of the fermentation broth can reach 93mg/L, the relative percentage content of taxol is 10.5%, the yield of taxol is 7.81mg/L, which is higher than the yield disclosed in the prior art (Qiude et al. separation of Taxus yunnanensis endophyte, journal of bacteriology 1994(4): 314-316; Li J, et al. Ambuic acid, ahighly functionalized Cyclohexene with anti inflammatory behaviour strain, and Monochaetia sp. Phytochemistry,2001,56(5): 468-468; Zhongpo Boyuki, Pingqiu Wen et al. Yew. paclitaxel producing bacteria separation research [ J ]. journal of microbiology, 2. 1, 18, eight, microbiology report, 2005,45 (3): 355-358.).

3. A CCK-8 reagent method is adopted to test the inhibition effect of the metabolic extract in the Aspergillus flavus BP6T2 fermentation liquor on Hela tumor cells, and the test shows that the inhibition effect on the proliferation of the Hela tumor cells is enhanced along with the increase of the concentration of the extract, the inhibition rate is 39.90% when the concentration of the extract is 0.0310 mu g/mL, the inhibition effect is strongest when the Hela cells are treated by 3.104 mu g/mL, and the inhibition rate is 66.20%.

4. the taxol is produced by fermenting the endophyte of the plant of the invention, and can be produced in a short time and on a large scale without being limited by resources, environment, conditions, equipment and the like.

Drawings

FIG. 1 shows the chemical structure of paclitaxel.

FIG. 2 is a photograph of a lawn of Aspergillus flavus BP6T2, the colony color is grass green, and the colony texture is velvet.

FIG. 3 is a photomicrograph of Aspergillus flavus BP6T2, wherein the conidial heads are loose and radial, the apical capsule is nearly spherical, and the monolayer and the bilayer of the small peduncle exist at the same time.

FIG. 4 is a high performance liquid chromatogram of a paclitaxel standard, with the peak indicated by the arrow being paclitaxel.

FIG. 5 is a high performance liquid chromatogram of a metabolic extract of Aspergillus flavus BP6T2, with the peak indicated by the arrow being a taxol analog.

Detailed Description

example 1: separation and purification of endophyte of torreya grandis

1. Pretreatment of torreya grandis tree material

Collecting roots, stems, leaves and barks of torreya grandis in a geological forest park of Shennongjia nationality of Hubei province, washing with sterile water, and drying with absorbent paper; sterilizing with 75% (v/v) ethanol for 5min and 2% (v/v) hypochlorous acid for 8min, washing with sterile water, cutting the collected root, stem and bark into 2-3cm pieces, and cutting the leaf into sections.

2. Separation of endophyte of torreya grandis

Placing the materials of each pretreated part in a PDA solid culture medium plate, and culturing at 28 ℃; PDA culture medium: 200g of potato, 20g of glucose and KH₂PO₄ 1.0g，MgSO₄·7H₂0.5g of O, 1000mL of water, and agar (15g/L) as a PDA solid medium.

3. Purification of endophyte of torreya grandis

When fine hyphae grow on the edge of the section of the inoculum in the plate, the hyphae are picked by an inoculating needle and planted on a newly prepared PDA solid plate, and the PDA culture medium is repeatedly purified until a pure culture is obtained.

example 2: endophytic fungus bacteriostatic activity detection method

1. Activating test bacteria (staphylococcus aureus and escherichia coli) by using an LB liquid culture medium, and respectively coating the activated test bacteria on the surface of an LB solid culture medium;

2. the purified endophyte agar medium blocks obtained in example 1 were cut out, placed in plates of staphylococcus aureus and escherichia coli, respectively, and cultured at 37 ℃ for 1-2 days, and the zone of inhibition around the agar blocks was observed.

Through the method, a strain with staphylococcus aureus (G) resistance is obtained through screening⁺) And Escherichia coli (G)^-) The active strain was designated as BP6T 2.

Example 3: identification of endophytic fungus BP6T2

Observed by the plate culture characteristics, the bacterial colony of the strain is grass green, and the bacterial colony is velvet (figure 2); the heads of conidiospores are loose and radial, the apical sacs are nearly spherical, and the single-layer and double-layer layers of small peduncles exist simultaneously when observed under a microscope (figure 3), and the conidiospores can be preliminarily identified as Aspergillus flavus (Aspergillus flavus) BP6T2 according to the fifth aspergilli and related sexual types of the Chinese funguses (edited by Zizu Hokko, and the fifth aspergilli and related sexual types thereof [ M ]. Beijing: scientific Press, 1997, 5-10.).

Example 4: extraction of fat-soluble substances in aspergillus flavus BP6T2 fermentation liquor

1. Activating the Aspergillus flavus BP6T2 obtained in example 2 in a PDA liquid culture medium (activation condition: 28 ℃, 180rpm, culture for 5-7 days) to obtain a seed solution;

2. inoculating into PDA liquid culture medium according to the inoculation amount of 5% (v/v), culturing at 28 deg.C and 180rpm for 7-10 days;

3. Filtering and collecting filtrate, adding 1/3 volume of ethyl acetate into the filtrate, carrying out countercurrent extraction for 3 times, and collecting an upper-layer organic phase;

4. Removing organic solvent from the organic phase at 35 deg.C in a rotary evaporator, dissolving the substance adhered to the wall with methanol, volatilizing, drying, and determining the content of liposoluble extract of the fermentation liquid by gravimetric method; the fat-soluble extract content in the Aspergillus flavus BP6T2 fermentation liquor is determined to be about 93mg/L on average.

