Atractylodes macrocephala endophytic fungus and application thereof

Abstract

The invention discloses a bighead atractylodes rhizome endophytic fungus and application thereof, and belongs to the field of plant microbial engineering. The endophytic fungi are separated from a compositae rhizoma atractylodis plant bighead atractylodes rhizome living body by adopting an endophytic fungi separation and purification technology, belong to hypocrea, arbuscular and fusarium fungi, are classified and named as fusarium sporotrichioides AM608, and have a strain preservation number of CGMCC (China General Microbiological Culture Collection Center) No.41083. The endophytic fungi can be used for preparing an endophytic fungi strain. The invention also discloses an application of the endophytic fungus: a fermentation product of the endophytic fungus has a remarkable growth inhibition effect on cancer cells A549, KB and MCF-7, the inhibition rates are respectively 69.60%, 83.22% and 73.39%, and the IC50 values are respectively 5.8 mu g / mL, 2.7 mu g / mL and 3.6 mu g / mL. The endophytic fungus provided by the invention has a remarkable inhibition effect on the growth of cancer cells, and a wide application prospect is brought to the field of research and development of antitumor drugs.

Description

Technical Field

[0001] The invention relates to the field of microbial engineering, in particular to an endophytic fungus of Atractylodes macrocephala and application thereof in inhibiting the growth of cancer cells. Background Art

[0002] Atractylodes macrcephalakoidz is a perennial herbaceous plant of the Compositae family. Its dried rhizomes can be used as medicine and are mainly distributed in Zhejiang, Hunan, Sichuan, Anhui, etc. Atractylodes was first recorded in the Shennong's Herbal Classic in the Han Dynasty and was called "Shu" together with Atractylodes lancea and was listed as a top grade product. Atractylodes macrocephala is one of the famous Chinese medicinal materials "Zhejiang Eight Flavors". As a Chinese medicine for strengthening the spleen and replenishing qi, it is mainly used for spleen deficiency, poor appetite, phlegm and fluid, palpitations, edema, spontaneous sweating, abdominal distension and diarrhea, fetal movement disorder, etc. Most studies have shown that the main chemical components of Atractylodes macrocephala include volatile oils, polysaccharides and lactones. Atractylodes macrocephala has multiple pharmacological effects such as regulating gastrointestinal function, anti-aging, anti-cancer, improving immunity, improving and treating diabetes, protecting the liver, improving memory, regulating lipid metabolism, lowering blood sugar, anti-platelet, antibacterial, anti-inflammatory and analgesic, adjusting water metabolism, immune regulation, etc. It is now also used in clinical practice as an auxiliary treatment for chronic wasting diseases such as hypertension, so as to reduce the adverse reactions caused by western medicine and improve the quality of life of patients.

[0003] Cancer is an important disease that affects human health and has become the second leading cause of death in the world. At present, the prevention and treatment of cancer has become a very important research topic. The relevant national departments have proposed to promote cancer screening and early diagnosis and early treatment strategies and improve the level of cancer diagnosis and treatment as the main measures to prevent and treat cancer. Whether "early detection and early treatment" can be achieved is of great significance to the prevention and treatment of cancer. In terms of cancer treatment, the main methods currently include surgical resection, radiotherapy and chemotherapy, but they all have their own defects to a greater or lesser extent. Therefore, early detection of cancer and effective treatment of cancer are of great significance to conquering cancer. However, a series of anti-tumor drugs have many side effects on human health, and even many anti-tumor drugs cannot effectively treat certain diseases. Instead, they will produce drug resistance or cause tumor deterioration. Therefore, it is urgent to find newer and more effective anti-tumor drugs in nature.

