Compound with triterpenoid saponin structure as well as preparation method and application of compound

Abstract

The invention relates to a compound with a triterpenoid saponin structure as well as a preparation method and application thereof, and the structure of the compound with the triterpenoid saponin structure is shown in the following formula (I) or (II): # imgabs0 #, wherein R1 represents glucosyl or methyl; r2 represents a methyl group, a hydroxymethyl group, a glucosyl group or a glucodisaccharide group; r3 represents a glucosyl group or a hydroxymethyl group; r4 represents hydrogen or hydroxyl; and R5 represents a hydroxyl group or a glucose triglycosyl group. According to the invention, active ingredients of pimpinella amygdaliana are extracted, a series of novel compounds with triterpenoid saponin structures are obtained for the first time, and further cell viability verification and function verification prove that the compounds with the triterpenoid saponin structures have no cytotoxicity and can promote differentiation of osteoblasts; animal experiments prove that the compound with the triterpenoid saponin structure can be used for treating osteoporosis, and is clear in effect and safe.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines. Specifically, it relates to a compound with a triterpenoid saponin structure, and particularly to a compound with a triterpenoid saponin structure, its preparation method and uses. Background Art

[0002] Pimpinella candolleana Weight et Arn. is the root or whole herb of the plant of the genus Pimpinella in the Umbelliferae family. In Miao medicine, it is called "mountain angelica" and "goat odor". It is a plant of the genus Pimpinella distributed in the southwestern region of China. Its root or whole herb is used as medicine, with the effects of warming the middle-jiao to dispel cold and promoting qi flow to relieve pain. Folk medicine research shows that the underground part of this plant is used as medicine and has a good therapeutic effect on wind-cold colds; while the fresh above-ground part is mostly used to treat diseases such as cold pain in the epigastric and abdominal regions. In recent years, this medicine has been developed into Huangxuan Yigan Powder for the treatment of chronic hepatitis B and put into use. However, the research on the material basis and pharmacodynamics of Pimpinella candolleana is relatively less. Only 2 pieces of literature have reported the flavonoid and steroid components such as ursolic acid and luteolin, which have activities such as antioxidant, antibacterial and hypoglycemic in vitro.

[0003] Triterpenoid saponins are composed of triterpenoid sapogenins, sugars, uronic acids and other organic acids. More than 30 types have been discovered. Except for a few acyclic triterpenoids, bicyclic triterpenoids and tricyclic triterpenoids, they are mainly tetracyclic triterpenoids and pentacyclic triterpenoids. Triterpenoid saponins have currently become the most active and fastest-developing research field in natural products, with a variety of biological activities and pharmacological effects, such as anti-inflammatory, anti-tumor, hypoglycemic, etc. And the research on the anti-osteoporosis of triterpenoid saponin components has also been reported. Chen Dihua et al. extracted 9,19-cyclolanostane-type triterpenoid saponin components from the traditional Chinese medicine Cimicifuga foetida L., which have pharmacological activities such as antagonizing calcium ions, inhibiting bone loss and analgesic and anti-inflammatory effects. Yu Dayong et al. found through research that the triterpenoid saponin components extracted from Achyranthes bidentata Blume can inhibit the differentiation of osteoclasts, so as to achieve the purpose of preventing and treating osteoporosis. Yang Yue et al. proved through experiments that the active components in the total saponins of Aconitum carmichaelii Debx. can effectively negatively regulate the differentiation and bone resorption of osteoclasts induced by RANKL, providing a basis for the treatment of osteoporosis with Aconitum saponins.

[0004] Searching for and obtaining new compounds with triterpenoid saponin structures and studying their pharmacological effects have great significance. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a compound with a triterpenoid saponin structure, its preparation method and uses.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a compound having a triterpenoid saponin structure, and the structure of the compound is shown by the following formula (I) or (II):

[0008]

[0009] Wherein, R1 represents a glucosyl group or a methyl group; R2 represents a methyl group, a hydroxymethyl group, a glucosyl group or a glucosyl disaccharide group; R3 represents a glucosyl group or a hydroxymethyl group; R4 represents hydrogen or a hydroxyl group; R5 represents a hydroxyl group or a glucosyl trisaccharide group.

[0010] As a preferred embodiment, the structure of the compound is shown by the following formulas CP-1 to CP-8:

[0011]

[0012]

[0013] In a second aspect, the present invention provides a method for preparing a compound having a triterpenoid saponin structure, comprising the following steps:

[0014] A. After drying and pulverizing the whole herb of Pimpinella candolleana, adding ethanol with a volume fraction of 50-90% for extraction, and concentrating the obtained extract under reduced pressure until the alcohol smell disappears to obtain an extract paste;

[0015] B. After diluting the extract paste with water, extracting successively with petroleum ether, ethyl acetate and n-butanol, and recovering the solvents under reduced pressure to obtain a petroleum ether fraction extract, an ethyl acetate fraction extract and an n-butanol fraction extract respectively;

[0016] C. Removing the pigment from the n-butanol fraction extract, loading it onto a normal-phase silica gel column with 100-200 mesh, and performing gradient elution with a mixed solution of dichloromethane and methanol. After detecting the eluate by thin-layer chromatography, collecting the elution fractions containing the triterpenoid saponin structures shown by formulas (I) and (II);

[0017] D. Further eluting and subdividing the elution fractions containing the triterpenoid saponin structures shown by formula (I) or (II) to obtain the compounds having the triterpenoid saponin structures shown by each of formulas (I) or (II).

