CN110585231A - Application of platycodon grandiflorum polysaccharide in preparation of medicine for treating cell damage caused by Cr (VI) - Google Patents
Application of platycodon grandiflorum polysaccharide in preparation of medicine for treating cell damage caused by Cr (VI) Download PDFInfo
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- CN110585231A CN110585231A CN201910908106.4A CN201910908106A CN110585231A CN 110585231 A CN110585231 A CN 110585231A CN 201910908106 A CN201910908106 A CN 201910908106A CN 110585231 A CN110585231 A CN 110585231A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses application of platycodon grandiflorum polysaccharide in preparing a medicine for treating cell damage caused by Cr (VI). The invention researches the effect of platycodon grandiflorum polysaccharide in cell damage by establishing a Cr (VI) -induced chick embryo fibroblast cell line (DF-1 cell) cell damage model, and discovers for the first time that platycodon grandiflorum polysaccharide (50-200 mu g/ml) with a certain concentration can antagonize the cell damage caused by Cr (VI). The invention expands the medical application of the platycodon grandiflorum polysaccharide and is beneficial to the development of a medicine which takes the platycodon grandiflorum polysaccharide as an effective component and has new indications.
Description
Technical Field
The invention relates to the field of medical application of platycodon grandiflorum polysaccharide, in particular to application of platycodon grandiflorum polysaccharide in preparing a medicine for treating cell damage caused by Cr (VI).
Background
Chromium (Cr) is a common component of organic matter and is ubiquitous in the natural environment. Chromium has a variety of applications in industry, including chemical, refractory, and metallurgical industries. The valence states range from-2 to +6, with trivalent and hexavalent chromium being the most common valence states. Hexavalent chromium is most easily absorbed in all valence states. It was reported that hexavalent chromium salts remained ten times as long as trivalent chromium salts in rats. Hexavalent chromium ions enter the blood and soon bind to globin in hemoglobin via the erythrocyte membrane. Under the victory condition, the chromium is absorbed and then enters the blood circulation to be combined with beta globulin and transported to the action site, and is usually transported to subcellular structures such as nucleus and mitochondria in the cell. Hexavalent chromium has multiple manifestations of toxic effects on humans and animals, the most common of which include liver and kidney damage and even tumors. In conclusion, hexavalent chromium shows toxic effects in gene, nervous, immune and cancer aspects.
Platycodon grandiflorum (Jacq) a.dc) is a perennial herb belonging to the genus Platycodon of the family platycodonaceae. The main chemical components of the platycodon include platycodin, volatile oil, steroids, flavonoids, polysaccharide and the like. Radix Platycodi can be used for treating pharyngalgia, hoarseness, cough with excessive phlegm, chest distress, pyocutaneous disease with no ulcer, sore throat relieving, lung ventilating, phlegm eliminating, and pus discharge. The radix Platycodi can also be used as food, and has rich nutrition, and contains various essential microelements and amino acids. Modern pharmacological researches find that platycodon grandiflorum has various biological activities, such as improvement of insulin resistance, oxidation resistance, inflammation diminishing, enhancement of body immune function, tumor resistance and the like.
The polysaccharide is one of the main active ingredients of platycodon grandiflorum, and researches show that the platycodon grandiflorum polysaccharide has the functions of bacteriostasis, tumor resistance, blood sugar reduction, inflammation resistance, organism immunity regulation, oxidation resistance and the like. However, no report on the antagonism of the cell damage caused by Cr (VI) has been found for platycodon grandiflorum polysaccharide.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide the application of platycodon grandiflorum polysaccharide in the preparation of drugs for treating cell damage caused by cr (vi).
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, the application of platycodon grandiflorum polysaccharide in preparing a medicament for treating cell damage caused by Cr (VI) is provided.
