A new use of polysaccharide

Abstract

This invention belongs to the field of medicine and discloses the use of polysaccharides from Pteris racemosus var. peltulae in the preparation of a drug for treating gastric cancer. Extracted from Pteris racemosus var. peltulae, the drug is used as an anti-gastric cancer drug, inhibiting the viability and proliferation of gastric cancer cells while having no significant effect on the viability of normal gastric epithelial cells. Furthermore, the polysaccharide can enhance the anti-gastric cancer activity of the chemotherapy drug cisplatin, inhibit gastric cancer cell viability, promote gastric cancer cell apoptosis, and induce gastric cancer cell cycle arrest.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to a use of polysaccharide. 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 assembly fern leaf 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, a polysaccharide of Pteris racemosus var. peltulae is used in the preparation of a drug for treating gastric cancer. Preferably, the polysaccharide of Pteris racemosus var. peltulae is used in combination with cisplatin.

[0011] Preferably, the polysaccharide of Pteris racemosus var. peltulosa powder is prepared by the following method: heating an aqueous solution of Pteris racemosus var. peltulosa powder in a water bath at 95° C. and extracting in a water bath for 2 hours; collecting the filtrate; centrifuging at 12,000 r / min for 15 minutes, and collecting the supernatant polysaccharide crude extract; adding ammonium sulfate with a crude extract concentration of 30% (the crude extract mass is weighed using an electronic scale) and the same volume of tert-butanol 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 ultrahigh-speed centrifuge at 12,000 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 using a freeze dryer to obtain the polysaccharide of Pteris racemosus var. peltulosa.

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

[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 a capsule, tablet, oral liquid, granule, pill, powder, pill or paste. The non-oral preparation is one or more of an injection, cream, patch, ointment or spray.

[0019] Pharmaceutically acceptable excipients include, for example, various organic or inorganic carriers commonly used as pharmaceutical materials, which serve as excipients, lubricants, binders, disintegrants, and thickeners in solid preparations; and as solvents, dispersants, solubilizers, suspending agents, tonicity agents, buffers, analgesics, etc. in liquid preparations, and are formulated in appropriate amounts. Furthermore, additives such as preservatives, antioxidants, colorants, and sweeteners may be used as needed according to conventional methods.

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

[0021] The present invention provides a new anti-tumor use of polysaccharides from Pteris racemosus peltula variant. Compared with the existing traditional Chinese medicine polysaccharides related to gastric cancer treatment, this method extracts Pteris racemosus peltula variant 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 chemotherapy drug cisplatin to exert its anti-gastric cancer activity, inhibit the viability of gastric cancer cells, promote gastric cancer cell apoptosis 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 therapeutic effect 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 colony formation 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 gastric cancer cell viability according to an embodiment of the present invention.

[0027] Figure 5 This is a graph showing the effect of CRVP combined with CIS on gastric cancer cell apoptosis 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 facilitate a clearer understanding of the present invention for those skilled in the art. It should be noted that the following examples do not limit the scope of the present invention and are provided for illustrative purposes only. Unless otherwise specified, the raw materials, reagents, and devices mentioned in the following examples are commercially available or obtained by known methods.

[0030] Material:

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

[0032] Experimental Example 1: CRVP Extraction

[0033] Weigh 100g of dried algae powder, add 2L of grade tertiary water, stir thoroughly, and extract in a 95°C waterbath for 2h. Stir every 30 minutes to ensure full contact between the algae powder and the extract. After extraction, filter through gauze and collect the filtrate. Add 2L of grade tertiary water to the filter residue for a second extraction. Combine the two extracts and concentrate the filtrate to 1 / 5 of its original volume using a rotary evaporator at 45°C. Centrifuge at 12,000 rpm for 15 minutes in an ultrahigh-speed centrifuge to collect the supernatant, a crude polysaccharide extract. A three-phase method is used to remove pigments, proteins, and lipids. Inorganic salts and an organic solution are added to the crude extract to form a three-phase system (i.e., sulfuric acid-tert-butyl alcohol-water). The upper phase, the tert-butyl alcohol phase, primarily extracts less polar substances such as pigments and lipids; the middle phase is the protein extraction layer; and the lower phase, the ammonium sulfate phase, primarily contains ammonium sulfate and water-soluble substances such as polysaccharides. The specific operation is to add ammonium sulfate at a concentration of 30% of the crude extract mass and the same volume of tert-butyl alcohol as the crude extract to the crude extract, mix thoroughly, and stir with a magnetic stirrer for 1 hour. The mixture is then placed in an ultrahigh-speed centrifuge at 12,000 rpm for 10 minutes, and the lower layer of solution is collected. The collected solution is dialyzed for 3 days using an 8000D dialysis bag, and then dried using a freeze dryer to obtain a polysaccharide powder.

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

[0035] 1. Cell seeding: 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% 10000 U / mL penicillin and 10 mg / mL streptomycin); after counting, the cells were plated at 5×10 cells per well. 3 The cells were seeded at a density of 100 μL in a 96-well culture plate, and 100 μL was added to each well; the cells were cultured overnight in a 37° C., 5% CO 2 incubator to allow the cells to adhere to the wall.

[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 also set up and cultured for 24 hours.

