CN109276576B - Application of ganoderma leucocontextum polysaccharide in preparation of antitumor drugs - Google Patents
Application of ganoderma leucocontextum polysaccharide in preparation of antitumor drugs Download PDFInfo
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- 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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Abstract
The invention discloses application of ganoderma leucocontextum polysaccharide in preparation of antitumor drugs, and the preparation method of the ganoderma leucocontextum polysaccharide comprises the following steps: (1) extracting; (2) precipitating with ethanol; (3) freeze-drying; (4) and (5) separating and purifying. The invention has the beneficial effects that: provides a new application of the ganoderma leucocontextum polysaccharide, can effectively inhibit the growth of tumors, can be used for preparing antitumor drugs, can also be used as a synergist of the antitumor drugs such as paclitaxel and the like, and has good medicinal value.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of ganoderma leucocontextum polysaccharide in preparation of antitumor drugs.
Background
Cancer is the second leading cause of death worldwide, causing 880 million deaths in 2015, which has become a serious public health problem worldwide. Screening single or auxiliary antitumor compounds from natural products is still an important way for discovering antitumor drugs. Polysaccharides are high molecular polymers which are connected by aldehyde groups and ketone groups through glycosidic bonds, are not only used as energy sources and supporting tissues in organisms, but also are more important to participate in life phenomena and physiological processes such as cell recognition, neural development, tumor metastasis and the like, and a plurality of polysaccharides from natural sources have the functional activities of regulating immunity, resisting tumors, reducing blood sugar and the like. Ganoderma lucidum has a long medicinal history in east Asia countries as a large fungus for both food and medicine. The ganoderma lucidum polysaccharide is proved by modern pharmacology to have a plurality of functions of protecting liver, resisting tumor, reducing blood pressure, reducing blood fat, regulating organism immunity and the like.
Ganoderma leucocontextum (Ganoderma leucocontextum) is Ganoderma lucidum which is different from known Ganoderma lucidum of Ganoderma lucidum in Zaxi Baochun of Bomi county in 2011 and is identified as a novel Ganoderma lucidum species (Li T et al.2014) according to morphological and molecular biological results after four years of research. The polysaccharide of the white-flesh ganoderma lucidum fruiting body is detected, and the result shows that the content of the main component polysaccharide of the individual strain is 6-8 times higher than that of the commercially available ganoderma lucidum.
At present, research on ganoderma leucocontextum is limited to cultivation and identification of ganoderma leucocontextum and an extraction method and application of an extract, but the research on the ganoderma leucocontextum in the aspects of inhibiting tumor growth and serving as an antitumor drug synergist is rarely related, so that the research on the ganoderma leucocontextum has a remarkable significance in the aspect of intensive research on the ganoderma contextum.
Disclosure of Invention
In order to solve the technical problems, the invention provides a new application of ganoderma leucocontextum polysaccharide, namely an application of ganoderma leucocontextum polysaccharide serving as an active ingredient in preparing an anti-tumor medicament, the ganoderma contextum polysaccharide has the effect of inhibiting tumor growth, and can be used together with an anti-tumor medicament taxol to enhance the effect of inhibiting tumor growth.
The preparation method of the ganoderma leucocontextum polysaccharide specifically comprises the following steps:
(1) extraction: crushing the fruiting body of ganoderma leucocontextum, soaking in water, heating, extracting under reflux for multiple times, combining filtrates, and concentrating the filtrate to obtain a concentrated solution;
(2) alcohol precipitation: precipitating the concentrated solution with ethanol, standing overnight, and centrifuging to obtain precipitate;
(3) freeze-drying: dissolving the precipitate with pure water, freeze drying to obtain crude polysaccharide, and drying for storage;
(4) separation and purification: dissolving the obtained ganoderma leucocontextum crude polysaccharide in water, performing step-by-step elution through an ion exchange column, and performing freeze drying again to obtain the ganoderma leucocontextum polysaccharide.
Preferably, the preparation method of ganoderma leucocontextum polysaccharide further comprises the following steps:
(1) extraction: crushing the white-meat ganoderma lucidum fruiting body, and mixing the materials according to a material-liquid ratio of 1: soaking the raw materials in water for half an hour in a proportion of 10-20, heating the raw materials at 80-100 ℃, performing reflux extraction for three times, combining filtrates, and concentrating the filtrates to one seventh to one tenth of the original solution to obtain a concentrated solution;
(2) alcohol precipitation: carrying out alcohol precipitation on the concentrated solution until the final concentration of the alcohol solvent is 40-80%, standing overnight, and then centrifuging to obtain a precipitate;
(3) freeze-drying: dissolving the precipitate with pure water, freeze drying to obtain crude polysaccharide, and drying for storage;
(4) separation and purification: dissolving the obtained ganoderma leucocontextum crude polysaccharide in water, performing step-by-step elution through an ion exchange column, and performing freeze drying again to obtain the ganoderma leucocontextum polysaccharide.
