A bacterium called Crystallomyces hypericus and its applications
By culturing and extracting Crystallomyces hypericus, an extract with anticancer activity was prepared, which solved the problem of the lack of effective anticancer drugs in the existing technology, especially with significant therapeutic effects on cervical cancer, pancreatic cancer and neurocarcinoma.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- KUNMING UNIVERSITY
- Filing Date
- 2026-02-11
- Publication Date
- 2026-07-14
AI Technical Summary
There is a lack of effective anticancer drugs in the current technology, especially for the treatment of cervical cancer, pancreatic cancer and neurocancer.
Crystallomyces hypericus was cultured in liquid potato and rice media, and its secondary metabolites were extracted using methanol and ethyl acetate to prepare an extract with anticancer activity.
The prepared extract showed significant inhibitory effects on human cervical cancer, pancreatic cancer, and neurocellular cancer cells, providing new raw materials for anticancer drugs.
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Figure CN121674225B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology and its application technology, specifically to a... Crystallomyces hypericus Bacteria and their applications. Background Technology
[0002] Cancer, a major disease posing a serious threat to human health worldwide, has long maintained high incidence and mortality rates. Statistics from the World Health Organization (WHO) clearly reveal the grim situation regarding cancer incidence and mortality. In recent years, the global incidence of cancer has continued to rise, severely impacting numerous countries and regions.
[0003] Fungi in special habitats, such as those growing in extreme environments, face unique survival pressures such as high temperature, low temperature, high pressure, high salt, and nutrient deficiency. In order to adapt to these environments, they may have evolved unique metabolic pathways and physiological mechanisms. Therefore, extracts prepared from these fungi may have special uses. Summary of the Invention
[0004] To address the problems existing in the prior art, one of the objectives of this invention is to provide a solution. Crystallomyces hypericus The bacterium (HJB-JST-2) was deposited on August 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42087. The suggested classification name is... Crystallomyces hypericus .
[0005] The second objective of this invention is to provide a method for... Crystallomyces hypericus Extracts prepared from bacteria.
[0006] Preferably, the method for preparing the extract is as follows:
[0007] (1) Crystallomyces hypericus The bacteria were cultured in liquid potato broth to obtain a seed culture solution.
[0008] (2) The seed culture solution is inoculated into rice culture medium for culture to obtain a culture medium with attached bacteria. The culture medium with attached bacteria is extracted by cold soaking with methanol (the amount of methanol is not special, just enough to completely submerge the solid). The solid and liquid are separated to obtain methanol extract. The methanol extract is concentrated to obtain methanol concentrate.
[0009] (3) Extracting the methanol concentrate with ethyl acetate to obtain the ethyl acetate layer extract is the extract.
[0010] Preferably, the number of times the methanol is used for cold extraction in step (2) is ≥3.
[0011] Preferably, the amount of ethyl acetate used in step (3) is equal to the volume of methanol concentrate.
[0012] The third objective of this invention is to provide a method for... Crystallomyces hypericus Application of extracts obtained from bacterial preparation in the preparation of anticancer drugs.
[0013] Preferably, the cancer is cervical cancer, pancreatic cancer, neurogenic cancer, or liver cancer.
[0014] Compared with the prior art, the present invention provides a Crystallomyces hypericus The bacteria and their applications have the following beneficial effects:
[0015] This invention provides a new genus of fungi Crystallomyces hypericus Experiments have verified that the bacteria, when cultured in potato liquid medium and rice medium, are effective against [the bacteria]. Crystallomyces hypericus After the bacteria were cultured, the secondary metabolites produced by the strain were extracted with methanol and ethyl acetate. The resulting extract of the secondary metabolites showed significant inhibitory effects on human cervical cancer cells, human neurocarcinoma cells, and human pancreatic cancer cells. Therefore, the extract can be used to prepare anticancer drugs, providing new raw materials for anticancer treatment. Attached Figure Description
[0016] Figure 1 Photographs of six cultures (CDP, DPY, PDA, SDA, YPD) on a plate.