[0018] As a preferred embodiment, in step A, the number of extractions is 2-5 times, the extraction temperature is 80-90 °C, and the extraction time for each extraction is 1-3 h.

[0019] As a preferred embodiment, in step A, ethanol with a volume fraction of 70-90% is added for extraction.

[0020] As a preferred embodiment, in step B, the extract paste is diluted with 2-6 times the amount of water; more preferably, it is diluted with 3-5 times the amount of water.

[0021] As a preferred embodiment, in step B, petroleum ether, ethyl acetate and n-butanol in amounts of 2 - 6 times are respectively used for extraction, and the extraction times for each solvent are 2 - 4 times; more preferably, petroleum ether, ethyl acetate and n-butanol in amounts of 3 - 5 times are respectively used for extraction, and the extraction times for each solvent are 3 times.

[0022] As a preferred embodiment, in step C, in the mixed solution of dichloromethane and methanol used for gradient elution, the volume ratios of dichloromethane to methanol are successively 1:0, 100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1 and 1:1.

[0023] As a preferred embodiment, in step D, the specific steps for further fractionation by elution are: the elution fractions containing the triterpenoid saponin structures shown in formulas (I) and (II) are fractionated by gradient elution with an aqueous methanol solution, and the elution sub-fractions containing the triterpenoid saponin structures shown in each of formulas (I) or (II) are obtained.

[0024] In the aqueous methanol solution used for the gradient elution fractionation, the volume ratios of methanol to water are successively 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9.

[0025] As a preferred embodiment, the method further includes a step of subjecting the compounds with the triterpenoid saponin structures shown in each of formulas (I) or (II) obtained in step D to purification by preparative high performance liquid chromatography to obtain the compounds with the triterpenoid saponin structures shown in each of formulas (I) or (II).

[0026] As a preferred embodiment, the purification is carried out by elution with an aqueous methanol solution; the volume concentration of methanol in the aqueous methanol solution is 50 - 80%, and the volume of the aqueous methanol solution is 8 - 12 L. More preferably, the volume concentration of methanol is 55 - 75%, and the volume of the aqueous methanol solution is 10 L.

[0027] In a third aspect, the present invention provides the use of a compound with the triterpenoid saponin structure as described above in the preparation of a drug or health product for promoting the proliferation and differentiation of osteoblasts or treating osteoporosis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] By extracting the active ingredients from Pimpinella candolleana, the present invention for the first time obtains a series of new compounds with triterpenoid saponin structures, the structures of which are shown in formula I or II. Further, through cell activity verification and function verification, it is proved that the compounds with the triterpenoid saponin structures have no cytotoxicity and can promote the osteogenic differentiation of osteoblasts; through animal experiments, it is proved that the compounds with the triterpenoid saponin structures can be used for treating osteoporosis, and the efficacy is clear and it has safety. Description of the Drawings

[0030] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments read in conjunction with the accompanying drawings:

[0031] Figure 1 Key for the novel triterpenoid saponin compounds CP-1 to CP-8 derived from Pimpinella candolleana 1 H- 1 H COSY (thick red lines) and HMBC signals (blue arrows).

[0032] Figure 2 Key NOESY signals (blue dashed arrows) for the novel triterpenoid saponin compounds CP-1 to CP-8 derived from Pimpinella candolleana

[0033] Figure 3 Effects of treatments with different concentrations of CP-1 to CP-8 on osteoblast activity and differentiation; wherein, Figure 3 A shows the results of the CCK8 assay, indicating that CP-1 to CP-8 do not affect the proliferation of osteoblasts at different administration concentrations (10 μM, 20 μM, and 50 μM) and treatment times (24 h, 48 h, and 72 h) (n = 6); Figure 3 B shows the results of the ALP assay staining; Figure 3 C shows the results of alizarin red S staining, note: scale bar, 100 μm; Figure 3 D shows the quantitative analysis of the positive area of alizarin red S staining (n = 3, *P < 0.05, ****P < 0.0001).

[0034] Figure 4 Line graph showing the changes in body weight of mice in each group during modeling and drug administration; wherein, OVX: ovariectomized modeling group; Sham: sham operation group; OVX + AS: ovariectomized + alendronate treatment group (10 mg / kg); OVX + CP-1 (30 mg / kg): ovariectomized + low-dose CP-1 group; OVX + CP-1 (60 mg / kg): ovariectomized + medium-dose CP-1 group; OVX + CP-1 (120 mg / kg): ovariectomized + high-dose CP-1 group (n = 6).

[0035] Figure 5 HE staining images of the heart, liver, spleen, lungs, and kidneys of mice in each group (n = 3); note: scale bar, 200 μm.

