Novel gypenosides based on gypenosides and preparation and application thereof

Abstract

A new gypenoside based on gypenoside and its preparation and application, namely (4ζ)-α-terpineol 8-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside], the molecular formula is C 21 H 36 O 10 , the structural formula is as follows: the application is obtained by extraction, purification and separation from gypenoside, has the activity of reducing lipid and reducing cholesterol, has the purpose of being prepared into lipid-lowering and cholesterol-lowering drugs, meanwhile, the application is simple in operation and high in feasibility, the prepared compound is high in purity, and has the prospect of industrial application.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of compound applications, and particularly relates to a new gypenoside based on gypenoside and a preparation method and application thereof. BACKGROUND

[0002] The main chemical components of gypenoside are saponins (Gypenosides, GPs), flavonoids, polysaccharides, amino acids and various trace elements. Gypenoside is the main active component of gypenoside, and the content is more than 2%, even up to 15%. So far, 574 compounds have been identified from gypenoside, among which gypenoside LVI, gypenoside XLVI, ginsenoside Rd, ginsenoside Rb1, gypenoside XVII and gypenoside A are main saponin components. Studies have proved that gypenoside has pharmacological effects such as anti-tumor, blood glucose reduction, anti-aging, cardiovascular system regulation, immune regulation and liver protection, and can be used for treating tumors, hyperlipidemia, non-alcoholic fatty liver disease and oral diseases in clinical practice. SUMMARY

[0003] The application provides a new gypenoside based on gypenoside and a preparation method and application thereof, which is obtained by extraction, purification and separation from gypenoside, has lipid-lowering and cholesterol-lowering activities, and has the use of being prepared into lipid-lowering and cholesterol-lowering drugs. The application has the advantages of simple operation, high feasibility, high purity of the prepared compound and industrial application prospect.

[0004] The application is achieved by the following technical scheme:

[0005] The application relates to a new gypenoside based on gypenoside, that is, (4ζ)-α-terpineol 8-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside], which has a molecular formula of C 21 H 36 O 10 and a structural formula as shown in the following formula:

[0006] .

[0007] The application relates to a preparation method of the new gypenoside, which is obtained by elution and chromatography from gypenoside extract.

[0008] The elution and chromatography specifically include the following steps.

[0009] In step 1, the extract is prepared into a concentrated solution, and then is loaded into a macroporous resin and eluted with water and ethanol respectively, and then the ethanol part is collected and concentrated and dried to obtain gypenoside extract.

[0010] Step 2, the gynostemma pentaphyllum extract is mixed with silica gel, and then is loaded on a silica gel column, gradient elution is carried out by using dichloromethane-methanol solution as an eluent, the eluent is collected, and each fraction is combined and grouped according to thin layer chromatography analysis and high performance liquid chromatography analysis.

[0011] The elution is carried out at an elution ratio of 9:1, 4:1, 3:1 or 2:1.

[0012] Step 3, the target compound is taken from a group according to the liquid phase result, is dissolved in methanol, is loaded on a sephadex column, and is isocratically eluted with methanol as an eluent to remove impurities such as pigments.

[0013] Step 4, the fraction containing the compound is collected and subjected to ODS column chromatography and gradient elution with a methanol-water solution.

[0014] The elution is carried out at an elution ratio of 1:1 to 9:1.

[0015] Step 5, the fraction containing the compound is collected and subjected to ODS column chromatography and gradient elution with a methanol-water solution.

[0016] The elution is carried out at an elution ratio of 7:3 to 9:1.

[0017] Step 6, the compound is obtained by separation and purification through liquid chromatography with an acetonitrile-water solution.

[0018] The separation and purification are carried out at an elution ratio of 3:7.

[0019] The gynostemma pentaphyllum extract is preferably obtained by crushing and sieving dried gynostemma pentaphyllum, heating and refluxing in an ethanol solution and concentrating extraction.

[0020] The refluxing is carried out by using 50% to 90% ethanol solution at a material-liquid ratio of 1:6 to 1:10, under the condition of 85 DEG C, for 2 to 4 times, and each time for 1 to 2 hours, and the extract is obtained by concentrating the extraction solution.

[0021] The application relates to the application of the new gypenoside, and the new gypenoside is used for preparing a medicine for reducing cholesterol and triglyceride.