[0004] Endophytes refer to fungi that live inside cells or in the intercellular spaces of various organs and tissues of healthy plants at certain or all stages of their life cycle, and the infected host plants do not show obvious external disease symptoms. The earliest widely accepted concept of endophytes was proposed by Petrini, which refers to those fungi that live in plant tissues at a certain period of their life cycle and do not cause obvious disease symptoms to plant tissues. This definition includes those saprophytes that live on the surface at a certain stage of their life cycle, pathogens and mycorrhizal fungi that do not harm the host. In 1898, Vogl et al. isolated the world's first endophytic fungus from ryegrass (Loffum temulentum L.). Since then, plant endophytic fungi have gradually received widespread attention as a new microbial resource and opened the prelude to the study of endophytic fungi. Since endophytic fungi live in plant tissues or plant organs without external symptoms of infection, its role and research have not attracted people's attention. Endophytic fungi have potential beneficial properties for humans. All plant endophytic fungi extracts have antioxidant, anticancer and antibacterial activities, and show at least one biological activity that is the same or similar to the host. For example, in 1993, Stierle et al. isolated an endophytic fungus Taxomyces andreanae that can produce paclitaxel from the bark phloem of Taxus brevifolia. Since then, the study of endophytic fungi has gradually become a hot topic for scholars at home and abroad. Some scholars have isolated an endophytic fungus mainly composed of Paecilomyces and Aspergillus from the bark of mangrove plants, and its fermentation broth has obvious inhibitory activity against human oral epithelial cancer cells. Therefore, endophytic fungi have been regarded as an important source of active secondary metabolites.

[0005] However, there are no reports on the application of endophytic fungi in Atractylodes macrocephala to inhibit the growth of cancer cells. Summary of the invention

[0006] The purpose of the present invention is to provide an endophytic fungus of Atractylodes macrocephala and its application in inhibiting the growth of cancer cells in view of the deficiencies of the prior art.

[0007] In the first aspect of the present invention, a plant endophytic fungus - Atractylodes macrocephala endophytic fungus is provided, belonging to the Hypocreales, the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the plant of the Atractylodes macrocephala endophytic fungus, which is obtained by separation and purification technology of endophytic fungus, and is identified as Fusarium sporotrichioides AM608 by endophytic fungus ITS molecule. The strain has been deposited, the strain deposit number is CGMCC No.41083, the deposit date is January 23, 2024, the depository is the China General Microbiological Culture Collection Management Center (CGMCC), the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the zip code is 100101.

[0008] The solid culture characteristics of the endophytic fungi of the present invention are:

[0009] Cultured in the dark at 28℃ on potato dextrose agar (PDA), the colony is white, the inner hyphae are dense, the middle part is sparse, dry, and thin hyphae can be seen. Concentric rings can be vaguely seen. The hyphae grow vigorously, the back center is white, the middle part is dark red, and the Figure 1 .

[0010] The morphological characteristics of the endophytic fungus described in the present invention under a microscope are: the mycelium is transparent, thin-walled, smooth on the surface, slightly twisted, branched, with visible inclusions, and oblong spores scattered near some mycelium. Figure 2 .

[0011] The electrophoresis diagram of the endophytic fungus ITS amplification of the present invention is shown in Figure 3 , the ITS molecular sequence is shown in SEQ ID NO.1.

[0012] The sequencing results were compared with the gene sequences in the NCBI website database by Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The DNA sequences of Fusarium sporotrichioides (MN452651.1), Fusarium sporotrichioides (MN452649.1), and Fusarium sporotrichioides (MN452647.1) were the most similar, with similarities of 100.00%. ClustalX1.81 was used to align the MEGA7.0 software, and the neighbor-joining method was used to construct the phylogenetic tree. Figure 4 .

[0013] The second aspect of the present invention provides the use of the above-mentioned endophytic bacteria fermentation product in the preparation of anticancer drugs, which inhibit the growth of tumor cells.

[0014] In the above application, the AM608 fungal fermentation product has a significant inhibitory effect on cancer cells. The fungal fermentation product is obtained by oscillating fermentation in PDB liquid culture medium. The formula of PDB liquid culture medium is: 200g potatoes, 20g glucose, 1000ml distilled water, natural pH.

