CN114053309B - Marine protozoan Phaliota extract with antitumor activity - Google Patents
Marine protozoan Phaliota extract with antitumor activity Download PDFInfo
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- CN114053309B CN114053309B CN202111262512.1A CN202111262512A CN114053309B CN 114053309 B CN114053309 B CN 114053309B CN 202111262512 A CN202111262512 A CN 202111262512A CN 114053309 B CN114053309 B CN 114053309B
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- 239000000284 extract Substances 0.000 title claims abstract description 47
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 20
- 241000722826 Ardisia Species 0.000 claims abstract description 15
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 15
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 8
- 206010008342 Cervix carcinoma Diseases 0.000 claims abstract description 7
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims abstract description 7
- 201000010881 cervical cancer Diseases 0.000 claims abstract description 7
- 201000007270 liver cancer Diseases 0.000 claims abstract description 7
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 230000035755 proliferation Effects 0.000 claims abstract description 6
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims abstract description 5
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims abstract description 5
- 241001628505 Phlebopus Species 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- 241000209140 Triticum Species 0.000 claims description 16
- 235000021307 Triticum Nutrition 0.000 claims description 16
- 235000013339 cereals Nutrition 0.000 claims description 14
- 241000238631 Hexapoda Species 0.000 claims description 11
- 239000013535 sea water Substances 0.000 claims description 9
- 241001647939 Ardisia crispa Species 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 5
- 239000013049 sediment Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 7
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- 230000004083 survival effect Effects 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 4
- 230000004663 cell proliferation Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000002246 antineoplastic agent Substances 0.000 description 9
- 229940041181 antineoplastic drug Drugs 0.000 description 9
- 239000003814 drug Substances 0.000 description 7
- 229940079593 drug Drugs 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 239000005445 natural material Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 241000242757 Anthozoa Species 0.000 description 1
- 241000206761 Bacillariophyta Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 235000014653 Carica parviflora Nutrition 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 241000243142 Porifera Species 0.000 description 1
- 241000251555 Tunicata Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
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- 230000000340 anti-metabolite Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
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- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/68—Protozoa, e.g. flagella, amoebas, sporozoans, plasmodium or toxoplasma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract
The invention discloses a marine protozoon panosoma extract with anti-tumor activity, which relates to the technical field of protozoon extracts and comprises a preparation method of the marine panosoma extract, and the preparation method comprises the following steps: A. separating and purifying the population of the Phlebopus from the environment; B. expanding culture of Phantom; C. the preparation of the extract has the beneficial effects that: the extract of marine protozoon Ardisia with anti-tumor activity can specifically produce inhibition effects on cervical cancer Hela cells, human non-small cell lung cancer A549 cells and liver cancer HepG2 cells, and the MTT method is adopted to determine the half inhibition concentration IC 50 when the survival rate (or death rate) of each tumor cell is 50%, and the result is shown in Table 1, the half inhibition concentration is 24.53-30.24 mug/mL, the marine Ardisia extract effectively inhibits proliferation of the tumor cells, and the activity of inhibiting cell proliferation is higher.
Description
Technical Field
The invention relates to the technical field of protozoan extracts, in particular to an extract of marine protozoan Phaliota with anti-tumor activity.
Background
Malignant tumor is a potential killer of human life, and along with the development of science and technology, tumor chemotherapy has advanced to a certain extent, and the service life of patients with leukemia, malignant lymphoma and the like is obviously prolonged, but no effective treatment method for most solid tumors exists. The traditional cytotoxin medicines comprise alkylating agents, antimetabolites, metal platinum medicines, antitumor hormone medicines and antitumor antibiotics, and have the clinical positions of the using values, but have the defects of low treatment efficiency, large adverse reaction, easy generation of drug resistance and the like. The screening and developing of novel antitumor drugs from natural resources has become a trend of searching for novel, efficient and low-toxicity antitumor drugs.
