New application of polysaccharide

Abstract

The invention belongs to the field of medicine, and discloses application of caulerpa racemosa zhengye variant polysaccharide in preparation of a medicine for treating gastric cancer, caulerpa racemosa zhengye variant is extracted and used as an anti-gastric cancer medicine, and the caulerpa racemosa zhengye variant polysaccharide has an inhibition effect on cell viability and proliferation of gastric cancer cells; meanwhile, no obvious influence is caused on the activity of normal gastric mucosa epithelial cells. In addition, the polysaccharide can promote a chemotherapeutic drug cis-platinum to exert anti-gastric cancer activity, inhibit gastric cancer cell viability, promote gastric cancer cell apoptosis and induce gastric cancer cell cycle arrest.

Description

Technical Field

[0001] The invention relates to the field of medicine, in particular to the use of polysaccharides. Background Art

[0002] Gastric cancer is a common malignant tumor of the digestive system. It originates from the gastric mucosal epithelium and has clinical manifestations such as loss of appetite, abdominal pain, fatigue, anemia, and weight loss. Early gastric cancer can be cured through radical surgery, but due to the lack of typical symptoms in the early stages of the disease, most patients are not clinically diagnosed until the disease progresses to the middle and late stages, missing the opportunity for radical surgery. Chemotherapy is an indispensable part of gastric cancer treatment. However, the following problems exist during chemotherapy:

[0003] Toxic side effects: Since chemotherapy drugs have low selectivity between tumor cells and normal cells, patients are prone to toxic side effects such as hepatotoxicity, gastrointestinal toxicity and nephrotoxicity.

[0004] Drug resistance: Long-term use of chemotherapy drugs can easily lead to drug resistance in gastric cancer cells, thereby reducing the effectiveness of treatment.

[0005] Insufficient efficacy: Chemotherapy drugs have limited therapeutic effects on patients with advanced or metastatic gastric cancer, and it is difficult to control tumor progression.

[0006] Therefore, it is particularly important to explore new treatment strategies and drug development ideas. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a use of a polysaccharide extracted from Pteris racemosus in the treatment of gastric cancer, aiming to solve the following problems:

[0008] (1) Reduce toxic and side effects during treatment: The polysaccharide of the total assembled Pteris peltata variant is derived from natural edible seaweed and has the characteristics of wide sources, low toxic and side effects, and wide physiological activity. This polysaccharide can effectively inhibit the growth of gastric cancer cells without affecting the activity of normal gastric mucosal cells.

[0009] (2) Improving the anti-gastric cancer effect of chemotherapy drugs: When the polysaccharide is used in combination with cisplatin, the dosage of cisplatin can be reduced while promoting the inhibitory effect of cisplatin on gastric cancer cells.

[0010] According to one aspect of the present invention, there is provided a use of a polysaccharide of Pteris racemosus var. peltata in the preparation of a drug for treating gastric cancer. Preferably, the polysaccharide of Pteris racemosus var. peltata is used in combination with cisplatin.

[0011] Preferably, the polysaccharide of Pteris racemosus var. peltolacca is prepared by the following method: heating an aqueous solution of Pteris racemosus var. peltolacca powder in a water bath, wherein the water bath heating condition is to extract in a water bath at 95°C for 2 hours; collecting the filtrate; centrifuging at 12000 r / min for 15 minutes, and collecting the supernatant polysaccharide crude extract; adding ammonium sulfate with a crude extract mass concentration of 30% (the crude extract mass is weighed with an electronic scale) and tert-butanol with the same volume as the crude extract to the crude extract, mixing well, and stirring with a magnetic stirrer for 1 hour; placing the mixed solution in an ultra-high-speed centrifuge at 12000 r / min, centrifuging for 10 minutes, and collecting the solution in the lower layer; dialyzing the collected solution with an 8000D dialysis bag for 3 days; and drying the resulting solution with a freeze dryer to obtain the polysaccharide of Pteris racemosus var. peltolacca.

[0012] Preferably, the step of heating the aqueous solution of Pteris racemosus var. peltata powder in a water bath is repeated at least once, the water bath heating condition is 95° C. for extraction for 2 hours, and the filtrate is collected.

[0013] Preferably, the filtrates are combined and concentrated to 1 / 5 of the original volume using a rotary evaporator at 45°C.

[0014] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0015] Preferably, the drug is an oral preparation or a non-oral preparation.

[0016] Preferably, the oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, pills or pastes.

[0017] Preferably, the non-oral preparation is one or more of an injection, a cream, a patch, an ointment or a spray.

[0018] The oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, pills or pastes. The non-oral preparation is one or more of injections, creams, patches, ointments or sprays.