[0022] The application is characterized in that the concentration of the new gypenoside is 100 muM to 500 muM. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The new gypenoside is 13 C-NMR chart (C5D5N, 600MHz) chart;

[0024] Figure 2 The new gypenoside is 1 H-NMR chart (C5D5N, 600MHz) chart;

[0025] Figure 3 For new monoterpenoids 1 H- 1 HCOSY diagram;

[0026] Figure 4 The HMQC diagram for the new monoterpene;

[0027] Figure 5 HMBC diagram of the new monoterpene;

[0028] Figure 6 The NOESY diagram for the new monoterpenoid;

[0029] Figure 7 The image shows the HR-ESI-MS (positive ion mode) of the new monoterpene.

[0030] Figure 8 The effects of novel monoterpenoids on the survival rate of HepG2 cells and TC and TG levels in high-cholesterol model cells;

[0031] In the figure: #P<0.05; **P<0.0; ***P<0.001;

[0032] Figure 9 The effects of new monoterpenoids on the expression of NPC1L1, HMGCS1, SREBP2, and SREBP1 proteins in HepG2 cells;

[0033] In the figure: #P<0.05; ##P<0.01; ###P<0.001; *P<0.05. Detailed Implementation

[0034] Example 1

[0035] This embodiment relates to a method for preparing a novel monoterpene glycoside based on Gynostemma pentaphyllum, including:

[0036] Step 1: Weigh the dried Gynostemma pentaphyllum raw material, crush and sieve it, and extract it three times with 70% ethanol at 85℃ for 1 hour each time, using a material-to-liquid ratio of 1:8. Filter, discard the residue, and combine the extracts. Concentrate under reduced pressure using a rotary evaporator at 45℃ and 0.10 MPa to obtain a crude extract of Gynostemma pentaphyllum. Place the extract in a petri dish, dry it, and store it in a desiccator.

[0037] Step 2, the gynostemma pentaphyllum crude extract was first subjected to macroporous resin column chromatography, macroporous resin D101 was selected, and water and 95% ethanol were used for elution. The water phase was mainly composed of sugars and had little saponin content, so it was discarded. The 95% ethanol phase was mainly composed of saponins, and was concentrated and vacuum dried to obtain the gynostemma pentaphyllum ethanol phase (492.7 g) for further enrichment of gynostemma pentaphyllum saponins. The gynostemma pentaphyllum ethanol phase (200 g) was subjected to silica gel column chromatography, and silica gel with a particle size of 300-400 was selected. Gradient dichloromethane-methanol (9:1; 4:1; 3:1; 2:1) was used for elution, and the composition of each fraction was tested by thin layer chromatography. Fractions 1-12 were obtained by combining the fractions. (Fr1: 0.6507 g; Fr.2: 7.6954 g; Fr.3: 1.9001 g; Fr.4: 6.2102; Fr.5: 6.6017 g; Fr.6: 47.2588 g; Fr.7: 25.5523 g; Fr.8: 27.7608 g; Fr.9: 15.3051 g; Fr.10: 66795 g; Fr.11: 17.5061 g; Fr.12: 5.2192 g) The composition of Fr.1-Fr.12 was further analyzed by high performance liquid chromatography.

[0038] The chromatography method is as follows: chromatographic column: Agilent Eclipse XDB-C18 (4.6 mm x 250 mm, 5 μm); mobile phase: 0.3% formic acid aqueous solution (A)-acetonitrile (B), gradient elution (0-15 min, 30%B→40%B; 15-35 min, 40%B→60%B; 35-35.5 min, 60%B→90%B; 35.5-40 min, 90%B→90%B); flow rate: 1 mL·min-1; detection wavelength: 203 nm; column temperature: 35°C; injection volume: 10 μL.

[0039] Step 3, according to the chromatographic results, 30 g of Fr.6 was dissolved in pure methanol and separated by silica gel column chromatography. Isocratic elution was performed with dichloromethane-methane (5:1) to obtain Fr.6-1~Fr.6-6. Fr.6-5 was first purified by Sephadex column, isocratic elution with methanol, and further purified by ODS column, gradient elution with 50%~90% methanol-water to obtain Fr.6-5-1~Fr.6-5-5. Fr.6-5-5 was purified by preparative high performance liquid chromatography, and the purification isocratic was 30% acetonitrile-water, the flow rate was 3 mL / min, and the detection wavelength was 210 nm to obtain a new compound (tR: 12.47 min).