[0036] Figure 6 Statistical analysis of the trabecular bone structure and bone parameters of the femurs of mice in each group; wherein, Figure 6 A shows representative images of the three-dimensional reconstruction of Micro-CT of mice, upper part: three-dimensional reconstruction of trabecular bone; middle part: sagittal plane of the overall three-dimensional reconstruction of the femur; lower part: cross-section of the femur; Figure 6 B shows BMD (bone mineral density);Figure 6 C is BV / TV (bone volume density); Figure 6 D is BS / TV (bone surface area and tissue volume ratio); Figure 6 E is Tb.N (trabecular bone number); Figure 6 F is Tb.Pf (trabecular bone pattern factor; Figure 6 G is the quantitative analysis result of Tb.Sp (trabecular bone separation) (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0037] Figure 7 It is the comparison of the contents of BMP-2, OCN, OPG, RANKL, and OPG / RANKL ratio in the sera of mice in each group after drug intervention; among them Figure 7 A is BMP-2; Figure 7 B is OCN; Figure 7 C is OPG; Figure 7 D is RANKL; Figure 7 E is the ratio of OPG / RANKL (n = 6, *P < 0.05, **P < 0.01). Specific implementation manners

[0038] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0039] In the specific implementation manner of the present invention, a preparation method of a compound with a triterpenoid saponin structure is provided, including the following steps:

[0040] A. After drying and pulverizing the whole herb of Pimpinella candolleana, add ethanol with a volume fraction of 50-90% and extract at 80-90°C for 2-5 times, with each extraction time being 1-3 h. Combine the obtained extraction liquids and concentrate under reduced pressure until the alcohol smell disappears to obtain an extract;

[0041] B. Dilute the extract with 2-5 times the amount of water, and then extract with 2-6 times the amount of petroleum ether, ethyl acetate, and n-butanol respectively for 2-4 times. Recover the solvents under reduced pressure to obtain the petroleum ether fraction extraction liquid, ethyl acetate fraction extraction liquid, and n-butanol fraction extraction liquid respectively;

[0042] C. Remove the pigments from the extract of the n-butanol fraction, load it onto a normal-phase silica gel column with 100 - 200 mesh, and perform gradient elution with a mixed solution of dichloromethane and methanol (the volume ratios of dichloromethane to methanol are successively: 1:0, 100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, and 1:1). After detecting the eluate by thin-layer chromatography, collect each elution fraction;

[0043] D. Dissolve the three elution fractions collected by eluting with dichloromethane and methanol at a volume ratio of 8:1, 4:1, and 2:1 in methanol, and perform gradient elution and subdivision with a methanol aqueous solution (in the methanol aqueous solution used, the volume ratios of methanol to water are successively 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9). After detecting by thin-layer chromatography, combine the eluates with similar compositions, and recover the solvent under reduced pressure to obtain each elution sub-fraction.

[0044] E. Dissolve each elution sub-fraction collected by eluting and subdividing with a methanol aqueous solution at a volume ratio of 6:4 and 3:7 in methanol, and purify it by preparative high-performance liquid chromatography. Specifically, elute with a methanol aqueous solution with a volume fraction of 50 - 80% and a volume of 8 - 12 L to obtain the compounds with the triterpenoid saponin structure shown as CP-1 to CP-8 respectively.

[0045] The technical solution of the present invention will be described in detail below with specific embodiments.

[0046] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources or prepared by conventional methods without special instructions.

[0047] Example 1

[0048] This example provides a method for preparing a compound with a triterpenoid saponin structure, and the specific steps are as follows:

[0049] 1. Dry and pulverize the whole herb of Pimpinella candolleana (40.0 kg), add ethanol with a volume fraction of 80% (80 L), and extract at 85°C for 3 times, each extraction for 2 h. Combine the extracts and concentrate under reduced pressure to obtain about 4.0 kg of the extract of Pimpinella candolleana.

[0050] 2. Suspend the total extract in 10 L of water, and extract 3 times successively with 50 L of petroleum ether, ethyl acetate, and n-butanol respectively. Recover the solvent under reduced pressure to obtain the petroleum ether fraction (1.5 kg), ethyl acetate fraction (500 g), and n-butanol fraction (200 g) respectively.

[0051] 3. First, 200 g of the n-butanol fraction of Pimpinella candolleana was decolorized with MCI (90% methanol), and the concentrate was obtained as 150 g of an extract. The extract was loaded onto a normal-phase silica gel column (100 - 200 mesh, Shanghai Titan Technology Co., Ltd.) and eluted with a gradient of dichloromethane:methanol. The specific ratios (volume ratios) were: 1:0, 100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, and 1:1, and the elution flow rate was 50 mL / min. The eluate was detected by HSGF 254 thin-layer chromatography (Shanghai Titan Technology Co., Ltd.). The eluates with similar compositions were combined, and the solvent was recovered under reduced pressure to obtain 10 elution fractions.

[0052] 4. Next, for further fractionation, approximately 20 g of the 6th elution fraction obtained in step 3 (dichloromethane:methanol = 8:1) was dissolved in methanol and loaded onto a medium-pressure preparative column (model FLASH SYSTEM CHEETAH) and eluted with a gradient of methanol:water. The specific ratios (volume ratios) were: 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. Thin-layer chromatography was used for real-time monitoring, and the eluent type was gradually increased. When it reached methanol-water (9:1), it was detected that the compounds had basically all eluted, and the column chromatography was stopped. The eluates with similar compositions were combined by thin-layer chromatography detection, and the solvent was recovered under reduced pressure to obtain 9 elution sub-fractions.