[0019] Pharmaceutically acceptable excipients include various organic or inorganic carrier substances commonly used as preparation materials, which are used as excipients, lubricants, binders, disintegrants, thickeners in solid preparations, and solvents, dispersants, solubilizers, suspending agents, tonicity agents, buffers, analgesics, etc. in liquid preparations, and are appropriately prepared in appropriate amounts. In addition, additives such as preservatives, antioxidants, colorants, sweeteners, etc. can be used according to conventional methods as needed.

[0020] Preferred examples of excipients include, for example, lactose, white sugar, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, etc. Preferred examples of lubricants include, for example, magnesium stearate, calcium stearate, talc, colloidal silicon dioxide, etc. Preferred examples of binders include, for example, crystalline cellulose, white sugar, D-mannitol, dextrin, hydroxypropyl cellulose, polyvinyl pyrrolidone, etc. Preferred examples of disintegrants include, for example, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, sodium carboxymethyl starch, etc. Preferred examples of thickeners include, for example, natural gums, cellulose derivatives, acrylic acid polymers, etc. Preferred examples of solvents include, for example, water for injection, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, etc. Preferred examples of dispersants include, for example, Tween 80, HCO 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc. Preferred examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. Preferred examples of suspending agents include stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, etc. Preferred examples of surfactants include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose. Preferred examples of tonicity agents include sodium chloride, glycerol, D-mannitol, etc. Preferred examples of buffers include phosphates, acetates, carbonates, citrates, etc. Preferred examples of analgesics include benzyl alcohol, etc. Preferred examples of preservatives include parabens, chlorobutanol, benzyl alcohol, phenylethyl alcohol, dehydroacetic acid, sorbic acid, etc. Preferred examples of the antioxidant include sulfites, ascorbic acid, and the like.

[0021] The present invention provides a new anti-tumor use of polysaccharides from the peltaceous leaf variant of Pteris racemosus. Compared with the existing traditional Chinese medicine polysaccharides related to gastric cancer treatment, this method extracts the peltaceous leaf variant of Pteris racemosus and uses it as an anti-gastric cancer drug, which has an inhibitory effect on the cell viability and proliferation of gastric cancer cells; at the same time, it has no obvious effect on the viability of normal gastric mucosal epithelial cells. In addition, the polysaccharide can promote the anti-gastric cancer activity of the chemotherapy drug cisplatin, inhibit the viability of gastric cancer cells, promote the apoptosis of gastric cancer cells and induce gastric cancer cell cycle arrest. The present invention is expected to be developed into a new anti-tumor drug, especially an anti-gastric cancer drug. In addition, the present invention is conducive to the development of new anti-gastric cancer uses of Pteris racemosus polysaccharides, and the development of anti-tumor treatment methods used in combination with traditional chemotherapy drugs, so as to achieve the effect of increasing the efficacy while reducing the dosage of chemotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 This is a graph showing the effect of CRVP on GES-1 cell viability according to an embodiment of the present invention.

[0024] Figure 2 This is a graph showing the effect of CRVP on GES-1 cell viability according to an embodiment of the present invention.

[0025] Figure 3 This is a graph showing the effect of CRVP on the cloning of gastric cancer cells AGS according to an embodiment of the present invention.

[0026] Figure 4 This is a graph showing the effect of CRVP combined with CIS on the vitality of gastric cancer cells according to an embodiment of the present invention.

[0027] Figure 5 This is a graph showing the effect of CRVP combined with CIS on apoptosis of gastric cancer cells according to an embodiment of the present invention.

[0028] Figure 6 This is a graph showing the effect of CRVP on the cell cycle of gastric cancer cells according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods.

[0030] Material:

[0031] Caulerpa racemosa var peltata belongs to the genus Caulerpa, is a tropical and subtropical green algae, and is distributed in the East my country Sea and South China Sea. The Caulerpa racemosa var peltata used in this patent was collected from the sea near Zhanjiang City, Guangdong Province.