[0040] The new compound (white powder): HR-ESI-MS gives quasi-molecular ion peak m / z 447.2206 [M-H] + (C 21 H 35 O 10 , calculated value 447.2705), further combined 1 H-NMR spectrum and 13 C-NMR spectrum (Table 1) to determine the molecular formula of the compound C 21 H 36 O 10 . The compound's 13 C - NMR (Table 1) shows signals at d (C) 98.6 (d), 75.2 (d), 78.6 (d), 71.5 (d), 76.7 (d), 70.0 (t), which belong to the internal β-glucopyranose, and the compound's 13 C - NMR (Table 1) shows signals at d (C) 98.6 (d), 75.2 (d), 78.6 (d), 71.5 (d), 76.7 (d), 70.0 (t), which belong to the internal β-glucopyranose, and the compound's C 133.7 (C-1), 121.4 (C-2), which indicates that there is a double bond part at C-1, C-2 position. The HMBC correlation data also support this conclusion, such as δ H 5.37 (H-1) and δ C 121.4 (C-2) correlation. The NMR spectrum of the new compound is very similar to the known compound, and the structure of the aglycone part is consistent. But the C(6') of the new compound is connected with xylose, while the C(6') of the known compound is connected with arabinose. By acid hydrolysis of the compound to obtain the monosaccharide of the compound and derivatize it to monosaccharide compound, and derivatize the monosaccharide standard to obtain monosaccharide standard derivative, by comparing the chromatographic retention time of the monosaccharide derivative of the compound and the monosaccharide standard derivative, it is determined that the monosaccharide in the compound is D-glucose (t R = 15.3 min), D-xylose (t R = 17.7 min).

[0041] In summary, the isolated compound is identified as (4ζ)-α-terpineol 8-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside], the structural formula is, and it is confirmed as a new compound. 1 H NMR and 13 C NMR signal assignment is shown in Table 1. The nuclear magnetic and mass spectrometry results are shown in Figures 1 to 7 .

[0042]

[0043] Table 1 New compounds of 13 C- and 1 H-NMR data (600 MHz; C5D5N)

[0044] Position New compound δ (C) δ (H) 1 133.7(s) 2 121.4(d) 5.37(m) 3 24.2(t) 1.81 (m), 2.18 (m) 4 44(d) 1.59(m) 5 23.5(t) 1.25 (m), 1.83 (m) 6 31.1(t) 1.81 (m), 2.02 (m) 7 22.9(q) 1.58(s) 8 79.3(s) 9 27.2(q) 1.31(s) 10 24.1(q) 1.42(s) Suger moiety 1’ 98.6(d) 4.73(m) 2’ 75.2(d) 3.68(m) 3’ 78.6(d) 4.12 (t, J = 8.3) 4’ 71.5(d) 4.02(m) 5’ 76.7(d) 4.01(m) 6’ 70.0(t) 4.34 (dd, J = 11.0, 2.0), 4.20 (dd, J = 8.3, 3.3) 1’’ 105.8(d) 4.96 (d, J = 2.7) 2’’ 74.8(d) 3.91 (d, J = 4.3) 3’’ 78.1(d) 4.02(m) 4’’ 71.0(d) 4.13 (dd, J = 8.3, 3.3) 5’’ 67.0(d) 4.20 (m), 3.60 (d, J = 5.1)

[0045] Example 2

[0046] This example detects the effect of new compounds on cell survival rate and improves the activity of high cholesterol model cells, specifically including:

[0047] Step 1, take the HepG2 cells in the logarithmic growth phase, with a density of 1*10 4 cell / cm 2 inoculated in 96-well plates, then given 10 mg / L cholesterol + 1 mg / L 25-OH cholesterol for 24 hours to establish an in vitro liver cell model. The success of the cholesterol model was evaluated by measuring the intracellular cholesterol content and the expression of cholesterol metabolism-related proteins.

[0048] Step 2, detect the effect of new compounds on the proliferation ability of normal HepG2 cells. Take the HepG2 cells in the logarithmic growth phase, with a density of 5*10 3 cell / cm 2 inoculated in 96-well plates, and cultured in a cell incubator for 24 h.

[0049] Step 3, discard the culture medium, and add new compounds with concentrations of 50, 100, 200, and 400 μM respectively for 24 h, then detect the cell survival rate of HepG2 cells by CCK-8 method.

[0050] As Figure 2 shown by the detection results, the growth and reproduction ability of HepG2 cells is not inhibited by the new compounds between 50 and 400 μM concentrations, and the concentration range of 50-400 μM is the drug safety window.