Preferably, the time for the three reflux extractions in step (1) is 2h, and 1h, respectively.
Preferably, the concentrated solution after alcohol precipitation in the step (2) is left at 4 ℃ overnight and/or centrifuged at 4000-8000 rpm for 10-30 min.
Further preferably, the concentrated solution after alcohol precipitation in the step (2) is centrifuged at 6000rpm for 10 min.
Preferably, the alcohol precipitation in the step (2) is carried out until the final concentration of the alcohol solvent is 75%.
Preferably, the white Ganoderma lucidum is white Ganoderma lucidum Ganoderma leucocotextum HMGIM-Z110122, and the preservation number is CCTCC NO: M2016087.
The white-flesh lucid Ganoderma is collected from broad-leaved forests in Zaxigang county of Bomi county in Tibet area, is identified as a new white-flesh lucid Ganoderma strain, is organized and separated to obtain an original strain, is named as white-flesh lucid Ganoderma ganodera leucocotextum HMGIM-Z110122, is preserved in China center for type culture preservation (CCTCC for short, with the address of eight Lopa Gaojiashan mountain in Wuhan city, Hubei province) in 3 and 7 days of 2016, and has the preservation number of CCTCC NO: M2016087.
Further, the invention also provides the ganoderma leucocontextum polysaccharide, which is obtained by the preparation method.
The invention also provides an anti-tumor pharmaceutical composition, wherein the anti-tumor pharmaceutical composition contains the ganoderma leucocontextum polysaccharide with effective dose.
Preferably, the anti-tumor pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, and the excipients can comprise acid, alkali, salt, hydrate or ester, and other excipients.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a new application of ganoderma leucocontextum polysaccharide, which can effectively inhibit tumor growth, can be used for preparing antitumor drugs, cancer-prevention foods or health-care products, and can be used as a synergist of the antitumor drugs, thereby having good medicinal value.
Drawings
FIG. 1 is a graph showing a comparison of tumor weights of the respective experimental groups in Experimental example 1;
FIG. 2 is a graph showing a comparison of tumor weights of the respective experimental groups in Experimental example 2.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The reagents and apparatus described in the following examples are commercially available unless otherwise specified.
Example 1
The preparation method of the ganoderma leucocontextum polysaccharide comprises the following steps:
1) extraction: crushing the white-meat ganoderma lucidum fruiting body, and mixing the materials according to a material-liquid ratio of 1: soaking the raw materials in water for half an hour in a proportion of 10-20, heating the raw materials at 80-100 ℃, performing reflux extraction for three times, wherein the reflux extraction time is 2 hours, 2 hours and 1 hour respectively, combining filtrates, and concentrating the filtrate to one tenth of the original solution to obtain a concentrated solution;
2) alcohol precipitation: precipitating the concentrated solution with ethanol until the final concentration of the alcohol solvent is 75%, standing overnight at 4 deg.C, and centrifuging at 6000rpm for 10min to obtain precipitate;
3) freeze-drying: dissolving the precipitate with pure water, freeze drying to obtain crude polysaccharide, and drying for storage;
4) separation and purification: dissolving the obtained ganoderma leucocontextum crude polysaccharide in water, performing step-by-step elution through an ion exchange column, and performing freeze drying again to obtain the ganoderma leucocontextum polysaccharide.
Experimental example 1
1. Reagent and instrument
Cell lines: breast cancer mouse cell line (4T 1);
mice: BALB/c mice.
2. Cell culture
Mouse breast cancer 4T1 was cultured in DMEM medium containing 1% penicillin, 1% streptomycin and 10% Fetal Bovine Serum (FBS) and cultured in Thermo Forma3110 tissue culture box (Thermo, Germany) at 37 ℃ and 5% CO2 to logarithmic growth phase.