The marine environment is rich in biological resources to generate active natural substances, which can be used for treating human diseases, especially malignant tumors. Marine organisms living in extreme environments need to adapt to different environmental conditions than land-based organisms, thereby producing unique natural chemicals with diversity of species and high complexity. To date, over 36,000 compounds have been isolated from marine microorganisms such as fungi, bacteria and microalgae in the ocean, and from marine organisms such as macroalgae, sponges, corals, molluscs and tunicates, each year growing at rates that find hundreds of novel marine natural compounds. Drugs based on marine natural substances have begun to be used in clinical trials for the treatment of various diseases, and a number of anticancer drugs from several classes of marine compounds have been approved for use.
Protozoa are cell wall-free eukaryotic single-cell animals, which, although simple in structure, have all the functions necessary for life support and for continued offspring. Some antitumor actives have been extracted from certain marine protozoa, such as the use of Sulfavant A derivatives from diatoms, to improve antigen-specific immune protection and delay the onset and progression of melanoma. The medicine can be used as a synergist of antitumor medicines. The research of protozoan antitumor active ingredients has important significance.
Disclosure of Invention
Aiming at the limitations of low treatment efficiency, large adverse reaction, easy drug resistance generation and the like of the existing antitumor drugs, the invention provides the marine protozoan Ardisia extract with antitumor activity, and provides resources for searching novel, efficient and low-toxicity antitumor drugs.
In order to achieve the above purpose, the invention is realized by the following technical scheme: an extract of marine protozoon Ardisia with anti-tumor activity, comprising a preparation method of the extract of marine Ardisia, comprising the following steps:
A. Isolation and purification of the Phlebopus population from the environment: collecting partial seawater from seawater by using a vessel, collecting protozoa communities in a laboratory by using a specific collecting instrument and a specific mode, proportioning wheat grain culture solution, and expanding groups by using the wheat grain culture solution;
B. Expanding culture of Phantom fructicola: placing a single population inoculated with the Phragmitis insect into a liquid culture medium for culturing, enabling the Phragmitis insect to be cultured in an enlarged way to reach a 5L scale, placing a test tube sample filled with the liquid culture medium into a centrifugal machine for centrifugation, setting the rotating speed of the centrifugal machine at 3000rpm for 10min, opening the centrifugal machine, and taking out a cultured test tube to collect insect bodies;
C. Preparing extract: extracting with methanol for 3 times repeatedly, adding methanol into the sediment of the water sample removed after centrifugation, homogenizing the sediment by adopting a high-speed dispersing machine, setting the centrifugation speed at 12000rpm for 10min, taking a supernatant methanol sample, concentrating into an extract by adopting a rotary evaporator, and redissolving the extract into 5mL by using methanol.
Preferably, the extract is stored at 4 ℃, and the extract is placed in a refrigerator, and the temperature is set to be 4 ℃.
Preferably, the operation steps in the step a are as follows: sucking a Philippine insect under a microscope, and performing amplification culture with wheat grain culture solution to obtain a single population of Philippine insect.
Preferably, the wheat grain culture solution comprises the following components: 5 wheat grains are added into 100mL of filtered seawater, and 7-10 d wheat grains are replaced once.
Preferably, in the step B, the single population of the Ardisia armigera is cultured for 55-65 days by using a low-speed shaking table at the temperature of 25 ℃.
The application of the extract of the marine protozoon Phaliota to the preparation of antitumor drugs is provided, wherein the extract of the marine protozoon Phaliota has the effect of inhibiting proliferation of tumor cells.
Preferably, the tumor cells are cervical cancer Hela cells, human non-small cell lung cancer A549 cells and liver cancer HepG2 cells.
Preferably, the effect of inhibiting proliferation of tumor cells is determined by MTT assay at a half inhibitory concentration IC 50 at which the survival (or mortality) of each tumor cell is 50%.