Preferably, the platycodon grandiflorum polysaccharide is prepared by the following method:
weighing radix Platycodi root decoction pieces, adding 8-10 times by weight of water, soaking overnight, leaching at 80 deg.C for 3-5 hr, filtering, concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), centrifuging at 3000rpm for 10min to remove impurities to obtain leaching solution;
adding 95 vol% ethanol into the leaching solution while stirring to make ethanol final concentration 60%, standing, centrifuging, separating precipitate, and vacuum drying at 60 deg.C to obtain PGPS60c(ii) a Adding 95 vol% ethanol into the supernatant, stirring to reach final ethanol concentration of 80%, standing, centrifuging, separating precipitate, and vacuum drying at 60 deg.C to obtain PGPS80c；
Mixing PGPS60cAnd PGPS80cMixing according to the weight ratio of 2:1 to prepare the platycodon grandiflorum polysaccharide.
More preferably, the standing time is 24 hours.
Preferably, the PGPS60cThe content of polysaccharide is 53-55%, and the content of protein is 0.7-0.8%; the PGPS80cThe content of polysaccharide is 62-64%, and the content of protein is 3.0-3.2%.
Preferably, the concentration of the platycodon grandiflorum polysaccharide is 50-200 mu g/ml.
In a second aspect of the present invention, there is provided a medicament for treating cell damage caused by cr (vi), the medicament comprising platycodon grandiflorum polysaccharide as an active ingredient at a concentration of 50-200 μ g/ml.
Preferably, the platycodon grandiflorum polysaccharide is composed of PGPS60cAnd PGPS80cThe weight ratio of the components is 2: 1.
The invention has the beneficial effects that:
the invention researches the effect of platycodon grandiflorum polysaccharide in cell damage by establishing a Cr (VI) -induced chick embryo fibroblast cell line (DF-1 cell) cell damage model, and discovers for the first time that platycodon grandiflorum polysaccharide (50-200 mu g/ml) with a certain concentration can antagonize the cell damage caused by Cr (VI). The invention expands the medical application of the platycodon grandiflorum polysaccharide and is beneficial to the development of a medicine which takes the platycodon grandiflorum polysaccharide as an effective component and has new indications.
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FIG. 1: and (3) inspecting the proliferation effect of the platycodon grandiflorum polysaccharide on DF-1 cells.
FIG. 2: the CCK-8 method measures the viability of cells treated differently.
FIG. 3: and (5) detecting results by a confocal microscope.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, the platycodon grandiflorum polysaccharide, which is one of the main active ingredients of platycodon grandiflorum, has the functions of bacteriostasis, tumor resistance, blood sugar reduction, inflammation resistance, body immunity regulation, oxidation resistance and the like, but reports on the antagonism of cell damage caused by cr (vi) of the platycodon grandiflorum polysaccharide.
Polysaccharides are macromolecular compounds formed by connecting a plurality of monosaccharide molecular chains, and the pharmacological activity of the polysaccharides is different due to different molecular weights of the polysaccharides, different arrangement modes among monosaccharides in polysaccharide molecules and the like. The method comprises the steps of firstly extracting each graded platycodon grandiflorum polysaccharide by adopting a step-by-step alcohol precipitation method, and investigating the antagonistic action of each graded platycodon grandiflorum polysaccharide on cell damage caused by Cr (VI). Through experiments, PGPS60cAnd PGPS80cHas one effect on cell damage caused by Cr (VI)Antagonistic action of the compounds, PGPS60cAnd PGPS80cThe platycodon grandiflorum polysaccharide mixed according to the weight ratio of 2:1 has the optimal antagonistic effect on cell damage caused by Cr (VI), and has a remarkable synergistic promotion effect.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available.
