Gypenoside and preparation method thereof, and application of gypenoside in preparation of anti-pancreatic cancer drugs

Abstract

This invention discloses an acidic polysaccharide from Gynostemma pentaphyllum, AGPA12, its purification and extraction method, and its application in the preparation of anti-pancreatic cancer drugs. AGPA12 is mainly composed of mannose, glucuronic acid, rhamnose, glucose, galactose, and fucose, with a molar ratio of 1.05:1.74:6.29:1.40:69.48:19.03. The polysaccharide was extracted using an α-amylase-assisted water extraction and alcohol precipitation method, and purified by DEAE-Sepharose FF ion exchange chromatography and Sepharose CL6B gel chromatography. In vitro experiments showed that AGPA12 had significant inhibitory effects on the proliferation and migration of human pancreatic cancer AsPC1 cells, and its mechanism was related to the regulation of the MAPK signaling pathway and the expression of apoptosis-related proteins. This invention provides a new approach for the development of Gynostemma pentaphyllum polysaccharides and their application in anti-pancreatic cancer drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a Gynostemma pentaphyllum acidic polysaccharide, its preparation method, and its application in the preparation of anti-pancreatic cancer drugs. Background Technology

[0002] Gynostemma pentaphyllum ( Gynostemma pentaphyllum (Thunb.) Makino is a herbaceous plant belonging to the genus Gynostemma of the Cucurbitaceae family, also known as Southern Ginseng, Seven-Leaf Ginseng, Heavenly Grass, Root-Growing Plant, Five-Leaf Ginseng, or Seven-Leaf Ginseng. Gynostemma pentaphyllum is a traditional medicinal and edible plant, rich in saponins, polysaccharides, flavonoids, and other active ingredients, possessing various pharmacological effects such as anti-tumor, antioxidant, and lipid-lowering properties. Among these, polysaccharides, as one of its main active components, show potential in immunomodulation and anti-tumor effects. Current research on Gynostemma pentaphyllum polysaccharides mainly focuses on extraction process optimization and general activity screening; in-depth research and applications targeting its structural characteristics and specific tumors (such as pancreatic cancer) are still relatively rare.

[0003] Pancreatic cancer is a highly malignant digestive tract tumor with a poor prognosis. Existing chemotherapy drugs have significant toxic side effects and are prone to drug resistance. Therefore, developing novel, highly effective, and low-toxicity anti-pancreatic cancer drugs is of great importance. Plant polysaccharides, due to their multi-target and low-toxicity characteristics, have become one of the hot topics in anti-tumor drug research and development. Summary of the Invention

[0004] The present invention aims to provide a Gynostemma pentaphyllum acidic polysaccharide AGPA1 with a well-defined structure. 2. Its efficient extraction and purification method, and its application in the preparation of anti-pancreatic cancer drugs.

[0005] The Gynostemma pentaphyllum acidic polysaccharide AGPA1 described in this invention 2. Its monosaccharide composition and molar ratio are: mannose: glucuronic acid: rhamnose: glucose: galactose: fucose = 1.05: 1.74: 6.29: 1.40: 69.48: 19.03.

[0006] Furthermore, the molecular weight of AGPA1-2 is approximately 6.72 kDa. Structural analysis shows that its main chain is primarily composed of α... 1,3,6 Gal、α 1,6 Gal and α 1,3,4,6 Composed of Gal, with branches containing α 1,3 Gal、α 1,5 Ara, α t Ara and α t Gal and other residues.

[0007] The present invention also provides the Gynostemma pentaphyllum acidic polysaccharide AGPA1. The preparation method of 2 includes the following steps: S1. Crush the Gynostemma pentaphyllum raw material, soak it in water, and add α. Amylase was hydrolyzed at 50-70℃ for 1-5 h, and the extract was obtained by filtration after inactivation. S2. Concentrate and centrifuge the extract, add 70%~90% ethanol to the collected supernatant, let it stand to precipitate, collect the precipitate and dry it to obtain Gynostemma pentaphyllum total polysaccharide AGP. S3. Dissolve the total polysaccharides of Gynostemma pentaphyllum and pass them through DEAE. Separation was performed using a Sepharose FF ion exchange column, followed by elution with water and NaCl gradients of varying concentrations. The 0.29 M NaCl eluent was collected. S4. Process the portion obtained in step S3 using Sepharose CL. The sample was further purified using a 6B gel chromatography column, and the main peak component was collected, dialyzed, and lyophilized to obtain the Gynostemma pentaphyllum acidic polysaccharide AGPA1-2.