The invention provides a marine protozoan Ardisia crispa extract with anti-tumor activity, which has the following beneficial effects:
1. The extract of marine protozoon Ardisia with anti-tumor activity can specifically produce inhibition effects on cervical cancer Hela cells, human non-small cell lung cancer A549 cells and liver cancer HepG2 cells, and the MTT method is adopted to determine the half inhibition concentration IC 50 when the survival rate (or death rate) of each tumor cell is 50%, and the result is shown in Table 1, the half inhibition concentration is 24.53-30.24 mug/mL, the marine Ardisia extract effectively inhibits proliferation of the tumor cells, and the activity of inhibiting cell proliferation is higher.
2. The extract of marine protozoan Phaliota with antitumor activity can be used for preparing antitumor drugs, and is derived from marine protozoan, and the raw materials can be continuously obtained through enlarged culture, so that the extract has the advantages of environmental friendliness and low application cost. The application of the composition in developing medicaments for treating cervical cancer cells, lung cancer cells and liver cancer cells is extremely wide.
Detailed Description
In the following, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments.
Example 1
An extract of marine protozoon Ardisia with anti-tumor activity, comprising a preparation method of the extract of marine Ardisia, comprising the following steps:
A. Isolation and purification of the Phlebopus population from the environment: collecting partial seawater from seawater by using a vessel, collecting protozoa communities in a laboratory by using a specific collecting instrument and a specific mode, proportioning wheat culture liquid, and performing group expansion operation by using the wheat culture liquid, wherein the operation steps are as follows: sucking a Phantom under a microscope, and performing expanded culture by using wheat grain culture solution, wherein the wheat grain culture solution comprises the following components: 5 wheat grains are added into 100mL of filtered seawater, and 7-10 d wheat grains are replaced once. . Culturing the Ardisia armigera by using wheat grain culture solution to obtain a single population of Ardisia armigera;
B. expanding culture of Phantom fructicola: inoculating a single species of the Phragmitis to a liquid culture medium, culturing the single species of the Phragmitis for 55-65 days by using a low-speed shaking table at 25 ℃, expanding the culture to 5L scale, centrifuging the obtained sample, selecting a centrifuge with a centrifugal speed of 3000rpm, centrifuging for 10min, and collecting the insect bodies;
C. preparing extract: extracting with methanol for 3 times repeatedly, wherein methanol is added into the sediment of the water sample removed after centrifugation, the sediment is beaten into homogenate by a high-speed dispersing machine, the rotation speed is 12000rpm, the supernatant methanol sample is taken after 10min of operation, the supernatant methanol sample is concentrated into extract by a rotary evaporator, then the extract is redissolved to 5mL by methanol, the extract is preserved under the condition of 4 ℃, the extract is placed in a refrigerator, the temperature is set to be 4 ℃, the temperature is not too high or too low, and the 4 ℃ is the preservation temperature of the extract.
Application of sea Ardisia crispa extract in preparing antitumor drug is provided, wherein tumors are human cervical cancer, human lung cancer and human liver cancer.
Example two
Based on the first embodiment, the MTT method is used for measuring the anti-tumor activity of the marine Phaliota extract
1. In the embodiment, an in vitro cytotoxicity experiment is adopted to detect the survival rate of the isolated cultured human tumor cells after the marine Phaliota extract is added, so as to determine the anti-tumor activity of the marine Phaliota extract.
2. The selected cell lines were: human cervical cancer Hela cells, human non-small cell lung cancer a549 cells, and human liver cancer HepG2 cells were assayed for 50% survival (or mortality) of each tumor cell using MTT assay at half inhibitory concentration IC 50 (all available from ATCC).
3. The semi-inhibition concentration IC 50 of the marine Phaliota extract on the tumor cells is shown in Table 1, and from Table 1, it can be seen that the marine Phaliota extract effectively inhibits proliferation of the tumor cells, has higher activity of inhibiting cell proliferation, and can be used for preparing antitumor drugs.