Example 1: preparation of platycodon grandiflorum polysaccharide
Weighing radix Platycodi root decoction pieces, adding 10 weight times of water, soaking overnight, leaching at 80 deg.C for 4 hr, filtering, concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), centrifuging at 3000rpm for 10min to remove impurities to obtain leaching solution;
adding 95 vol% ethanol into the leaching solution while stirring to make ethanol final concentration 60%, standing for 24 hr, centrifuging, separating precipitate, and vacuum drying at 60 deg.C to obtain PGPS60c。
Adding 95 vol% ethanol into the supernatant, stirring to reach final ethanol concentration of 80%, standing, centrifuging, separating precipitate, and vacuum drying at 60 deg.C to obtain PGPS80c。
Mixing PGPS60cAnd PGPS80cMixing the raw materials according to the weight ratio of 2:1 to prepare the platycodon grandiflorum polysaccharide PGPSt.
Example 2: examination of proliferation of DF-1 cells by Platycodon grandiflorum polysaccharide
1. The test method comprises the following steps:
in order to study the effect of the platycodon grandiflorum polysaccharide on DF-1 cells, the platycodon grandiflorum polysaccharide prepared in example 1 was prepared into solutions with different concentrations, and DF-1 cells were treated with the platycodon grandiflorum polysaccharide solutions with different concentrations for 12h, and the change in cell viability was examined.
2. And (3) test results:
the test results are shown in FIG. 1, and it can be seen from FIG. 1 that the platycodon polysaccharides prepared in example 1 have no toxic effect on cells at concentrations below 400. mu.g/ml.
Example 3: research on protective effect of platycodon grandiflorum polysaccharide on DF-1 cell damage caused by Cr (VI)
1. The test method comprises the following steps:
the cell survival rate is detected by a CCK-8 method to research the protective effect of platycodon grandiflorum polysaccharide on DF-1 cell damage caused by Cr (VI).
Culturing DF-1, and culturing the cells (5X 10) when the cell fusion degree reaches more than 80%5One/well) to 96-well plates (100. mu.L per well) and cultured for 24 h. The experiment was divided into five treatments, respectively: con group, Cr group, treatment 1 group, treatment 2 group, and treatment 3 group; wherein Con group is blank control and is not treated; cr group cells were treated with 40. mu.M Cr (VI) for 6 h; treatment of group 1 with 40. mu.M Cr (VI) and 200. mu.g/ml PGPS60cTreating the cells for 6 h; treatment of group 2 with 40. mu.M Cr (VI) and 200. mu.g/ml PGPS80cTreating the cells for 6 h; treatment of 3 groups with 40. mu.M Cr (VI) and 200. mu.g/ml Platycodon Grandiflorum Polysaccharide (PGPS)60cAnd PGPS80cMix at a weight ratio of 2: 1) the cells were treated for 6 h.
PGPS60cAnd PGPS80cWere prepared according to the method of example 1.
After 6h, 10. mu.L of CCK-8 reagent was added to each well. Incubate in incubator for 2 h. Care was taken not to generate bubbles in the wells that would affect the OD readings. Absorbance at 450nm was measured with a microplate reader. The cell viability was calculated as follows,
cell survival rate ═ [ (test-well blank)/(control-blank) ] × 100%.
2. And (3) test results:
the results are shown in FIG. 2, and the results show that Cr (VI) has toxic effect on DF-1 and can obviously reduce the survival rate of cells. PGPS60cAnd PGPS80cThe platycodon grandiflorum polysaccharide serving as two grades can antagonize cytotoxicity caused by Cr (VI), but the antagonistic activity is different; to convert PGPS60cAnd PGPS80cThe platycodon grandiflorum polysaccharide prepared by mixing according to the weight ratio of 2:1 has the optimal effect of antagonizing cytotoxicity caused by Cr (VI).
Example 4: effect of Platycodon grandiflorum polysaccharide on Cr (VI) -induced autophagosome aggregation
1. The test method comprises the following steps:
DF-1 cells were fixed with 4% paraformaldehyde solution (PFA) and blocked with 5% BSA containing 0.4% Triton X-100. Then, cells were incubated with primary antibody against Tubulin, LC3 overnight at 4 ℃ and fluorescent secondary antibody was incubated for 1 hour at room temperature and then stained with DAPI for 5 minutes. And finally observed by a Leica TCS SPE confocal microscope.