[0017] Figure 2 for Crystallomyces hypericus Microscopic photograph of PDA culture medium.
[0018] Figure 3 for Crystallomyces hypericus Phylogenetic tree. Detailed Implementation
[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0020] The instruments, equipment, and materials used in this invention are shown below. Reagents not specifically described are conventional reagents in the art, and methods not specifically described are conventional methods in the art.
[0021] Constant temperature and humidity incubator (Shandong Boke Scientific Instruments Co., Ltd., model: BJPX-11-400), SPS-500B constant temperature and humidity incubator (Beijing Yatai Kelong Instrument Technology Co., Ltd.), SW-C-2D (practical vertical novel) double-person single-sided clean bench (Suzhou Purification Equipment Co., Ltd.), vertical pressure steam sterilizer (Shanghai Dongya Pressure Vessel Manufacturing Co., Ltd., model: YXQ-LS-50G), DMSO, DMEM culture medium (Biological Industries), fetal bovine serum, double antibiotics (Biosharp), HepG2, Hela, SY5Y, Miacapa, Panc-1 cells (Kunming Institute of Botany), glucose, maltose, sodium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, sucrose, peptone, tryptone, yeast extract, yeast extract (Shanghai Maclean Biochemical Technology Co., Ltd.).
[0022] The formulation of the culture medium used in this invention is as follows:
[0023] (1) Rice solid culture medium: rice and water are mixed and sterilized. The solid-liquid ratio of rice to water is 3:7, and the unit is g:mL.
[0024] (2) Potato liquid culture medium: 200g / L potato, 20g / L glucose.
[0025] (3) CDP liquid culture medium: sodium nitrate 3g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, ferrous sulfate 0.01g / L, sucrose 30g / L, peptone 10g / L, potassium chloride 0.5g / L.
[0026] (4) DPY liquid culture medium: dextrin 20g / L, peptone 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 0.5g / L.
[0027] (5) Modified SDB liquid medium: peptone 20 g / L, maltose 10 g / L, glucose 10 g / L.
[0028] (6) YPD liquid culture medium: yeast extract 10g / L, tryptone 20g / L, glucose 20g / L.
[0029] Note: All culture media must be sterilized before use. The only difference between liquid and solid culture media is whether or not agar is added. When 15 g / L of agar is added to the culture medium, the corresponding solid culture medium is obtained. Example 1
[0030] Isolation and identification of strains
[0031] High-throughput screening was performed on microorganisms from the flower buds of *Hypericum* (family Clusium, family Hypericaceae) in Cangshan Mountain, Dali, Yunnan, China, to isolate a pure culture strain. First, it was identified using ITS and 28S rRNA assays. Sequence comparison was then performed using the BLAST tool on the NCBI website. The query sequence showed a low match with the target sequence in the database. Using sequences with higher similarity from NCBI, the strain was further searched to determine its parent species. Stilbosporaceae Science, through paper mulberry, discovered that it does not belong to Stilbosporaceae This indicates that this strain of fungus is related to... Stilbosporaceae The department indicates a kinship relationship; further investigation reveals that this department belongs to... Diaporthales Order, and construct a virtual tree of all families under this order and this fungus (see...) Figure 3 The discovery that the fungus exhibited a single branch indicated that it belonged to a new family, and it was named accordingly. Crystallomyces hypericus The nucleotide sequence of the 16S rRNA of this bacterium is shown in SEQ ID NO.1, and the microscopic image is shown below. Figure 2 In the image, A is the ascocarp on PDA medium; B is the ascocarp and tiny crystals on PDA medium (crystals under high magnification); CD is the longitudinal section of the ascocarp; E is the ascocarp that releases asci and ascospores; F is the ascus; and G is the ascospore. Example 2
[0032] Crystallomyces hypericus The specific steps for preparing the bacterial extract are as follows:
[0033] (1) The strain was inoculated into a 1L conical flask containing 400mL potato liquid culture medium (solvent is water, solute is 200g / L potato boiled for 20min and filtered, glucose 20g / L) and cultured at 25℃ for 7 days at 180rpm / min to obtain seed culture medium.