[0053] 5. Next is the preparation process. Approximately 2.5 g of the 6th elution sub-fraction (methanol-water = 6:4) and 1 g of the 7th elution sub-fraction (methanol-water = 7:3) obtained in step 4 were each dissolved in chromatographically pure methanol. Another very small amount was dissolved and diluted to approximately 10 mg / ml, and high-performance liquid chromatography (HPLC, model Agilent Technologies 1200 series) was used for detection. The method was 10% - 100% MeOH - 40 min. The detection results were as follows: For the 6th elution sub-fraction, a large absorption peak was observed at 24 min (detection wavelength 210 nm), and for the 7th elution sub-fraction, large absorption peaks were observed at 25.0 min and 25.5 min respectively (detection wavelength 210 nm). The aforementioned 6th and 7th elution sub-fractions dissolved in chromatographically pure methanol were each purified by preparative high-performance liquid chromatography. The 6th elution sub-fraction was eluted with 58% methanol (10 L) to obtain compound CP-2 (100.0 mg, purity 99%); the 7th elution sub-fraction was eluted with 65% methanol (10 L) to obtain compound CP-5 (65.1 mg, purity 98%) and CP-7 (20.0 mg, purity 98%).

[0054] 6. Take about 30 g of the 8th elution fraction obtained in Step 3 (dichloromethane:methanol = 4:1). After dissolving it in methanol, subject it to medium-pressure preparation and start eluting from methanol:water (1:9). The elution steps are the same as in Step 4. Use thin-layer chromatography for real-time monitoring and gradually increase the eluent formulation. When it reaches methanol-water (9:1), it is detected that the compound has basically all flowed out, and stop column chromatography. Combine the eluents with similar compositions through thin-layer chromatography detection, and recover the solvent under reduced pressure to obtain 9 elution sub-fractions.

[0055] 7. Next is the preparation process. Take about 4 g of the 6th elution sub-fraction (methanol-water = 6:4) and 1 g of the 7th elution sub-fraction (methanol-water = 7:3) obtained in Step 6 and dissolve them in chromatographically pure methanol. Take a very small amount and dissolve it, dilute it to about 10 mg / ml, and use high-performance liquid chromatography (HPLC) for detection. The method is 10%-100% MeOH - 40 min. The detection results are as follows: For the 6th elution sub-fraction, a relatively large absorption peak can be observed at 24.5 min (detection wavelength 210 nm); for the 7th elution sub-fraction, a relatively large absorption peak can be observed at 26 min (detection wavelength 210 nm). Purify the aforementioned 6th and 7th elution sub-fractions dissolved in chromatographically pure methanol respectively by preparative high-performance liquid chromatography. The 6th elution sub-fraction is eluted with 62% methanol (10 L) to obtain compound CP-1 (2.5 g, purity 99%); the 7th elution sub-fraction is eluted with 67% methanol (10 L) to obtain compound CP-8 (20 mg, purity 98%).

[0056] 8. Take about 15 g of the 9th elution fraction obtained in Step 3 (dichloromethane:methanol = 2:1). After dissolving it in methanol, subject it to medium-pressure preparation and start eluting from methanol:water (1:9). The elution steps are the same as in Step 4. Use thin-layer chromatography for real-time monitoring and gradually increase the eluent formulation. When it reaches methanol-water (9:1), it is detected that the compound has basically all flowed out, and stop column chromatography. Combine the eluents with similar compositions through thin-layer chromatography detection, and recover the solvent under reduced pressure to obtain 9 elution sub-fractions.

[0057] 9. Next is the preparation process. Approximately 2 g (methanol - water = 6:4) of the 6th elution sub - fraction and 1 g (methanol - water = 7:3) of the 7th elution sub - fraction obtained in step 8 were dissolved using chromatographically pure methanol. Another very small amount was taken for dissolution, diluted to approximately 10 mg / ml, and detected using high - performance liquid chromatography (HPLC). The method was 10% - 100% MeOH - 40 min. The detection results were as follows: The 6th elution sub - fraction had two relatively large absorption peaks at 26.5 min and 27 min respectively (detection wavelength 210 nm), and the 7th elution sub - fraction was observed to have a relatively large absorption peak at 28 min (detection wavelength 210 nm). The aforementioned 6th and 7th elution sub - fractions dissolved with chromatographically pure methanol were respectively purified using preparative high - performance liquid chromatography. The 6th elution sub - fraction was eluted with 68% methanol (10 L) to obtain compound CP - 4 (60.5 mg, purity 99%) and CP - 6 (120 mg, purity 99%). The 7th elution sub - fraction was eluted with 70% methanol (10 L) to obtain compound CP - 3 (10.2 mg, purity 98%).

[0058] The structures of the compounds obtained in the aforementioned steps are as follows:

[0059]

[0060]

[0061] The key of CP - 1 to CP - 8 1 H - 1 The H COSY and HMBC signals are as Figure 1 shown, and the key NOESY signals are as Figure 2 shown.

[0062] Each compound was analyzed for its hydrogen spectrum and carbon spectrum using a Bruker 400 MHz nuclear magnetic resonance spectrometer. The characterization results are shown in Tables 1 and 2 below.

[0063] Table 1 H - NMR data of CP 1 - 8 (δ in ppm, J in Hz, 400 MHz) 1

[0064]

[0065]

[0066] a) Measured in 400 MHz CD3OD. b) Measured in 400 MHz pyridine - d5.