[0008] Preferably, as an improvement, in step S1, α The amount of amylase added is 1% to 3% of the raw material mass, and the enzymatic hydrolysis pH is 5.5. 6.5, enzymatic hydrolysis time is 2 4 h.

[0009] Preferably, as an improvement, the final concentration of ethanol in step S2 is 80%, and the precipitation time is 8~24 h.

[0010] Preferably, as an improvement, in step S3 DEAE The elution conditions for the Sepharose FF column are: washing with water for 4 column volumes, and eluting with different concentrations of NaCl for 8 column volumes.

[0011] Preferably, as an improvement, in step S4, Sepharose CL The elution mobile phase for column 6B was 0.2 M NaCl, and the flow rate was 0.4–0.6 mL / min.

[0012] In vitro activity studies have shown that the AGPA1-2 described in this invention can significantly inhibit the proliferation and migration of human pancreatic cancer AsPC-1 cells and induce apoptosis. The mechanism is related to downregulating the expression of matrix metalloproteinases such as MMP2 / MMP9, regulating the Bax / Bcl-2 ratio, activating Caspase9 and Caspase3, and modulating the MAPK signaling pathway. Therefore, the AGPA1-2 described herein can be used to prepare drugs for treating pancreatic cancer. Attached Figure Description

[0013] Figure 1 Flowchart of total polysaccharide extraction process from Gynostemma pentaphyllum; Figure 2 Schematic diagram of the separation and purification process of Gynostemma pentaphyllum polysaccharides; Figure 3 AGPA1 was separated and purified using a Sepharose CL-6B gel chromatography column; Figure 4 Analysis of molecular weight and purity of AGPA1-1, AGPA1-2 and AGPA1-3; Figure 5 AGPA1 2 pairs of AsPC 1. Effect of cell viability (MTT assay) (n=5, compared with the control group, P < 0.05 P < 0.01); Figure 6 AGPA1 2 pairs of AsPC 1. Effects on cell migration (scratch assay) (n=3; compared with group 0, P < 0.05 P < 0.01; Bar = 100 μm; A. Changes in cell state in each group after 24 h of AGPA1-2 treatment; B. Statistical analysis of scratch healing area. Figure 7 AGPA1 2 pairs of AsPC 1. Effect of apoptosis-related protein expression (Western Blot) (n=3, compared with the 0 mg / mL group, P < 0.05 (P < 0.01) A. Migration-related protein bands; B. Statistical analysis of migration-related protein expression levels; Figure 8 : AO / PI double staining to detect the effect of AGPA1-2 on apoptosis of AsPC-1 cells (Bar=100 μm). Figure 9 Effect of AGPA1-2 on the expression levels of apoptosis-related proteins in AsPC-1 cells (n=3, compared with the 0 mg / mL group, P < 0.05 P < 0.01); Figure 10 Effects of AGPA1-2 on the expression levels of MAPKs-related proteins in AsPC-1 cells (n=3, compared with the 0 mg / mL group, P < 0.05 P < 0.01). Detailed Implementation

[0014] The following detailed description illustrates the specific implementation method: Example 1: Extraction of total polysaccharides from Gynostemma pentaphyllum Process flow as follows Figure 1 As shown, weigh 1 kg of dried Gynostemma pentaphyllum, pulverize it, add 20 L of distilled water, adjust the pH to 6.0, and add 1.5% α-propanediol. Amylase (w / w) was used for enzymatic hydrolysis at 60℃ for 3 h, followed by inactivation at 100℃ for 20 min. The mixture was filtered, and the filtrate was concentrated. The supernatant was collected after centrifugation at 4000 rpm (for a total of 20 min). Four volumes of 95% ethanol were added (final concentration 80%), and the mixture was allowed to stand overnight. The precipitate was collected after centrifugation at 4000 rpm (for a total of 20 min), reconstituted with distilled water, and the ethanol was evaporated by heating. The precipitate was then freeze-dried to obtain total polysaccharides from Gynostemma pentaphyllum (AGP).