[0034] (2) 5 mL of seed culture solution was inoculated into a 500 mL culture flask containing rice solid culture medium (containing 30 g of rice and 70 mL of water), and cultured statically in an incubator at 25 °C for 30 days to obtain fermentation product. The fermentation product was soaked in methanol three times and concentrated to obtain methanol extract. Ethyl acetate was extracted three times in equal proportion with methanol extract and concentrated to obtain ethyl acetate layer as extract. Example 3
[0035] Test of the anticancer activity of the extract prepared in Example 2
[0036] This experiment used MTT assay to detect cytotoxic activity. The principle is as follows: the dehydrogenase present in the mitochondria of living cells can reduce the yellow thiazolyl blue MTT to insoluble blue-purple formazan. In dead cells, this enzyme disappears, and MTT is not reduced. The formazan was dissolved in DMSO (dimethyl sulfoxide), and the absorbance (OD) was measured at 570 nm using a microplate reader. The OD value is directly proportional to the number of living cells.
[0037] The tumor cell lines used in this invention are: human cervical cancer cells (HeLa), human liver cancer cells (Hepg-2), human pancreatic cancer cells (Miapaca), human neurocellular cancer cells (SY5Y), and human pancreatic cancer cells (Panc-1).
[0038] Experimental methods: Tumor cells in the logarithmic growth phase were digested with trypsin, then pipetted into single-cell suspensions, and diluted with culture medium to a concentration of 1×10⁻⁶. 5 Cells / mL were seeded into 96-well plates (100 μL / well) and incubated at 37°C and 5% CO2 for 24 hours. 1 μL of the extract prepared in Example 2 was added to the experimental group, and 1 μL of diluted DMSO (1000-fold diluted with 1640 medium) was added to the cell-free wells of the blank control group. Three replicates were set up for each group. After incubation at 37°C and 5% CO2 for 48 hours, 10 μL of freshly prepared MTT solution containing 5 mg / mL was added to each well, and the plate was incubated at 37°C for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well to dissolve the formazan. The absorbance (OD) was measured at 570 nm using a microplate reader. 570 ), calculate the inhibition rate (%). Calculation formula: Inhibition rate (%) = (1 - OD) Sample / OD Control ) × 100%. IC 50 The value represents the drug concentration at which the inhibition rate is 50%. The IC50 was calculated using Probit regression analysis in SPSS 29.0 software. 50 value.
[0039] The results showed that the extract prepared in Example 2 had a significant inhibitory effect on human liver cancer cells, human cervical cancer cells, human neurocarcinoma cells and human pancreatic cancer cells, with IC50 values of 32, 29, 18, 21 and 23 mg / mL, respectively; the specific data are shown in Table 1 below.
[0040] Table 1. Cytotoxic activity (mg / mL) of the extract prepared in Example 2.
[0041]
[0042] Comparative Example 1
[0043] Crystallomyces hypericus The specific steps for preparing the bacterial extract are as follows:
[0044] (1) The strain was inoculated onto a 9 cm plate containing CDP solid medium and cultured at 25°C for 7 days to obtain bacterial blocks. See the plate photo below. Figure 1 .
[0045] (2) Inoculate the bacterial block into a 1L conical flask containing CDP liquid medium and culture it at 25℃ for 14 days at a speed of 180rpm / min to obtain the fermentation product. Extract the fermentation product three times with ethyl acetate and fermentation broth in a certain ratio, and concentrate the ethyl acetate extract to obtain the extract.
[0046] Comparative Example 2
[0047] Crystallomyces hypericus The specific steps for preparing the bacterial fermentation broth are as follows:
[0048] (1) The strain was inoculated onto a 9cm plate of DPY solid medium and cultured at 25℃ for 7 days to obtain bacterial blocks. See the plate photo below. Figure 1 .