[0037] 3. MTT assay: After the incubation period, add 10 μL of 0.5 mg / mL MTT solution to each well and continue incubation for 4 hours. 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) and incubate overnight. After the incubation period, measure the absorbance of each well at 570 nm using a microplate reader. Compare the absorbance values ​​of each experimental group with those of the control group to calculate the cell survival rate. Cell survival rate (%) = absorbance value of experimental group / absorbance value of blank group × 100

[0038] Different concentrations of CRVP (0-400 μg / mL) were set to treat gastric mucosal epithelial cells for 24 hours and then the cell viability of GES-1 was detected using the MTT assay. 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 seeding: 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% 10000 U / mL penicillin and 10 mg / mL streptomycin); after counting, the cells were plated at 5×10 cells per well. 3 The cells were seeded at a density of 100 μL in a 96-well culture plate, and 100 μL was added to each well; the cells were cultured overnight in a 37° C., 5% CO 2 incubator to allow the cells to adhere to the wall.

[0041] 2. Drug Treatment: Prepare 25 mg / mL, 100 mg / mL, and 400 mg / mL CRVP solutions, respectively. Add appropriate amounts of CIS to each of these solutions to create a stock solution with a CIS concentration of 200 μM. This stock solution is then serially diluted with complete culture medium to produce combinations of CRVP and CIS at varying concentrations. After cell attachment, remove the original culture medium and replace it with 100 μL of culture medium containing CRVP and various CIS combinations. Simultaneously, establish experimental groups containing only CRVP and no CIS, and control groups containing neither CRVP nor CIS, and culture the cells for 24 hours.

[0042] 3. MTT assay: After the incubation period, add 10 μL of 0.5 mg / mL MTT solution to each well and continue incubation for 4 hours. 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) and incubate overnight. After the incubation period, measure the absorbance of each well at 570 nm using a microplate reader. Compare the absorbance values ​​of each experimental group with those of the control group to 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 viability of gastric cancer cells (AGS, HGC-27, MKN45)

[0044] 0, 25, 100, and 400 μg / mL CRVP were combined with different doses of CIS to treat AGS, HGC-27, and MKN45 cells for 24 hours, and the MTT assay was used to detect the viability of gastric cancer cells under each combined action. Figure 4 As shown in Figure 2, within a certain dose range, the combined action of CRVP and CIS significantly reduced the viability of gastric cancer cells compared to CIS alone, and the higher the concentration of the combined polysaccharide, the more significant the reduction. Figure 2 In addition, Figure 4 The horizontal axis represents the concentration change of CIS. When the horizontal axis of each broken line is "0", the corresponding vertical axis is the activity 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 added to the culture medium), a CIS group (5 μM CIS added to the culture medium), a CRVP group (400 μg / mL CRVP added to the culture medium), and a CIS+CRVP group (400 μg / mL CRVP and 5 μM CIS 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 2. Figure 5 As shown, CRVP significantly increased apoptosis in AGS cells (p<0.05). Furthermore, the combined application of CIS and CRVP significantly increased the apoptosis rate in AGS cells compared to CIS alone (p<0.05). Furthermore, the combined application of CIS and CRVP promoted apoptosis in HGC-27 cells compared to CIS alone, but the effect was 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 added to the culture medium), a CIS group (5 μM CIS added to the culture medium), a CRVP group (400 μg / mL CRVP added to the culture medium), and a CIS+CRVP group (400 μg / mL CRVP and 5 μM CIS 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 the effect of CIS alone, the combined effect of CRVP and CIS can promote the G2 arrest in AGS cells and S arrest in HGC-27 cells.

[0049] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a polysaccharide of Pteridium racemosa var. peltulae in the preparation of a drug for treating gastric cancer, wherein the polysaccharide of Pteridium racemosa var. peltulae is prepared by the following method: Heat the aqueous solution of Pteris racemosus var. peltula powder in a water bath at 95°C for 2 hours; collecting the filtrate; Centrifuge at 12000 r / min for 15 minutes and collect the supernatant polysaccharide crude extract; To the crude extract was added 30% crude extract mass concentration of ammonium sulfate and the same volume of tert-butanol crude extract, mixed well, and stirred with a magnetic stirrer for 1 hour; The mixture was placed in an ultra-high-speed centrifuge at 12000 r / min and centrifuged for 10 minutes, and the lower layer of solution was collected; The collected solution was dialyzed using an 8000Da dialysis bag for 3 days; The obtained solution was dried using a freeze dryer to obtain the Pteris racemosa var. peltula polysaccharide.

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 Repeat the steps of heating the aqueous solution of the Pteris racemosus var. peltula powder in a water bath at least once, wherein the water bath heating condition is 95° C., extracting in a water bath for 2 hours, and collecting the filtrate.

4. The use according to claim 3, characterized in that The filtrates were combined and concentrated to 1 / 5 of the original volume using a rotary evaporator at 45°C.

5. The use according to claim 1, characterized in that The drug further contains pharmaceutically acceptable excipients.

6. The use according to claim 1, characterized in that The medicine is an oral preparation or a non-oral preparation.

7. The use according to claim 6, characterized in that The oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders or pastes.

8. The use according to claim 6, characterized in that The non-oral preparation is an injection.

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