[0015] Based on the above process, this study used the traditional water extraction and alcohol precipitation method combined with α-amylase-assisted extraction of total polysaccharide AGP from Gynostemma pentaphyllum. Each extraction of Gynostemma pentaphyllum raw material was 250 g, and a total of 4 extractions were performed, yielding a total of 132.89 g of total polysaccharide AGP from Gynostemma pentaphyllum, with an extraction rate of 13.28 ± 0.29%.

[0016] Table 1: Extraction of total polysaccharides from Gynostemma pentaphyllum

[0017] Example 2: Isolation and purification of Gynostemma pentaphyllum polysaccharides Separation and purification process as follows Figure 2 As shown, 1 g of AGP was dissolved in 40 mL of distilled water to obtain a polysaccharide solution of 25 mg / mL. The solution was centrifuged at 10000 r / min for 5 min, and the supernatant was collected and subjected to DEAE. Separation was performed using a Sepharose FF column (10 mm × 100 mm). The elution was carried out sequentially with distilled water, 0.29 M NaCl, and 0.57 M NaCl, each eluting four column volumes. Phenol was collected separately. Sugar content was determined by sulfuric acid method, and the 0.29 M NaCl eluent fraction (AGPA1) was combined, dialyzed, and lyophilized.

[0018] 200 mg of AGPA1 was dissolved in 6 mL of 0.2 M NaCl mobile phase solution. After complete dissolution, the solution was centrifuged at 10,000 rpm for 5 min. The supernatant sugar solution was filtered through a microporous membrane (0.45 μm) and then loaded onto a pre-equilibrated Sepharose CL-6B column for separation and elution. The mobile phase was 0.2 M NaCl, and the flow rate was 0.5 mL / min. The main peak fraction (AGPA1) was collected. 2), dialysis, freeze drying.

[0019] Based on the above procedure, the acidic sugar fraction AGPA1 was further purified using a Sepharose CL-6B gel chromatography column, and the results are as follows. Figure 3 As shown, AGPA1 was purified into three polysaccharide fractions by Sepharose CL-6B gel chromatography, namely AGPA1-1, AGPA1-2 and AGPA1-3, with yields of 2.12%, 7.50% and 12.43%, respectively.

[0020] Example 3: AGPA1 2 Structural Characterization AGPA1 was further separated using a Sepharose CL-6B column, and its molecular weight and purity were determined by HPGPC. The results are as follows: Figure 4 As shown, AGPA1 was separated into three fractions, AGPA1-1, AGPA1-2, and AGPA1-3, by a Sepharose CL-6B column, with yields of 2.12%, 7.50%, and 12.43%, respectively. The solvent peak was observed at 19.4 min. The AGPA1-2 signal peak was a single symmetrical peak, indicating that it is a homogeneous polysaccharide. AGPA1-1 and AGPA1-3, however, exhibited multiple elution peaks with uneven distribution, indicating non-uniform molecular weight. The molecular weight distribution of AGPA1-2 was measured to be approximately 6.72 kDa.

[0021] PMP pre-column derivative The monosaccharide composition was analyzed by HPLC, and the results are shown in Table 2. The monosaccharide composition of AGPA1-2 is Man:GlcA:Rha:Glc:Gal:Fuc = 1.05:1.74:6.29:1.40:69.48:19.03 (molar ratio).

[0022] Table 2: Monosaccharide composition of each fraction of AGP

[0023] As shown in Table 3, methylation analysis and nuclear magnetic resonance analysis show that this polysaccharide is mainly composed of Gal and Ara. The main chain is mainly composed of α-1,3,6-Gal, α-1,6-Gal and α-1,3,4,6-Gal, while the side chains are mainly composed of α-1,3-Gal (the largest proportion, 33.04%), α-1,5-Ara, α-t-Ara and α-t-Gal.

[0024] Table 3: Methylation analysis of AGPA1-2

[0025] The glycosidic bond configuration in the structure of AGPA1-2 was determined by two-dimensional HMBC NMR spectroscopy. The results suggest that the heteropolysaccharide is composed of galactose in the main chain and arabinose in the branched main chain. Its main structure is shown below: .

[0026] Physicochemical properties of Gynostemma pentaphyllum polysaccharides—determination of sugar content, protein content, and uronic acid content: Sugar content was determined using the sulfuric acid-phenol method, uronic acid content was determined using the m-phenylphenol method, and protein content was determined using the BCA method. The results are shown in Table 3.