[0067] Table 2 C - NMR data of CP 1 - 8 (δ in ppm, 400 MHz) 13 ​​

[0068]

[0069]

[0070] a) Measured in 400 MHz CD3OD. b) Measured in 400 MHz pyridine-d5.

[0071] Verification Example 1

[0072] This example verifies the biological activities of compounds CP-1 to CP-8 obtained by the aforementioned method. The specific experimental steps are as follows:

[0073] 1. MC3T3-E1 cell culture

[0074] Use α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, preheat to 37 °C for standby. After thawing the cryopreserved MC3T3-E1 cells (purchased from Shanghai Jining Industrial Co., Ltd.) in a 37 °C water bath, centrifuge (1000 rpm, 5 min), discard the supernatant, resuspend and inoculate in a 25 cm 2 culture flask, culture at 37 °C and 5% CO2, and change the medium every 2 days. When the cell confluence reaches 80%-90%, wash with PBS and add 0.25% trypsin-EDTA (containing 0.1% EDTA) to digest for 1-2 min. After terminating the digestion, passage at a ratio of 1:3.

[0075] 2. Drug administration protocol

[0076] Prepare the compound solutions. Weigh accurately compound CP-1 (4.13 mg), CP-2 (3.25 mg), CP-3 (4.21 mg), CP-4 (3.33 mg), CP-5 (3.17 mg), CP-6 (4.71 mg), CP-7 (3.18 mg), and CP-8 (3.99 mg) respectively. After verifying that the purity of each compound is greater than 98% by HPLC, add 500 μL of DMSO to dissolve them into a stock solution with a concentration of 5 mM, and then dilute the stock solutions of each compound to concentrations of 10 μΜ, 20 μM, and 50 μM respectively. After aliquoting, store them at -80 °C.

[0077] 3. CCK-8 assay

[0078] Seed the osteoblasts (the passage cells obtained in step 1) at a density of 1×10 4The cells were seeded into a 96-well plate at a density of cells / well, and different concentrations (10 μΜ, 20 μM, 50 μM) of CP-1 to CP-8 were added respectively. The control group was only added with the culture medium. Then it was placed back into the incubator and cultured for 24, 48, and 72 hours. 10 μL of CCK-8 reagent was added to each well, incubated on a shaker for 2 hours, and the absorbance was measured at a wavelength of 450 nm to calculate the cell survival rate. The results are as Figure 3 shown in Figure A. CP-1 to CP-8 did not significantly inhibit cell viability within the concentration range of 10 - 50 μM, indicating that CP-1 to CP-8 have no obvious cytotoxicity. Compounds CP-1, CP-2, CP-4, and CP-6 with relatively high contents were selected for subsequent studies.

[0079] 4. ALP Staining Experiment

[0080] The osteoblasts (passage cells obtained in step 1) were seeded into a 12-well plate at a density of 1×10 5 cells / well, and different concentrations (10 μΜ, 20 μM, 50 μM) of CP-1, CP-2, CP-4, and CP-6 were added respectively. After culturing for 7 days, the culture medium was discarded, washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, and then BCIP / NBT substrate was added for alkaline phosphatase (ALP) staining. 600 μL was added to each well, and the staining intensity was observed under a microscope. The results are as Figure 3 shown in Figure B. Compared with the control group, CP-1, CP-2, CP-4, and CP-6 significantly enhanced ALP activity at concentrations of 20 μM and 50 μM. Among them, the ALP activity of CP-1 increased most significantly at a concentration of 20 μM (the A / D value reached 400), indicating that it significantly promoted osteoblast differentiation. Therefore, CP-1 (20 μM) was selected for subsequent studies.

[0081] 5. Alizarin Red S Staining Experiment

[0082] The osteoblasts were seeded into a 6-well plate at a density of 5×10 5 cells / well, and different concentrations of CP-1 (10 μM, 20 μM, and 50 μM) were added respectively. After culturing for 21 days, it was washed and fixed with PBS, and then stained with 40 mM alizarin red S solution (pH 4.2) for 30 minutes. The formation of calcium nodules was observed, and the area of calcium nodules was quantitatively analyzed by ImageJ software. The results are as Figure 3 shown in Figure C-3D. Compared with the Control group, CP-1 showed an obvious promoting effect on mineralization at a concentration of 20 μM (the area of calcium nodules > 15%), indicating that CP-1 can promote osteoblast mineralization.

[0083] Verification Example 2

[0084] This example verified the anti-osteoporosis effect of the CP-1 compound. The specific experimental steps are as follows:

[0085] 1. Construction of OVX mouse model

[0086] Thirty-six 8-week-old female C57BL / 6 mice (purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) were used in this study. First, they were fed adaptively for one week. Subsequently, the mice were randomly divided into six groups of 6 each. The specific grouping was as follows: Sham operation group (Sham group), bilateral ovariectomy group (OVX group), ovariectomy + alendronate treatment group (OVX + AS group, dose 10 mg / kg), ovariectomy + CP-1 (prepared in Example 1) low-dose treatment group (OVX + CP-1(30 mg / kg) group, dose 30 mg / kg), ovariectomy + CP-1 (prepared in Example 1) medium-dose treatment group (OVX + CP-1(60 mg / kg) group, dose 60 mg / kg), ovariectomy + CP-1 (prepared in Example 1) high-dose treatment group (OVX + CP-1(120 mg / kg) group, dose 120 mg / kg). In the Sham group, the mice only had an abdominal incision and a small amount of adipose tissue removed, without ovariectomy. The mice in the other groups all underwent bilateral ovariectomy. All surgeries were performed under sterile conditions. The specific steps of the ovariectomy were as follows: First, the mice were anesthetized with 1.0% isoflurane. Subsequently, a longitudinal incision of about 1 cm was made in the midline of the mouse abdomen, and the skin and abdominal wall were carefully separated with forceps. During the operation, the pink plum-blossom-shaped ovarian tissue needed to be carefully found and excised. After complete bilateral ovariectomy, the abdominal wall and skin were sutured layer by layer with absorbable sutures to ensure the tightness of the surgical site and reduce the risk of postoperative infection. To prevent postoperative infection, the mice were treated with intraperitoneal injection of penicillin for the first 5 days after surgery. From the 7th day, drug intervention was started to explore its effect on the bone loss status of the mice.

[0087] 2. Drug administration intervention methods for each group

[0088] 2.1 Drug preparation

[0089] (1) 0.5% CMC-Na solution: Weigh 1.0 g of CMC-Na powder, dissolve it in 200 ml of physiological saline, stir well until completely dissolved, and store at 4°C.

[0090] (2) CP-1 administration solution: Accurately weigh 40 mg, 80 mg, and 160 mg of CP-1, add each to 20 mL of 0.5% CMC-Na solution, vortex thoroughly to mix evenly, and obtain CP-1 suspensions with concentrations of 2, 4, and 8 mg / mL respectively. Store in the dark at 4°C.

[0091] (3) Alendronate Sodium Administration Solution (AS): Weigh 10 mg of alendronate sodium powder and add it to 20 ml of 0.5% CMC-Na solution. Vortex to mix evenly to obtain a 0.5 mg / ml AS solution, and store it at 4°C.

[0092] 2.2 Administration

[0093] Seven days after modeling, each group of mice was given corresponding drug gavage every morning. According to the grouping, the Sham group was given 0.5% CMC-Na solution, the OVX group was given 0.5% CMC-Na solution, the OVX + AS group was given 0.5 mg / ml AS solution (dose: 10 mg / kg), the OVX + CP-1(L) group was given 2 mg / ml CP-1 suspension (dose: 30 mg / kg), the OVX + CP-1(M) group was given 4 mg / ml CP-1 suspension (dose: 60 mg / kg), and the OVX + CP-1(H) group was given 8 mg / ml CP-1 suspension (dose: 120 mg / kg). The body weight of the mice was recorded every 2 weeks and the administration volume was adjusted accordingly. After 8 weeks, the samples were taken for analysis.

[0094] 3. Animal Sampling

[0095] 3.1 Serum Sampling

[0096] Eight weeks after administration, the experimental mice were subjected to serum collection. First, the mice were anesthetized with isoflurane to ensure painlessness during the sampling process. Subsequently, the tail vein method was used to collect blood samples. The blood samples were placed in 1.5 ml EP tubes without anticoagulant and allowed to stand for 30 minutes to allow the blood to coagulate naturally. The coagulated blood samples were centrifuged (3000 rpm, 10 min) to separate the serum. The upper-layer serum was collected and transferred to a new sterile 1.5 ml EP tube and stored in a -80°C refrigerator for subsequent analysis.

[0097] 3.2 Visceral Sampling

[0098] After the serum samples were collected, 3 mice were randomly selected from each group and sacrificed by cervical dislocation. Immediately, an abdominal incision was made, the abdominal wall and muscle tissues were dissected, and the visceral organs including the heart, liver, spleen, lungs, and kidneys were exposed and removed. The removed organs were immediately rinsed with PBS to remove the surface blood and other impurities, and then fixed in 4% paraformaldehyde for subsequent histological section analysis.

[0099] 3.3 Femur Sampling

[0100] A transverse incision was made in the hip joint area of the mice to dissect the skin and muscle tissues and expose the bilateral femurs. The connection between the femur and the hip joint was cut with scissors to remove the complete femur. It was immediately rinsed with PBS and quickly transferred to 4% paraformaldehyde for fixation for subsequent experimental operations.

[0101] 4. Femoral Micro-CT Analysis

[0102] First, take the left femurs of the mice in each group, place the samples on the Micro-CT scanning bed for scanning, and set the scanning parameters as follows: the radial field of view is 100 mm, the maximum axial scanning range is 250 mm, the scanning speed is 4 s / bed, the reconstructed pixel size is 2 μm. After the scanning is completed, use Recon software to reconstruct the femurs, and use Cruiser software to quantitatively analyze multiple microstructural parameters of the femurs, including BMD (bone mineral density), BV / TV (bone volume density), BS / TV (bone surface area and tissue volume ratio), Tb.N (trabecular number), Tb.Pf (trabecular pattern factor), and Tb.Sp (trabecular separation).

[0103] 5. HE Staining of Organ Samples

[0104] 5.1 Embedding and Sectioning: First, dehydrate the organ samples obtained in 3.2 above through a gradient alcohol solution, and then immerse them in molten paraffin for embedding. During the sectioning process, select the maximum cross-sectional slice of the organ samples.