[0051] Further detection of the anti-high cholesterol activity of gypenoside, specifically including:

[0052] Step a, take the HepG2 cells in the logarithmic growth phase, with a density of 2*10 5 cell / cm 2 inoculated in 6-well plates, and cultured in a cell incubator for 24 h, then discard the culture medium.

[0053] Step b, the experiment was divided into 4 groups: blank control group: add 2 mL complete medium; model group: add 2 mL complete medium prepared cholesterol (10 mg / L) and 25-hydroxycholesterol (1 mg / L) mixed solution; new compound treatment group: add 2 mL serum-free medium prepared cholesterol (10 mg / L), 25-hydroxycholesterol (1 mg / L) and different concentrations of saponin compound (50, 100, 200 μmol / L) mixed solution. Each group of cells was cultured for 24 h and used for subsequent experiments.

[0054] Step c, detect the total cholesterol content in the cells. Add 1 mL anhydrous ethanol to each well of the cells, and ultrasonically break (power 20%, ultrasonic 3 s, interval 7 s, repeat 30 times) to extract the intracellular lipids. Centrifuge at 8000 g and 4°C for 10 min, take the supernatant, and place it on ice for detection. The total cholesterol content in the cells was determined by total cholesterol determination kit.

[0055] As shown in Figure 2 , the intracellular cholesterol of the model group was significantly increased, and the new compound could reduce the intracellular cholesterol content of HepG2 cells in a dose-dependent manner.

[0056] Secondly, the intracellular triglyceride content was detected: the logarithmic growth period HepG2 cells were inoculated in 6-well plates at 2×10 5 cells / well, and each group of cells was treated according to the grouping. The cell supernatant was aspirated, washed with phosphate buffer solution (PBS) for 3 times, 100 μL cell lysis solution was added, and the lysis solution was fully reacted by gently shaking. The cells were collected in a centrifuge tube, centrifuged at 2000 r / min and 4°C for 5 min, and the supernatant was taken. The intracellular TG content was determined according to the kit instructions.

[0057] As shown in Figure 8 , the intracellular triglyceride of the model group was significantly increased, and the new compound could reduce the intracellular triglyceride content of HepG2 cells in a dose-dependent manner.

[0058] Example 3

[0059] This example detects the improvement effect of the new compound on high cholesterol cell activity related proteins:

[0060] Cholesterol metabolism related proteins: NPC1L1, HMGCS1, SREBP-2 and SREBP-1. NPC1L1 is a key protein for intestinal absorption of dietary and biliary cholesterol. HMGCS1 is a key protein for regulating cholesterol synthesis. SREBP-2 is a key protein for regulating cholesterol synthesis and metabolism. SREBP-1 is a key protein for regulating fatty acid and triglyceride synthesis and metabolism.

[0061] In-Cell Western-Blot method for detecting cholesterol metabolism related proteins: logarithmic growth phase cells were trypsinized and centrifuged, resuspended into cell suspension after adding complete medium, inoculated in 96-well plates at a concentration of 1x10 4 After 24 h incubation in a cell incubator, the supernatant was discarded. The experiment was divided into model group, experimental group and control group, and complete medium containing modeling agent, complete medium containing modeling agent and different concentrations of compounds, and complete medium were added respectively. The experimental group was divided into high-dose group (200 μM) and low-dose group (100 μM) of new compounds. 150 μL of paraformaldehyde was added to each well for fixation, and the plate was placed at room temperature for 20 min. The paraformaldehyde solution was discarded, and 200 μL of PBS containing 1% Triton X-100 was added to each well, and the plate was shaken at room temperature for 5 min, repeated 4 times. 150 μL of blocking solution was added to each well, and the plate was shaken at room temperature for 1.5 h. The corresponding primary antibody was diluted with blocking solution at a ratio of 1:800, and 50 μL of diluted primary antibody was added to each well. The remaining well in each group was not added with primary antibody as a blank control. The 96-well plate was placed in a 4 °C refrigerator overnight or shaken at room temperature for 2 h. The primary antibody was recovered and reused. 200 μL of PBS containing 0.1% Tween 20 was added to each well and shaken for 5 min, repeated 5 times. The corresponding secondary antibody (1:800) was diluted with blocking solution containing 0.1% Tween 20, 50 μL was added to each well, and the plate was shaken at room temperature for 1 h in the dark. 200 μL of PBS containing 0.1% Tween 20 was added to each well and shaken for 5 min, repeated 5 times. Detection was performed on the Odyssey infrared imaging system.