[0027] Table 3: Physicochemical properties of Gynostemma pentaphyllum polysaccharides

[0028] Example 4: AGPA1 2. Study on anti-pancreatic cancer activity 1. MTT assay for the in vitro antitumor activity of Gynostemma pentaphyllum polysaccharides Human pancreatic cancer AsPC One cell was used as a model, and cell viability was detected by the MTT assay. The results showed that ( Figure 5 AGPA1-1, AGPA1-2, and AGPA1-3 isolated from AGPA1 also showed good inhibitory effects on ASPC-1 cell viability. Furthermore, analysis revealed that AGPA1-2 is a homogeneous polysaccharide; therefore, it was chosen as the focus of this study to further investigate its mechanism of action against pancreatic cancer.

[0029] 2. Effects of AGPA1-2 on AsPC-1 cell migration AsPC-1 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 3-5 × 10⁵ cells / well. When the cell density reached approximately 70%, streaking was performed. The cells were washed 1-2 times with PBS, and the streaks were observed and photographed under a microscope. The healing process was observed and photographed after 12 and 24 hours of treatment with AGPA1-2 at concentrations of 0, 100, 200, and 400 µg / mL, and the healed area was statistically analyzed using ImageJ.

[0030] The results are as follows Figure 6 As shown, compared to the control group, the scratch healing area gradually decreased with increasing drug concentration, indicating a gradual decrease in the number of migrating cells. Western blot results also showed that with increasing drug concentration, the expression of matrix metalloproteinases MMP2, MMP3, and MMP9 decreased. Figure 7 This suggests that AGPA1-2 may reduce ECM degradation and inhibit ASPC-1 cell migration by decreasing the expression of matrix metalloproteinases such as MMP2, MMP3, and MMP9.

[0031] 3. Effects of AGPA1-2 on AsPC-1 cell apoptosis The AO / PI double staining apoptosis detection kit is a commonly used method for detecting nuclear apoptosis in cells by double staining the cell nucleus with AO / PI, a technique frequently used in apoptosis morphology studies. AsPC-1 cells were treated with AGPA1-2 for 48 h and then subjected to AO / PI double staining. Apoptosis and necrosis were observed under a fluorescence microscope. The results are as follows: Figure 8 As shown, compared with the control group, the number of cells decreased relatively with the increase of drug concentration, while the number of red fluorescence increased, indicating that the ASPC-1 cell membrane was damaged and PI entered the apoptotic cell nucleus, thereby eliciting red fluorescence, indicating that AGPA1-2 induced apoptosis of ASPC-1 cells.

[0032] To further investigate the effect of AGPA1-2 on AsPC-1 cell apoptosis, AO / PI double staining experiments revealed that AGPA1-2 induced apoptosis in AsPC-1 cells. Therefore, the apoptosis mechanism was studied at the protein level. Western Blot results ( Figure 9 The results showed that with increasing drug concentration, the expression of the anti-apoptotic protein Bcl-2 decreased relatively, while the expression of the pro-apoptotic proteins Bid and Bax increased, and the relative expression of Cleaved-caspase3 and Cleaved-caspase9 proteins increased. This suggests that AGPA1-2 may induce apoptosis in AsPC-1 cells by reducing Bcl-2 expression, promoting Bid and Bax expression, activating Caspase9 expression, and further activating Caspase3.

[0033] 4. The impact of AGPA1-2 on the MAPK signaling pathway The mitogen-activated protein kinase signaling pathway, including important members such as ERK, p38, and JNK, is a crucial pathway for cell signaling. It plays a key role in gene expression regulation and cytoplasmic function, participating in various cell biological processes, such as proliferation, differentiation, apoptosis, and stress responses under both normal and pathological conditions. Treatment with AGPA1-2 for 24 hours yielded the following results: Figure 10 As shown, the phosphorylation levels of ERK1 / 2, p38, and JNK proteins all exhibited varying degrees of change. Specifically, the phosphorylation levels of ERK1 / 2, p38, and JNK proteins all increased with increasing drug concentration. These results suggest that AGPA1-2 may inhibit SW1990 cell migration and promote apoptosis by activating the expression of p38 and JNK phosphorylation in the MAPK signaling pathway.