2. And (3) test results:
the test result is shown in figure 3, wherein Control in the figure is blank Control and is not processed; cr represents cells treated with 40. mu.M Cr (VI); PGPSt represents cells treated with 200. mu.g/ml of PGPSt prepared in example 1; cr + PGPSt represents cells treated with 200. mu.g/ml of PGPSt prepared in example 1 and 40. mu.M of Cr (VI). The green spots represent autophagosomes observed in DF-1 cells exposed to 40. mu.MCr (VI); autophagosome aggregation was reduced after PGPSt (200. mu.g/ml) + Cr (VI) co-treatment.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (7)
1. Application of platycodon grandiflorum polysaccharide in preparing medicines for treating cell damage caused by Cr (VI).
2. The use of claim 1, wherein the platycodon grandiflorum polysaccharide is prepared by the following method:
weighing radix Platycodi root decoction pieces, adding 8-10 times by weight of water, soaking overnight, leaching at 80 deg.C for 3-5 hr, filtering, concentrating the filtrate to relative density of 1.05-1.15 (measured at 50 deg.C), centrifuging at 3000rpm for 10min to remove impurities to obtain leaching solution;
adding 95 vol% ethanol into the leaching solution while stirring to make ethanol final concentration 60%, standing, centrifuging, and separatingSeparating the precipitate, vacuum drying the precipitate at 60 deg.C to obtain PGPS60c(ii) a Adding 95 vol% ethanol into the supernatant, stirring to reach final ethanol concentration of 80%, standing, centrifuging, separating precipitate, and vacuum drying at 60 deg.C to obtain PGPS80c；
Mixing PGPS60cAnd PGPS80cMixing according to the weight ratio of 2:1 to prepare the platycodon grandiflorum polysaccharide.
3. Use according to claim 2, characterized in that the standing time is 24 h.
4. Use according to claim 2, wherein the PGPS is used60cThe content of polysaccharide is 53-55%, and the content of protein is 0.7-0.8%; the PGPS80cThe content of polysaccharide is 62-64%, and the content of protein is 3.0-3.2%.
5. The use according to claim 1 or 2, wherein the concentration of platycodon grandiflorum polysaccharide is 50-200 μ g/ml.
6. A medicament for treating cell damage caused by Cr (VI), which is characterized in that the medicament takes platycodon grandiflorum polysaccharide with the concentration of 50-200 mu g/ml as an active ingredient.
7. The medicament of claim 6, wherein the platycodon grandiflorum polysaccharide is composed of PGPS60cAnd PGPS80cThe weight ratio of the components is 2: 1.
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Cited By (2)
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| CN111358805A (en) * | 2020-03-18 | 2020-07-03 | 山东农业大学 | Application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy |
| CN112656807A (en) * | 2021-01-22 | 2021-04-16 | 山东德信生物科技有限公司 | Application of platycodon grandiflorum polysaccharide in preparation of medicine for degrading SOCS1/2 |
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| CN110200987A (en) * | 2019-06-13 | 2019-09-06 | 山东农业大学 | Application of the campanulaceae total starches in the drug that preparation treats by the CCCP Apoptosis induced |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111358805A (en) * | 2020-03-18 | 2020-07-03 | 山东农业大学 | Application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy |
| CN111358805B (en) * | 2020-03-18 | 2021-11-02 | 山东农业大学 | Application of platycodon grandiflorum polysaccharide in antagonizing fumonisin B1-induced apoptosis through autophagy |
| CN112656807A (en) * | 2021-01-22 | 2021-04-16 | 山东德信生物科技有限公司 | Application of platycodon grandiflorum polysaccharide in preparation of medicine for degrading SOCS1/2 |
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Application publication date: 20191220 |