[0015] The above application is mainly achieved through the following technical scheme: the endophytic fungus fermentation product is prepared according to the following steps:

[0016] (1) Inoculate the mother mycelium of AM608 from the cryopreserved tube into PDA medium and culture in a fungal incubator at 26°C and 65% humidity for 7 days;

[0017] (2) After the mycelium has grown all over the plate, punch holes to make bacterial cakes, inoculate 100 bacterial cakes into a conical flask containing 1.2 L of sterile PDB medium, and ferment and culture at 26°C and 150 rpm for 7 days;

[0018] (3) After the culture is completed, the mycelium and the bacterial liquid are filtered and processed separately. The mycelium is processed according to the following steps: Place it in a 50°C oven to dry to constant weight, weigh it, grind it into powder, and extract it by ultrasonic (25°C) for 1 hour with a ratio of methanol (mL): mycelium (g) = 20:1, filter it, and take the filtrate; The bacterial liquid is extracted and separated three times with a ratio of ethyl acetate: bacterial liquid = 1:1, and 750mL of bacterial liquid is used for extraction each time. The extract is taken and rotary evaporated (45°C) to obtain an extract;

[0019] (4) The mycelium filtrate was poured into the bacterial extract for concentration, rotary evaporated to a paste, and an appropriate amount of methanol was added for ultrasonic dissolution. The flask was transferred to a 100 mL round-bottom flask, and further rotary evaporated to an extract. The flask was sealed and stored in a refrigerator to obtain the AM608 fermentation product.

[0020] Furthermore, the anticancer drug is a drug for lung cancer, human oral epidermoid carcinoma or breast cancer.

[0021] The advantages of the present invention are:

[0022] The endophytic fungus Fusarium sporotrichioides AM608 of Atractylodes macrocephala described in the present invention can effectively inhibit the growth of cancer cells through the fermentation product of PDB liquid culture medium. The endophytic fungus of the present invention has a significant inhibitory effect on the growth of cancer cells, and provides a reference for the development of engineering bacteria for anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0024] Figure 1 :The colony morphology of Fusarium sporotrichioides AM608 on PDA solid medium;

[0025] Figure 2 :Mycelial morphology of Fusarium sporotrichioides AM608 under optical microscope;

[0026] Figure 3 :Gel electrophoresis of the ITS sequence of AM608 (Fusarium sporotrichioides) amplified with primers ITS5 and ITS4;

[0027] Figure 4 : Phylogenetic tree of Fusarium sporotrichioides AM608 based on ITS (NJ method);

[0028] Figure 5 : Fermentation product liquid prepared by Fusarium sporotrichioides AM608;

[0029] Figure 6 : The results of the inhibitory effect of Fusarium sporotrichioides AM608 on cancer cell growth (A: OD value of cancer cell A549 growth; B: OD value of cancer cell KB growth; C: OD value of cancer cell MCF-7 growth; D: IC value of AM608 on cancer cells A549, KB, and MCF-7 50 value). DETAILED DESCRIPTION

[0030] The endophytic fungus of the invention is a fungus separated from the living body of Atractylodes macrocephala produced in Pan'an, Zhejiang Province.

[0031] Embodiment 1:

[0032] The endophytic fungus is isolated and obtained according to the following steps: fresh Atractylodes tubers from Pan'an, Zhejiang Province are selected, first rinsed with natural water to remove surface impurities, and then rinsed with pure water. Weigh (1-1.5g) and then subjected to a three-step disinfection method. Under the aseptic conditions of a clean bench, the tubers are soaked in a 75vol% ethanol solution for 1min, then soaked in a 2.5vol% sodium hypochlorite solution for 3min, a 75vol% ethanol solution for 30s, and finally washed with sterile water 5 times, each time for 1min. After disinfection, take out the tubers, cut about 0.5cm×0.5cm in size, and inoculate them in a sterile isolation culture plate with penicillin sodium (50mg / L), with 4 tissue blocks per plate. Seal the plate with a sealing film, record the number, and culture it in a fungal incubator for 3-7d. Regularly observe the growth of fungal colonies, and screen out target single colonies according to their colony morphology and color. In a sterile clean bench, the tip of the target hyphae was picked and transferred to the freshly prepared PDA for re-cultivation, and the number was recorded. After the cultivation was completed, the appropriate colonies were observed and screened again, and the hyphae at the edge of the colonies were inoculated on a new plate. The screening, picking and culturing were repeated to purify the strains, and finally the endophytic fungus strain AM608 of the present invention was isolated from the tuber part. Figure 1 As shown, the colony is white, with dense hyphae inside and sparse and dry hyphae in the middle. Slender hyphae can be seen, and concentric rings can be vaguely seen. The hyphae grow vigorously, the center of the back is white, and the middle is dark red. Figure 1 Under the microscope, the mycelium is transparent, thin-walled, and smooth on the surface. The mycelium is slightly twisted and branched, and the contents are visible. Some mycelium has long oval spores scattered around it. Figure 2 , and stored at low temperature (4°C).

[0033] Take the AM608 strain cryopreservation tube, under sterile conditions, use an inoculation needle to pick up a small amount of mycelium and place it in a freshly prepared PDA medium for activation, and culture it at 28°C in the dark until mature, for ITS sequence amplification and molecular identification. Extract the strain DNA according to the modified CTAB method, and use primers ITS5 and ITS4 to amplify its ITS sequence by PCR. PCR reflects the cycle parameter amplification steps: 1. 95°C, 3min, initial denaturation; 2. 94°C, 40s, denaturation; 3. 52°C, 50s, annealing; 4. 72°C, 1min, extension; 5. Repeat 2-4 cycles 35 times; 6. 72°C, 10min, extension. The PCR product detected by agarose gel electrophoresis was sent to Shanghai Shenggong Company for sequence determination. The sequence measured by the PCR product was used as the target sequence, and the homologous sequence was searched in the GenBank database in NCBI. The reference sequence most similar to the target sequence was downloaded, and the phylogenetic analysis was performed using the neighbor-joining method (NJ) to determine the phylogenetic status of the strain to be identified, such as Figure 3 , 4The endophytic fungus strain of the present invention has a DNA sequence similarity of 100.00% with Fusarium sporotrichioides (MN452651.1), Fusarium sporotrichioides (MN452649.1), and Fusarium sporotrichioides (MN452647.1), and belongs to the Hypocreales, the Combretaceae, the Fusarium, and is classified and named as Fusarium sporotrichioides AM608 (Fusarium sporotrichioides). It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 23, 2024, and the culture collection number is CGMCC No. 41083.

[0034] Embodiment 2:

[0035] Inoculate the mother bacteria from the cryopreservation tube into the PDA medium and culture it in a fungal incubator. After the culture is completed, punch holes to inoculate 100 bacterial cakes into a conical flask (specification: 2L) containing 1.2L PDB. Place the 2L conical flask containing 100 bacterial cakes in a shaker for fermentation and culture. After the culture is completed, filter and separate the mycelium and the bacterial solution; the mycelium is first placed in a 50°C oven for drying, weighed, and then ground into powder with a grinder, and extracted with an ultrasonic (not exceeding 30°C) ratio of methanol (mL): mycelium (g) = 20:1 for 1h, and filtered to obtain the filtrate; extract and separate the liquid with a volume ratio of bacterial solution: ethyl acetate = 1:1, extract with 750mL bacterial solution each time, extract 3 times, and rotary evaporate (not exceeding 40°C); pour the filtrate in the mycelium into the bacterial solution, rotary evaporate again, add an appropriate amount of methanol and ultrasonic, transfer to a 100mL round-bottom flask, and rotary evaporate to an extract, such as Figure 5 As shown, seal and store in a 4°C refrigerator.