[0105] 5.2 Dewaxing of Sections: Immerse each section in xylene I and xylene II for 20 min in sequence, and then gradually dehydrate through absolute ethanol I, absolute ethanol II, and 75% ethanol, with each step lasting 5 min. Finally, wash with distilled water to remove the solvent.

[0106] 5.3 HE Staining: Place each section in hematoxylin stain (5 min), after treatment with the differentiation solution, and then perform blueing treatment. Subsequently, the sections continue to be dehydrated through the same gradient alcohol solution as in 5.2, and finally stained in eosin stain (5 min).

[0107] 5.4 Dehydration and Sealing: The stained sections are subjected to stepwise dehydration treatment, including absolute ethanol I (purchased from Sinopharm Chemical Reagent Co., Ltd.), absolute ethanol II (purchased from Sinopharm Chemical Reagent Co., Ltd.), absolute ethanol III (purchased from Sinopharm Chemical Reagent Co., Ltd.), and xylene I (purchased from Sinopharm Chemical Reagent Co., Ltd.), xylene II (purchased from Sinopharm Chemical Reagent Co., Ltd.), each for 5 min, and finally sealed with neutral gum.

[0108] 5.5 Microscopic Observation: Observe and photograph the sections under a microscope to evaluate their histological characteristics.

[0109] 6. ELISA Determination of Serum BMP-2, OCN, OPG, RANKL, and OPG / RANKL Contents

[0110] The contents of BMP-2, OCN, OPG and RANKL in mouse serum were detected according to the ELISA kit instructions, and the OPG / RANKL ratio was calculated.

[0111] 7. Statistical analysis

[0112] In this experiment, to ensure the accuracy and reliability of the results, all experimental data were measured at least three times. Statistical analysis and visualization of the data were performed using GraphPad Prism 9.0 software, and all results were expressed as mean ± standard deviation (Mean ± SD). For comparisons between two groups, Student’s t-test was used for statistical analysis; while the differences among multiple groups were tested by one-way analysis of variance (One-way ANOVA). The criterion for judging statistical significance was: P < 0.05 indicated statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0113] 8. Experimental results

[0114] 8.1 The CP-1 compound has good in vivo safety

[0115] To evaluate the in vivo safety of the CP-1 compound, this study analyzed by continuously observing the body weight changes of mice during model establishment and drug administration. The trend of body weight change is as Figure 4 shown. The results showed that the body weights of mice in each experimental group showed a gradually increasing trend, but the body weight differences between different groups did not reach statistical significance (P > 0.05). This indicates that at different treatment doses, the effect of CP-1 treatment on mouse body weight gain has no significant difference, further verifying that CP-1 has good safety within the dose range designed in this experiment.

[0116] To further illustrate that CP-1 has no obvious toxic effect on the visceral organs of OVX-induced mice, this study examined the visceral organs (heart, liver, spleen, lung and kidney) of experimental mice by HE staining. The results are as Figure 5As shown. Through histological observation by HE staining, it was found that CP-1 did not cause obvious toxic effects on the main organs within the experimental dose range. No inflammatory infiltration, cell damage or other significant pathological abnormalities were found in each tissue section. The specific manifestations were as follows: In the heart tissue, the myocardial fibers were arranged orderly, and no fracture or necrosis was observed; The liver section showed that the hepatocytes were arranged regularly, and no inflammatory cell infiltration or fatty degeneration was observed; The demarcation between the white pulp and red pulp of the spleen tissue was clear, and the tissue distribution was normal; The lung tissue showed that the alveolar structure was intact, and no obvious alveolar collapse, interstitial thickening or inflammatory cell aggregation and other pathological changes were found; In the kidney section, the glomerular structure remained intact, the renal tubular epithelial cells were arranged neatly, and there was no sign of damage. The above results showed that CP-1 showed good in vivo safety at the test dose and did not cause systemic toxic reactions.

[0117] 8.2 CP-1 compound alleviates bone loss in OVX mice

[0118] To visually evaluate the improvement effect of CP-1 compound on bone loss in OVX mice, after 8 weeks of administration, the left femur of the mice was scanned and detected by Micro-CT, and high-resolution three-dimensional reconstruction analysis of bone microstructure was carried out in combination with CTvox software. The results were as Figure 6 shown in Figure A. The three-dimensional reconstructed trabecular bone structure (upper part), sagittal plane image (middle part) and cross-sectional image (bottom part) clearly showed the changes in bone tissue of each experimental group. By observing the trabecular bone structure and the sagittal plane image of the femur, it was found that compared with the Sham group, the OVX group of mice showed significant bone loss, the trabecular bone structure was sparse and fractured, and the arrangement was disordered, and the number of trabecular bones was significantly reduced, while the trabecular bones of the mice in the Sham group were dense, evenly distributed and remained intact, verifying the successful construction of the OVX model. After intervention with alendronate sodium (AS) and CP-1 administration, CP-1 at each dose improved bone loss to varying degrees. Specifically, the number of trabecular bones in the medium-dose group (60 mg / kg) and high-dose group (120 mg / kg) of CP-1 increased significantly, and the arrangement was more compact and continuous. The effect was significantly better than that of the low-dose group (30 mg / kg) and the OVX group, and was close to the level of the Sham group. This indicated that CP-1 could reverse bone mass loss in the OVX model and significantly improve bone structure damage. Further observation of the cross-sectional image of the femur showed that the trabecular bone density and thickness of the OVX group of mice were significantly reduced, while in the medium- and high-dose groups of CP-1 (60 mg / kg and 120 mg / kg), these pathological changes were significantly reversed. Especially in the high-dose group of CP-1 (120 mg / kg), the trabecular bone space was reduced, the thickness increased, and the bone microstructure recovered to near the normal state.