[0062] As Figure 9As shown, compared with the control group, the model group has increased intracellular cholesterol content, and reduced NPC1L1, HMGCS1, SREBP-2 and SREBP-1 protein content. Compared with the model group, the drug group improves the high-fat and high-cholesterol state of the cells, significantly increases the NPC1L1 protein content, and has a concentration-dependent effect. The HMGCS1, SREBP-2 and SREBP-1 protein contents have an increasing trend. The results show that the lipid-lowering and cholesterol-lowering activity of the new compound may be related to the reduction of NPC1L1 content. Cholesterol, as a signal molecule, can bind to NPC1L1 protein and trigger the subsequent absorption process. The transcription of NPC1L1 is regulated by SREBP2, and the new compound has a trend of increasing SREBP2. At the same time, under the condition of high cholesterol in cells, cholesterol can inhibit related transcription factors (such as SREBP2), reduce HMGCS1 transcription, accelerate its degradation, and reduce its content. The new compound can moderately reverse this change and has a trend of increasing HMGCS1. In addition, under the condition of high cholesterol in cells, lipids and triglycerides are also abnormal, and the expression of related protein SREBP1 is inhibited. The new compound can moderately reverse this change and has a trend of increasing SREBP1, and there is a concentration-dependent effect.

[0063] Compared with the prior art, the performance index of the present application is improved in that a new monoterpene glycoside compound is isolated from Gynostemma pentaphyllum, which is first disclosed to directly intervene in the cholesterol synthesis and intestinal absorption pathway by significantly up-regulating the expression of NPC1L1 protein (in a concentration-dependent manner) and regulating the expression trend of key cholesterol metabolism proteins such as HMGCS1, SREBP-2 and SREBP-1. Compared with the existing total gypenosides or a single known saponin (such as TN-1) which only focuses on the regulation of sugar metabolism or general lipid-lowering effect, the present method realizes specific targeted intervention on the cholesterol metabolism network at the molecular mechanism level, and provides a new technical path for developing cholesterol metabolism regulators based on new structural compounds.

[0064] The above specific embodiments can be adjusted in different ways by those skilled in the art without departing from the principles and purposes of the present application, and the protection scope of the present application is subject to the claims and is not limited by the above specific embodiments. Each implementation within the scope is subject to the constraints of the present application.

Claims

1. A method for preparing a new gypenoside, characterized in that, The dried gynostemma is crushed and sieved, then extracted by heating refluxing in ethanol solution and concentrating to obtain gynostemma extract, which is eluted and chromatographed to obtain; The elution and chromatography specifically includes: Step 1, the extract is prepared into a concentrated solution, then loaded onto a macroporous resin, eluted with water and ethanol respectively, and then the ethanol eluate is collected, concentrated and dried to obtain gynostemma extract; Step 2, the gynostemma extract is mixed with silica gel, loaded onto a silica gel column, and gradient eluted with dichloromethane-methanol solution as eluent, then the eluate is collected, and each fraction is combined and grouped according to thin layer chromatography analysis and high performance liquid chromatography analysis; Step 3, the target compound is taken from the group according to the liquid chromatography result, dissolved in methanol, and loaded onto a sephadex column, and isocratic eluted with methanol as eluent to remove impurities; Step 4, the fraction containing the compound is collected and chromatographed on an ODS column with methanol-water solution for gradient elution; Step 5, the fraction containing the compound is collected and chromatographed on an ODS column again with methanol-water solution for gradient elution; Step 6: The new single glycoside was purified by liquid chromatography with acetonitrile-water solution, which is (4ζ)-α-terpineol 8-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside], with molecular formula of C 21 H 36 O 10 and structural formula: ， The new monopetasin 13 The C-NMR chart is shown in Figure 1. 1 The H-NMR chart is shown in Figure 2. 1 H- 1 The HCOSY chart is shown in Figure 3; the HMQC chart is shown in Figure 4; the HMBC chart is shown in Figure 5; and the NOESY chart is shown in Figure 6.

2. The method of claim 1, wherein, The refluxing is performed with 50%-90% ethanol solution at a material-liquid ratio of 1:6-1:10 at 85℃ for 2-4 times, each time for 1-2 hours, and the extract is obtained by concentrating the extractive.

3. Use of the new singuphoside prepared by the process according to claim 1 or 2, characterized in that, The gynostemma extract is used for preparing a medicine for reducing cholesterol and triglyceride.

4. Use according to claim 3, characterized in that, The concentration of the new gypenoside ranges from 100 μM to 500 μM.

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