[0036] The CCK-8 method was used to detect the growth inhibition of cancer cells. A549, KB, and MCF-7 cells cultured to the logarithmic growth phase were treated with trypsin digestion and then centrifuged (1000 rpm, 5 min). The supernatant was discarded and single-cell suspension was made by pipetting. After cell counting and dilution, the suspension was inoculated into a 96-well plate, and 5×10 cells were cultured in each well. 3Each group of cells was set up with no less than 3 replicate wells, and incubated overnight in complete medium at 37°C and 5% CO2. The medium was discarded, and 200 μL / well of low serum medium (1640+5% FBS) with gradient drug concentration was added. The fermentation product concentration was 20 μg / mL. Paclitaxel (PXL) was used as the positive group, cells without drugs were used as the control group, and no cells and medium were added, and only reagents were added as the blank group. The cells were cultured in an incubator at 37°C and 5% CO2 for a preset time, and then the cell proliferation was determined using CCK-8 reagent. The original culture medium in each 96-well plate was aspirated, and 100 μL of low-serum culture medium (1640 + 5% FBS) containing 10 μL of CCK-8 reagent was added to each well using a dispenser; the 96-well culture plate was then placed in an incubator at 37°C and 5% CO2 for 2 h; the 96-well plate was taken out, and its optical density (OD) at 450 nm was measured using an ELISA reader. The results are as follows: Figure 6 As shown, the OD of cancer cells A549, KB, and MCF-7 control groups 450 The OD values ​​of the paclitaxel-treated group were 1.25±0.21, 1.43±0.30, and 1.09±0.28, respectively. 450 The OD values ​​of the AM608-treated group were 0.43±0.07, 0.37±0.04, and 0.28±0.05, respectively. 450 The results show that the fermentation products of the endophytic fungi of the present invention have good ability to inhibit the growth of tumor cells. The IC 50 IC value of cancer cell A549 50 The value is 5.8 μg / mL, and the IC 50 The value was 2.7 μg / mL, and the IC 50 The value is 3.6μg / mL.

[0037] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the invention, and these equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. An endophytic fungus of Atractylodes macrocephala, characterized in that The endophytic fungus of Atractylodes macrocephala is isolated from the living body of Atractylodes macrocephala, a plant of the genus Atractylodes in the family Asteraceae, belongs to the order Hypocreales, family Combretaceae, genus Fusarium, and is classified and named Fusarium sporotrichioides AM608. It was deposited in the China General Microbiological Culture Collection Center on January 23, 2024, and the culture collection number is CGMCC No.41083.

2. A use of the endophytic fungus of Atractylodes macrocephala according to claim 1, characterized in that: The fermentation product of the endophytic fungus of Atractylodes macrocephala in PDB liquid culture medium is used for preparing anticancer drugs.

3. The use according to claim 2, characterized in that: The anticancer drug is a drug for lung cancer, human oral epidermoid carcinoma or breast cancer.

4. The use according to claim 2, characterized in that: The anticancer drug inhibits the growth of cancer cells A549, KB and MCF-7.

5. The use according to claim 2, characterized in that: The fermentation product is prepared according to the following steps: The mycelium of the endophytic fungus of Atractylodes macrocephala is made into a bacterial cake and inoculated into a sterilized PDB medium, and fermented and cultured for 7 days at 26° C. and 150 rpm; After the cultivation is completed, the mycelium and the bacterial liquid are filtered and processed separately; the mycelium is dried and ground into powder, and ultrasonic extraction is performed for several hours with methanol:mycelium=20mL:1g, and the filtrate is taken; the bacterial liquid is extracted and separated several times with ethyl acetate: bacterial liquid=1:1 volume ratio, and the extract is taken and rotary evaporated to extract; The obtained filtrate is poured into the extract for concentration, rotary evaporated to a paste state, and dissolved by ultrasonic addition of methanol, and further rotary evaporated to an extract state to obtain the fermentation product.

6. The use according to claim 2, characterized in that: The formula of PDB medium is: 200 g potato, 20 g glucose, 1000 mL distilled water, pH natural.

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