[0034] 5. Results In summary, the Gynostemma pentaphyllum polysaccharide sample AGPA1-2, prepared using an α-amylase-assisted extraction process, exhibited significantly enhanced antitumor activity against the pancreatic cancer AsPC-1 cell line. Systematic in vitro activity experiments confirmed that AGPA1-2 dose-dependently inhibited tumor cell proliferation, the scratch assay showed a significant decrease in cell migration within 48 hours, and AO / PI double staining revealed typical apoptotic body formation and changes in membrane permeability. Further Western blot experiments showed that AGPA1-2 inhibited cell invasion and migration by downregulating the expression of MMPs (molecular molecular weight protein) series proteins. Western blot experiments on apoptosis-related proteins revealed that AGPA1-2 affected the expression of apoptosis-related proteins such as Bcl-2, Bax, Bid, Caspase 9, and Caspase 3 to varying degrees. Bcl-2 family proteins, as core regulators of apoptosis, determine cell life and death through intermolecular dynamics. Studies have shown that the expression ratio of Bcl-2 to Bax constitutes a molecular switch for apoptosis; an increased Bax / Bcl-2 ratio significantly inhibits DNA repair and promotes programmed cell death. AGPA1-2 can relatively reduce the expression of the anti-apoptotic protein Bcl-2, increase the expression of the pro-apoptotic proteins Bid and Bax, and increase the relative expression of Cleaved-caspase3 and Cleaved-caspase9 proteins. These results indicate that AGPA1-2 has a good anti-AsPC-1 tumor cell activity.

Claims

1. A type of Gynostemma pentaphyllum acidic polysaccharide AGPA1-2, characterized in that: The polysaccharide is mainly composed of mannose, glucuronic acid, rhamnose, glucose, galactose and fucose, with a molar ratio of 1.05:1.74:6.29:1.40:69.48:19.03 for each monosaccharide.

2. The Gynostemma pentaphyllum acidic polysaccharide AGPA1-2 according to claim 1, characterized in that: The molecular weight of AGPA1-2 is 6.72 kDa.

3. The Gynostemma pentaphyllum acidic polysaccharide AGPA1-2 according to claim 1, characterized in that: The main chain of AGPA1-2 is mainly composed of α-1,3,6-Gal, α-1,6-Gal and α-1,3,4,6-Gal, and the side chains are mainly composed of α-1,3-Gal, α-1,5-Ara, α-t-Ara and α-t-Gal.

4. A method for preparing the Gynostemma pentaphyllum acidic polysaccharide AGPA1-2 according to claim 1, characterized in that... Includes the following steps: S1. Crush the Gynostemma pentaphyllum raw material, soak it in water, and add α. Amylase was hydrolyzed at 50-70℃ for 1-5 h, and the extract was obtained by filtration after inactivation. S2. Concentrate and centrifuge the extract, add 70%~90% ethanol to the collected supernatant, let it stand to precipitate, collect the precipitate and dry it to obtain Gynostemma pentaphyllum total polysaccharide AGP. S3. Dissolve the total polysaccharides of Gynostemma pentaphyllum and pass them through DEAE. Separation was performed using a Sepharose FF ion exchange column, followed by elution with water and NaCl gradients of varying concentrations. The 0.29 M NaCl eluent was collected. S4. Process the portion obtained in step S3 using Sepharose CL. The sample was further purified using a 6B gel chromatography column, and the main peak component was collected, dialyzed, and lyophilized to obtain the Gynostemma pentaphyllum acidic polysaccharide AGPA1-2.

5. The method according to claim 4, characterized in that: In step S1, α The amount of amylase added is 1% to 3% of the raw material mass, and the enzymatic hydrolysis pH is 5.

5. 6.5, enzymatic hydrolysis time is 2 4 h.

6. The method according to claim 4, characterized in that: In step S2, the final concentration of ethanol is 80%, and the precipitation time is 8-24 h.

7. The method according to claim 4, characterized in that: DEAE in step S3 The elution conditions for the Sepharose FF column are: washing with water for 4 column volumes, and eluting with different concentrations of NaCl for 8 column volumes.

8. The method according to claim 4, characterized in that: Sepharose CL in step S4 The elution mobile phase for column 6B was 0.2 M NaCl, and the flow rate was 0.4–0.6 mL / min.

9. The use of the Gynostemma pentaphyllum acidic polysaccharide AGPA1-2 according to claim 1 in the preparation of anti-pancreatic cancer drugs.

10. The application according to claim 9, characterized in that: The anti-pancreatic cancer drug is used to inhibit the proliferation, migration, or induce apoptosis of pancreatic cancer cells.

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