Example 5: high Performance Liquid Chromatography (HPLC) quantitative detection of aflatoxin analogs of Aspergillus flavus BP6T2 extract paclitaxel

1. Fat-soluble extract and mobile phase treatment obtained in example 4: centrifuging the concentrated product obtained in example 4 at 13200r/min for 2min, sucking the supernatant, filtering through a filter membrane with the pore diameter of 0.22 μm to remove impurities, and then ultrasonically exhausting the gas together with a prepared mobile phase (methanol/water is 65/35, V/V) for 3min and 10-15 min respectively;

2. The chromatographic conditions are as follows: column chromatography, ODS (C18) column 4.6X 250mm, 5 nm; column temperature, normal temperature; sample and mobile phase, methanol/water 65/35 (V/V); flow rate, 1.0 mL/min; sample size, 20 μ L; ultraviolet detection wavelength is 227 nm;

3. the Rt (retention time) of the paclitaxel standard (Sigam) is measured to be 13-14 min (figure 4);

4. the components found in the extract sample prepared in example 4 substantially matched the retention time of the paclitaxel standard, the peak time was 13.208min (fig. 5), and the paclitaxel analogue can be preliminarily identified;

5. Quantitative analysis: the relative peak area of taxol analogue in the Aspergillus flavus BP6T2 metabolic extract was determined to be 2491.2 (FIG. 5) using HPLC Wufeng liquid chromatography workstation software according to chromatogram, and the relative percentage content was about 8.4% on average.

example 6: determination of Aspergillus flavus BP6T2 fermentation liquor containing paclitaxel by liquid chromatography-mass spectrometry (HPLC-MS)

1. Liquid chromatography instrument model: shimadzu LC-30A UPLC

(1) Sample and mobile phase treatment: centrifuging the fat-soluble extract prepared in example 4 at 13200r/min for 2min, sucking supernatant, filtering through a filter membrane with the aperture of 0.22 mu m to remove impurities, and then ultrasonically exhausting the mixture together with a prepared mobile phase (acetonitrile/water is 65/35, V/V) for 3min and 10-15 min respectively;

(2) The chromatographic conditions are as follows: column chromatography, ODS (C18) column 4.6X 250mm, 5 nm; column temperature, normal temperature; sample and mobile phase, methanol/water 65/35 (V/V); flow rate, 1.0 mL/min; sample size, 20 μ L; ultraviolet detection wavelength is 227 nm;

2. mass spectrometer model AB sciex 4500QQQ (triple quadrupole liquid mass spectrometer)

The structure of the taxol analogue in the metabolic extract is measured by a liquid chromatograph-mass spectrometer (table 1), which shows that the parent ion Da is 876.300, the daughter ions Da are 307.900, 591.200 and 531.200 respectively, the source voltage is 150, the collision energy is 36, 36 and 40 respectively, the taxol analogue is completely consistent with the taxol standard of a taxane compound, and the taxol analogue in the aspergillus flavus BP6T2 fermentation liquor can be qualitatively identified as taxol.

TABLE 1 Mass Spectrometry parameters for paclitaxel standards and paclitaxel analogs in Aspergillus flavus BP6T2 fermentation broths

Q1Mass(Da)	Q3Mass(Da)	DP	CE	Dwell(msec)
					876.300	307.900	150	36	50
876.300	591.200	150	36	50
					876.300	531.200	150	40	50

Example 7: inhibition effect of aspergillus flavus BP6T2 fermentation liquor extract on Hela tumor cells

1. Spreading Hela cells (Hualianke biotechnology, Inc.) in logarithmic growth phase, and culturing the cells at 100 μ L/well (1-5 × 10)³Individual cells/well) were seeded in 96-well plates at 37 ℃ in 5% CO₂culturing for 24h in an incubator;

2. The extract of Aspergillus flavus BP6T2 fermentation broth containing paclitaxel of example 4 was prepared at different concentrations (Table 2), and 10. mu.L of the extract was added to the sample wells at 37 ℃ and 5% CO₂culturing for 24h in an incubator;

3. taking out the culture plate, removing the supernatant, adding 90 μ L of fresh culture solution (DMEM culture solution containing 10% fetal calf serum) and CCK-810 μ L into each well, and culturing for 4 hr under the same condition;

4. Reading A at 450nm with microplate reader₄₅₀Values, cell growth inhibition rate was calculated: hela cell Inhibition Ratio (IR) ═ control well a value-sample well a value)/control well a value × 100%.

The experiment proves that the Aspergillus flavus BP6T2 fermentation broth extract has obvious inhibition effect on tumor cells (Table 2), and the inhibition effect on cell proliferation is enhanced along with the increase of the concentration of the extract, wherein the inhibition effect is strongest when the extract is processed at the concentration of 3.104 mu g/mL, and the inhibition rate is 66.20%. At the lowest concentration of 0.0310. mu.g/mL, the inhibition rate was 39.90%, so the anti-Hela cell substance produced by Aspergillus flavus BP6T2 was presumed to be paclitaxel.

TABLE 2 inhibition of Hela cells by Aspergillus flavus BP6T2 fermentation broth extract

Sample concentration (μ g/mL)	A₄₅₀value of	Inhibition ratio (%)
			Blank control	1.153	----
0.0310	0.693	39.9％
			0.3104	0.615	46.7％
3.104	0.39	66.2％

Claims

1. A taxol-producing endophyte is characterized in that: the endophyte is Aspergillus flavus (A. flavus) ((A. flavus))Aspergillus flavus) BP6T2, the preservation number is CCTCC NO: m2016318.

2. The use of the endophyte of claim 1 in the preparation of a medicament for the treatment of a tumor cell, wherein the tumor cell is a cervical cancer cell.