[0032] Experimental Example 1: CRVP Extraction

[0033] Weigh 100g of dried algae powder, add 2L of tertiary water, stir evenly, and extract in a 95℃ water bath for 2h. Stir once every 30 minutes during the extraction period to allow the algae powder to fully contact with the extract. After the extraction is completed, filter with gauze and collect the filtrate. Add 2L of tertiary water to the filter residue for a second extraction. Combine the two extracts and use a rotary evaporator to concentrate the filtrate to 1 / 5 of the original volume at 45℃. Use an ultrahigh-speed centrifuge to centrifuge at 12000r / min for 15 minutes to collect the supernatant polysaccharide crude extract. The three-phase method is used to remove pigments, proteins, lipids and other substances. By adding inorganic salts and organic solutions to the crude extract, it forms a three-phase system (i.e., sulfuric acid-tert-butanol-water three-phase system). The upper phase is the tert-butanol phase, which mainly extracts less polar substances such as pigments and lipids; the middle phase is the protein extraction layer; the lower phase is the ammonium sulfate phase, and the main substances are water-soluble substances such as ammonium sulfate and polysaccharides. The specific operation is as follows: add ammonium sulfate with a mass concentration of 30% of the crude extract and tert-butyl alcohol with the same volume as the crude extract to the crude extract, mix well, and stir with a magnetic stirrer for 1 hour; then place the mixed solution in an ultra-high-speed centrifuge at 12000r / min, centrifuge for 10 minutes, and collect the lower layer of solution. The collected solution is dialyzed for 3 days with an 8000D dialysis bag, and then the obtained solution is dried with a freeze dryer to obtain polysaccharide powder.

[0034] Experimental Example 2: Effect of CRVP on the Viability of Normal Human Gastric Mucosal Cells (GES-1)

[0035] 1. Cell inoculation: Cells in the logarithmic growth phase were digested with 0.25% trypsin and prepared into a single cell suspension by adding RPMI 1640 complete medium (90% RPMI 1640, 10% FBS, 1% 10000U / mL penicillin and 10mg / mL streptomycin); after counting, cells were plated at 5×10 3 The cells were inoculated at a density of 100 μL in a 96-well culture plate, and 100 μL was added to each well. The culture medium was incubated at 37°C and 5% CO 2 Culture in an incubator overnight to allow cells to adhere.

[0036] 2. Drug treatment: After the cells adhered, the original culture medium was removed and replaced with 100 μL of RPMI1640 complete culture medium containing different concentrations of CRVP. A control group without CRVP was set up and cultured for 24 hours.

[0037] 3. MTT assay: After the culture is completed, add 10 μL of 0.5 mg / mL MTT solution to each well, continue to culture for 4 hours, and then add 100 μL of triple solution (10 g SDS, 5 mL isobutanol, 0.1 mL 10 M hydrochloric acid, dilute to 100 mL with double distilled water) for overnight culture. After the culture is completed, use an enzyme reader to measure the absorbance of each well at 570 nm, compare the absorbance value of each experimental group with that of the control group, and calculate the cell survival rate. Cell survival rate (%) = absorbance value of experimental group / absorbance value of blank group × 100

[0038] Different concentrations of CRVP were set from 0 to 400 μg / mL, and the gastric mucosal epithelial cells were treated for 24 hours, and the cell viability of GES-1 was detected using the MTT method. Figure 1 As shown in the data, within the concentration range of 0-400 μg / mL, CRVP had no significant effect on the cell viability of normal gastric mucosal epithelial cells GES-1 (p>0.05).

[0039] Experimental Example 4: Anti-gastric cancer activity of CRVP combined with cisplatin (CIS)

[0040] 1. Cell inoculation: Cells in the logarithmic growth phase were digested with 0.25% trypsin and prepared into a single cell suspension by adding RPMI 1640 complete medium (90% RPMI 1640, 10% FBS, 1% 10000U / mL penicillin and 10mg / mL streptomycin); after counting, cells were plated at 5×10 3 The cells were inoculated at a density of 100 μL in a 96-well culture plate, and 100 μL was added to each well. The culture medium was incubated at 37°C and 5% CO 2 Culture in an incubator overnight to allow cells to adhere.

[0041] 2. Drug treatment: Prepare 25 mg / mL, 100 mg / mL and 400 mg / mL CRVP solutions respectively, add appropriate amount of CIS to these three solutions to prepare a mother solution with a CIS concentration of 200 μM, and then dilute the mother solution with complete culture medium to obtain a combination of CRVP and different concentrations of CIS. After the cells adhere to the wall, remove the original culture medium and replace it with 100 μL of culture medium containing CRVP and different combinations of CIS. At the same time, set up an experimental group containing only CRVP without CIS and a control group containing neither CRVP nor CIS, and culture for 24 hours.