[0049] (2) Inoculate the bacterial block into a 1L conical flask containing DPY liquid medium and culture it at 25℃ for 14 days at a speed of 180rpm / min to obtain the fermentation broth. Extract the fermentation broth three times with ethyl acetate in equal proportions and concentrate the ethyl acetate extract to obtain the extract.
[0050] Comparative Example 3
[0051] Crystallomyces hypericus The specific steps for preparing the bacterial fermentation broth are as follows:
[0052] (1) The strain was inoculated onto a 9cm plate of potato solid medium and cultured at 25℃ for 7 days to obtain mycelial blocks. See the plate photo below. Figure 1 .
[0053] (2) Inoculate the bacterial block into a 1L conical flask containing potato liquid culture medium and culture it at 25℃ for 14 days at a speed of 180rpm / min to obtain fermentation broth. Extract the fermentation broth three times with ethyl acetate in equal proportion and concentrate to obtain the ethyl acetate extract, which is the extract.
[0054] Comparative Example 4
[0055] Crystallomyces hypericus The specific steps for preparing the bacterial fermentation broth are as follows:
[0056] (1) The strain was inoculated onto a 9 cm plate of SDB solid medium and cultured at 25°C for 7 days to obtain bacterial blocks. See the plate photo below. Figure 1 .
[0057] (2) Inoculate the bacterial block into a 1L conical flask containing modified SDB liquid medium and culture it at 25℃ for 14 days at a speed of 180rpm / min to obtain the fermentation broth. Extract the fermentation broth three times with ethyl acetate in equal proportions and concentrate the ethyl acetate extract to obtain the extract.
[0058] Comparative Example 5
[0059] Crystallomyces hypericus The specific steps for preparing the bacterial fermentation broth are as follows:
[0060] (1) The strain was inoculated onto a 9cm plate of YPD solid medium and cultured at 25℃ for 7 days to obtain bacterial blocks. See the plate photo below. Figure 1 .
[0061] (2) Inoculate the bacterial block into a 1L conical flask containing YPD liquid medium and culture it at 25℃ for 14 days at a speed of 180rpm / min to obtain the fermentation broth. Extract the fermentation broth three times with ethyl acetate in equal proportions and concentrate the ethyl acetate extract to obtain the extract.
[0062] The extracts prepared in Comparative Examples 1 to 5 were subjected to cell experiments using the same method as in Example 3. The results showed that the extracts prepared in Comparative Examples 1 to 5 all had cytotoxic activity >100 mg / mL against human cervical cancer cells (HeLa), human liver cancer cells (Hepg-2), human pancreatic cancer cells (Miapaca), human neurocellular carcinoma cells (SY5Y), and human pancreatic cancer cells (Panc-1), indicating that these extracts had no inhibitory effect on the above-mentioned cells.
[0063] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.
Claims
1. A kind Crystallomyces hypericus The application of fungal extracts in the preparation of anticancer drugs, wherein the cancer is cervical cancer, pancreatic cancer, neurogenic cancer, or liver cancer. Crystallomyces hypericus The preservation number of this fungus is CGMCC No. 42087, and its taxonomic name is: Crystallomyces hypericus ; The method for preparing the extract includes the following steps: (1) Crystallomyces hypericus The bacteria were cultured in liquid potato broth to obtain a seed culture solution; (2) The seed culture solution was inoculated into rice culture medium for culture to obtain a culture medium with attached bacteria. The culture medium with attached bacteria was extracted by cold soaking with methanol. The solid and liquid were separated to obtain methanol extract. The methanol extract was concentrated to obtain methanol concentrate. (3) Extract the methanol concentrate with ethyl acetate to obtain the ethyl acetate layer extract, which is then concentrated to obtain the extract. In step (3), the amount of ethyl acetate used is equal to the volume of methanol concentrate.
2. As described in claim 1 Crystallomyces hypericus The application of fungal extracts in the preparation of anticancer drugs is characterized by: In step (2), the number of times the methanol is used for cold extraction is ≥3 times.