3. Tumor model animal experiment
3.1 inoculating tumor cells in logarithmic growth phase to the skin of a BABL/C mouse, and randomly grouping the tumor cells into a model group, a paclitaxel group, a white ganoderma lucidum polysaccharide group and a paclitaxel + white ganoderma lucidum polysaccharide group;
3.2 on the next day, the medicine is respectively administered to the groups, the administration mode is intraperitoneal injection, the dose is 50mg/kg/day, the model group is physiological saline for injection, and the medicine is continuously administered for 4 weeks;
3.3 the day after the last administration, sacrifice mice by carbon dioxide, take tumors, measure tumor weight and size;
3.4 the effect of the ganoderma leucocontextum polysaccharide on inhibiting the tumor growth of mice and assisting the synergy of paclitaxel anti-tumor is calculated and analyzed, and the optimal dosage is determined.
4. Results display
TABLE 1
| Group of | Tumor weight (g) | Inhibition ratio (%) |
| Model set | 0.255±0.13 | 0 |
| Paclitaxel group | 0.178±0.08 | 30.2±3.0 |
| Polysaccharide group | 0.195±0.1 | 23.5±4.0 |
Note: tumor growth inhibition rate calculations were compared to the model group.
Experimental example 2
1. Reagent and instrument
Cell lines: breast cancer mouse cell line (4T 1);
mice: BALB/c mice.
2. Cell culture
Culturing mouse breast cancer 4T1 with DMEM culture solution containing 1% penicillin, 1% streptomycin and 10% Fetal Bovine Serum (FBS), placing at 37 deg.C and 5% CO2Is cultured in Thermo Forma3110 tissue culture boxes (Thermo company, germany) to logarithmic growth phase.
3. Tumor model animal experiment
3.1 inoculating tumor cells in logarithmic growth phase to the skin of a BABL/C mouse, and randomly grouping the tumor cells into a model group, a paclitaxel group, a white ganoderma lucidum polysaccharide group and a paclitaxel + white ganoderma lucidum polysaccharide group;
3.2 on the next day, the medicine is respectively administered to the groups, the administration mode is intraperitoneal injection, the dose is 100mg/kg/day, the model group is physiological saline for injection, and the medicine is continuously administered for 4 weeks;
3.3 the day after the last administration, sacrifice mice by carbon dioxide, take tumors, measure tumor weight and size;
3.4 the effect of the ganoderma leucocontextum polysaccharide on inhibiting the tumor growth of mice and assisting the synergy of paclitaxel anti-tumor is calculated and analyzed, and the optimal dosage is determined.
4. Results display
TABLE 2
| Group of | Tumor weight (g) | Inhibition ratio (%) | Increase efficiency (%) |
| Model set | 0.143±0.06 | 0 | |
| Paclitaxel group | 0.103±0.07 | 27.8±7 | |
| Polysaccharide group | 0.067±0.05 | 52.9±15 | |
| Polysaccharide plus paclitaxel | 0.034±0.01** | 75.6±21 | 66±14* |
Note: tumor growth inhibition rate calculation compared to model group;
the efficiency of the increase is that the polysaccharide and the paclitaxel are compared with the paclitaxel.
From fig. 1, 2 and tables 1, 2, it can be found that: the ganoderma leucocontextum polysaccharide has the function of inhibiting the tumor growth in a mouse, can obviously enhance the effect of the antitumor drug taxol on inhibiting the tumor growth, and is a ganoderma lucidum variety with development potential.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the scope of the present invention.
Claims (9)
1. The application of the Ganoderma leucocontextum polysaccharide as an active ingredient in the preparation of anti-breast cancer drugs is characterized in that the Ganoderma leucocontextum is Ganoderma leucoderma leucocotinum HMGIM-Z110122, and the preservation number is CCTCC NO: m2016087, the preparation method comprises the following steps:
(1) extraction: crushing the fruiting body of ganoderma leucocontextum, soaking in water, heating, extracting under reflux for multiple times, combining filtrates, and concentrating the filtrate to obtain a concentrated solution;
(2) alcohol precipitation: precipitating the concentrated solution with ethanol, standing overnight, and centrifuging to obtain precipitate;
(3) freeze-drying: dissolving the precipitate with pure water, freeze drying to obtain crude polysaccharide, and drying for storage;
(4) separation and purification: dissolving the obtained ganoderma leucocontextum crude polysaccharide in water, performing step-by-step elution through an ion exchange column, and performing freeze drying again to obtain the ganoderma leucocontextum polysaccharide.