[0042] 3. MTT assay: After the culture is completed, add 10 μL of 0.5 mg / mL MTT solution to each well, continue to culture for 4 hours, and then add 100 μL of triple solution (10 g SDS, 5 mL isobutanol, 0.1 mL 10 M hydrochloric acid, dilute to 100 mL with double distilled water) for overnight culture. After the culture is completed, use an enzyme reader to measure the absorbance of each well at 570 nm, compare the absorbance value of each experimental group with that of the control group, and calculate the cell survival rate. Cell survival rate (%) = absorbance value of experimental group / absorbance value of blank group × 100

[0043] (1) CRVP combined with CIS inhibits the vitality of gastric cancer cells (AGS, HGC-27, MKN45)

[0044] 0, 25, 100, and 400 μg / mL CRVP were used in combination with different doses of CIS to treat AGS, HGC-27, and MKN45 cells for 24 hours, and the MTT method was used to detect the viability of gastric cancer cells under each combination. Figure 4 As shown in the figure, within a certain dose range, compared with CIS acting alone on gastric cancer cells, the combined action of CRVP and CIS significantly reduced the activity of gastric cancer cells, and the higher the concentration of the combined polysaccharide, the more obvious the decrease. Figure 2 In addition, Figure 4 The horizontal axis in the middle represents the change in the concentration of CIS. When the horizontal axis in each broken line is "0", the corresponding vertical axis is the vitality of gastric cancer cells with only CRVP added.

[0045] (2) CRVP combined with CIS promotes apoptosis of gastric cancer cells (AGS, HGC-27)

[0046] A control group (no CRVP and CIS were added to the culture medium), a CIS group (5 μM CIS was added to the culture medium), a CRVP group (400 μg / mL CRVP was added to the culture medium), and a CIS+CRVP group (400 μg / mL CRVP and 5 μM CIS were added to the culture medium) were set up. After treating the cells for 48 hours, the apoptosis of gastric cancer cells was detected by Annexin V / PI double staining. The experimental results are shown in Figure 5 As shown, CRVP significantly increased the apoptosis of AGS cells (p<0.05); at the same time, the apoptosis rate of AGS cells when CIS and CRVP acted together was significantly higher than that when CIS acted alone on AGS cells (p<0.05). In addition, compared with CIS alone, the combined action of CIS and CRVP can promote the apoptosis of HGC-27 cells, but the effect is not significant.

[0047] (3) CRVP combined with CIS induces cell cycle arrest in gastric cancer cells (AGS, HGC-27)

[0048] A control group (no CRVP and CIS were added to the culture medium), a CIS group (5 μM CIS was added to the culture medium), a CRVP group (400 μg / mL CRVP was added to the culture medium), and a CIS+CRVP group (400 μg / mL CRVP and 5 μM CIS were added to the culture medium) were set up. After treating the cells for 24 hours, the DNA of gastric cancer cells was labeled with DNA-binding dye propidium iodide, and the DNA content of gastric cancer cells in each group was detected by flow cytometry. The experimental results are shown in Figure 2. Figure 6 As shown, CRVP can cause G2 arrest in AGS cells and S arrest in HGC-27 cells respectively; compared with CIS alone, the combined action of CRVP and CIS can promote G2 arrest in AGS cells and S arrest in HGC-27 cells.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of polysaccharide of Pteris racemosus peltata variant in the preparation of drugs for treating gastric cancer.

2. The use according to claim 1, characterized in that The polysaccharide of Pteris racemosus peltata variant is used in combination with cisplatin.

3. The use according to claim 1, characterized in that The polysaccharide of Pteris racemosus var. peltata is prepared by the following method: The aqueous solution of the powder of Pteris racemosus var. peltata was heated in a water bath, and the water bath heating condition was 95°C for extraction for 2 hours; collecting the filtrate; Centrifuge at 12000 r / min for 15 min, and collect the supernatant polysaccharide crude extract; Adding ammonium sulfate having a mass concentration of 30% of the crude extract and tert-butyl alcohol having the same volume as the crude extract to the crude extract, mixing well, and stirring with a magnetic stirrer for 1 hour; The mixed solution was placed in an ultra-high speed centrifuge at 12000 r / min, centrifuged for 10 minutes, and the lower layer of solution was collected; The collected solution was dialyzed using an 8000D dialysis bag for 3 days; The obtained solution was dried using a freeze dryer to obtain the Pteris racemosa var. peltata polysaccharide.

4. The use according to claim 3, characterized in that The step of heating the aqueous solution of the Pteris racemosus var. peltata powder in a water bath is repeated at least once, wherein the water bath heating condition is 95° C. and the extraction is carried out in a water bath for 2 hours, and the filtrate is collected.

5. The use according to claim 4, characterized in that The filtrates were combined and concentrated to 1 / 5 of the original volume using a rotary evaporator at 45°C.

6. The use according to claim 3, characterized in that: The drug also contains pharmaceutically acceptable excipients.

7. The use according to claim 3, characterized in that: The medicine is an oral preparation or a non-oral preparation.

8. The use according to claim 7, characterized in that The oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, pills or pastes.

9. The use according to claim 7, characterized in that: The non-oral preparation is one or more of an injection, a cream, a patch, an ointment or a spray.

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