4. TABLE 1 anti-tumor cell half-inhibitory concentration IC 50 results of marine Ardisia crispa extract
The present invention is not limited to the above-mentioned embodiments, and any person skilled in the art, based on the technical solution of the present invention and the inventive concept thereof, can be replaced or changed within the scope of the present invention.
Claims (1)
1. An extract of marine protozoon Ardisia with anti-tumor activity, comprising a preparation method of the extract of marine Ardisia, which is characterized by comprising the following steps:
A. Isolation and purification of the Phlebopus population from the environment: collecting partial seawater from the seawater by using a vessel, separating and obtaining single individuals of the Ardisia crispa in the seawater sample by using a microscope in a laboratory, proportioning wheat grain culture solution, and culturing by using the wheat grain culture solution to obtain a single population of the Ardisia crispa;
B. Expanding culture of Phantom fructicola: placing a single population of the Phragmitis insect into a liquid culture medium for culturing, enabling the Phragmitis insect to be cultured in an enlarged manner to reach a 5L scale, placing a test tube sample filled with the liquid culture medium into a centrifugal machine for centrifugation, setting the rotating speed of the centrifugal machine at 3000rpm for 10min, opening the centrifugal machine, and taking out a cultured test tube to collect insect bodies;
C. preparing extract: extracting with methanol for 3 times repeatedly, wherein the extraction method comprises adding methanol into the water sample, centrifuging, removing sediment, homogenizing with a high-speed disperser at 12000rpm for 10min, collecting supernatant methanol sample, concentrating with a rotary evaporator to obtain extract, and redissolving with methanol to 5mL;
the preservation condition of the extract is that the extract is preserved at 4 ℃;
The operation steps in the step A are as follows: sucking a Philippine insect under a microscope, and performing expanded culture with wheat grain culture solution to obtain a single population of Philippine insect;
in the step B, a single species of the Phragmitis is required to be cultivated for 55-65 days by using a low-speed shaking table at the temperature of 25 ℃;
The extract of marine protozoon Ardisia has proliferation inhibiting effect on cervical cancer Hela tumor cells, human non-small cell lung cancer A549 tumor cells and liver cancer HepG2 tumor cells.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1614414A2 (en) * | 2004-07-09 | 2006-01-11 | Robert Sabin | Compositions comprising a copper compound for treatment of mammalian diseases |
| CN101285038A (en) * | 2008-06-05 | 2008-10-15 | 华东理工大学 | New type culture medium for preparing new type antineoplastic activity compounds via marine aspergillus glaucus and method for preparing same |
| CN101586148A (en) * | 2009-06-26 | 2009-11-25 | 河北大学 | Method for degrading crude oil with protozoan infusorian |
| CN102925372A (en) * | 2012-08-27 | 2013-02-13 | 浙江工业大学 | Marine fungus rhodotorula minuta and application thereof in preparing anti-tumor medicines |
-
2021
- 2021-10-28 CN CN202111262512.1A patent/CN114053309B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1614414A2 (en) * | 2004-07-09 | 2006-01-11 | Robert Sabin | Compositions comprising a copper compound for treatment of mammalian diseases |
| CN101285038A (en) * | 2008-06-05 | 2008-10-15 | 华东理工大学 | New type culture medium for preparing new type antineoplastic activity compounds via marine aspergillus glaucus and method for preparing same |
| CN101586148A (en) * | 2009-06-26 | 2009-11-25 | 河北大学 | Method for degrading crude oil with protozoan infusorian |
| CN102925372A (en) * | 2012-08-27 | 2013-02-13 | 浙江工业大学 | Marine fungus rhodotorula minuta and application thereof in preparing anti-tumor medicines |
Non-Patent Citations (1)
| Title |
|---|
| 张文.《海洋药物导论 第 2 版》.上海科学技术出版社,2012,第60页. * |
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