2. The preparation method of Ganoderma leucocontextum polysaccharide is characterized in that Ganoderma leucocontextum is Ganoderma leucocontextum HMGIM-Z110122, and the preservation number is CCTCC NO: m2016087, the preparation method comprises the following steps:
(1) extraction: crushing the fruiting body of ganoderma leucocontextum, soaking in water, heating, extracting under reflux for multiple times, combining filtrates, and concentrating the filtrate to obtain a concentrated solution;
(2) alcohol precipitation: precipitating the concentrated solution with ethanol, standing overnight, and centrifuging to obtain precipitate;
(3) freeze-drying: dissolving the precipitate with pure water, freeze drying to obtain crude polysaccharide, and drying for storage;
(4) separation and purification: dissolving the obtained ganoderma leucocontextum crude polysaccharide in water, performing step-by-step elution through an ion exchange column, and performing freeze drying again to obtain the ganoderma leucocontextum polysaccharide.
3. The method for preparing ganoderma leucocontextum polysaccharide according to claim 2, further comprising the steps of:
(1) extraction: crushing the white-meat ganoderma lucidum fruiting body, and mixing the materials according to a material-liquid ratio of 1: soaking the raw materials in water for half an hour in a proportion of 10-20, heating the raw materials at 80-100 ℃, performing reflux extraction for three times, combining filtrates, and concentrating the filtrates to one seventh to one tenth of the original solution to obtain a concentrated solution;
(2) alcohol precipitation: carrying out alcohol precipitation on the concentrated solution until the final concentration of the alcohol solvent is 40-80%, standing overnight, and then centrifuging to obtain a precipitate;
(3) freeze-drying: dissolving the precipitate with pure water, freeze drying to obtain crude polysaccharide, and drying for storage;
(4) separation and purification: dissolving the obtained ganoderma leucocontextum crude polysaccharide in water, performing step-by-step elution through an ion exchange column, and performing freeze drying again to obtain the ganoderma leucocontextum polysaccharide.
4. The method for preparing ganoderma leucocontextum polysaccharide according to claim 3, wherein the time for the three times of reflux extraction in the step (1) is 2h, 2h and 1h respectively.
5. The method for preparing ganoderma leucocontextum polysaccharide according to claim 3, wherein the concentrated solution after alcohol precipitation in the step (2) is left overnight at 4 ℃ and/or centrifuged at 4000-8000 rpm for 10-30 min; and/or, the alcohol precipitation in the step (2) is carried out until the final concentration of the alcohol solvent is 75 percent.
6. The method for preparing ganoderma leucocontextum polysaccharide according to claim 5, wherein the ethanol precipitated concentrate in step (2) is centrifuged at 6000rpm for 10 min.
7. A ganoderma leucocontextum polysaccharide, wherein the ganoderma leucocontextum polysaccharide is prepared by the preparation method according to any one of claims 2 to 6.
8. An anti-breast cancer pharmaceutical composition, wherein the anti-breast cancer pharmaceutical composition comprises an effective amount of the ganoderma leucocontextum polysaccharide of claim 7.
9. The anti-breast cancer pharmaceutical composition of claim 8, further comprising one or more pharmaceutically acceptable excipients.
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| CN113122458A (en) * | 2020-06-11 | 2021-07-16 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Ganoderma leucocontextum Z160097 and cultivation method and application thereof |
| CN112094358B (en) * | 2020-09-22 | 2022-05-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Tibetan ganoderma lucidum polysaccharide GLP-1 with antioxidant effect, and preparation method and application thereof |
| CN112569336B (en) * | 2020-12-16 | 2023-08-11 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of russula cinerea glycoprotein in preparation of medicines or health foods for enhancing organism immunity and preventing and/or treating tumor diseases |
| CN113278535B (en) * | 2021-06-30 | 2023-05-23 | 四川省中医药科学院 | New strain ZL167 of ganoderma lucidum and new application thereof |
| CN113768141B (en) * | 2021-09-18 | 2022-10-18 | 广东粤微食用菌技术有限公司 | Anti-oxidation repairing ganoderma lucidum extract and preparation method thereof |
| CN114766285B (en) * | 2022-04-24 | 2023-10-27 | 云南省农业科学院生物技术与种质资源研究所 | Ganoderma lucidum strain L4495 and cultivation method and application thereof |
| CN115886242A (en) * | 2022-11-23 | 2023-04-04 | 河北岁神本草肉灵芝生物科技有限公司 | Ganoderma lucidum, high-concentration oral liquid containing ganoderma lucidum and preparation method of oral liquid |
| CN116270712A (en) * | 2023-02-24 | 2023-06-23 | 安徽中医药大学 | Application of ganoderma lucidum polysaccharide based on white meat in preparation of antitumor drugs |
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