[0119] To further quantify the effect of CP-1 on bone mass changes, the histomorphometric parameters of the femurs of experimental mice in each group were analyzed by Micro-CT scanning, including BMD (bone mineral density), BV / TV (bone volume density), BS / TV (bone surface area and tissue volume ratio), Tb.N (trabecular bone number), Tb.Pf (trabecular bone pattern factor), and Tb.Sp (trabecular bone separation). The relevant results are shown as Figure 6 B, Figure 6 C, Figure 6 D, Figure 6 E, Figure 6 F, Figure 6 G. Compared with the Sham group, the BMD, BV / TV, BS / TV, and Tb.N of mice in the OVX group were significantly decreased, indicating that the integrity of the trabecular bone structure was damaged and severe bone loss occurred. While Tb.Pf and Tb.Sp were significantly increased, suggesting a decrease in the number of trabecular bones and sparse arrangement, further verifying the success of the OVX model construction. After intervention with alendronate sodium (AS) and CP-1, compared with the OVX group, both the OVX+CP-1 (60mg / kg) group and the OVX+CP-1 (120mg / kg) group effectively improved the bone microstructure, manifested as significant increases in BMD, BV / TV, BS / TV, and Tb.N, while Tb.Pf and Tb.Sp were significantly decreased, indicating that CP-1 can effectively inhibit bone loss, improve the trabecular bone structure, and enhance the trabecular bone density. Compared with the positive control alendronate sodium (AS), the improvement effect of the high-dose CP-1 group (OVX+CP-1 (120mg / kg)) on multiple key bone mass parameters was equivalent to or even better than that of AS, especially for BV / TV and Tb.N, indicating that the high-dose CP-1 group (120mg / kg) has a bone protection effect equivalent to that of AS. In addition, compared with the low- and medium-dose CP-1 groups (OVX+CP-1 (30mg / kg) and OVX+CP-1 (60mg / kg)), the bone protection effect of the high-dose CP-1 group (120mg / kg) was more significant, showing a certain dose-dependence, especially in terms of increasing bone density and optimizing the trabecular bone arrangement. In summary, the quantitative analysis results by Micro-CT further confirmed that the CP-1 compound can effectively alleviate bone loss in OVX mice and significantly improve the trabecular bone structure.

[0120] 8.3 Determination of the Contents of BMP-2 and OCN in Serum

[0121] As a key regulatory factor for bone formation, an increase in the content of BMP-2 reflects the enhancement of osteoblast differentiation and bone formation activity. The results of ELISA for determining the serum BMP-2 content are shown as Figure 7As shown in Figure A, the content in the OVX group was lower than that in the Sham group, but there was no significant difference (P>0.05). After drug intervention, compared with the OVX group, the serum BMP-2 content in the positive control alendronate sodium (OVX+AS) group was significantly increased (P<0.01), and the medium-dose group (60mg / kg) and high-dose group (120mg / kg) of CP-1 could also significantly increase the content of BMP-2 (P<0.05). OCN is a specific marker for osteoblast maturation and is closely related to the bone mineralization level. The serum OCN content was measured by ELISA as Figure 7 shown in Figure B, which was significantly increased in the OVX group compared with the Sham group (P<0.01). After drug intervention, the OCN contents in the OVX+AS group, OVX+CP-1(60mg / kg) group, and OVX+CP-1(120mg / kg) group were all significantly higher than those in the OVX group, and the differences were statistically significant (P<0.05). Further comparison of the OCN contents in each dose group of CP-1 found that there was an obvious dose-dependence. Especially the high-dose group (120mg / kg) of CP-1 showed a significant advantage in increasing the OCN content (P<0.01).

[0122] 8.4 Determination of OPG, RANKL and OPG / RANKL Contents in Serum

[0123] The results of ELISA for measuring the serum OPG content showed ( Figure 7 Figure C) that the OVX model group was significantly lower than the Sham group, with statistical significance (P<0.01). After AS intervention, compared with the OVX group, the serum OPG content in the OVX+AS group was significantly increased (P<0.05). After CP-1 intervention, the OPG content increased with the increase of the CP-1 drug concentration, and only the high-dose group (120mg / kg) of CP-1 had statistical significance in increasing OPG (P<0.05). The results of ELISA for measuring the serum RANKL content showed ( Figure 7 Figure D) that the OVX group was significantly higher than the Sham group (P<0.0001). After drug intervention, compared with the OVX group, the OVX+AS group, the medium-dose group and high-dose group (60mg / kg and 120mg / kg) of CP-1 could extremely significantly reduce the serum RANKL content, and the differences were all statistically significant (P<0.0001).

[0124] The OPG / RANKL ratio is crucial for maintaining the balance of osteoblast and osteoclast differentiation. Therefore, we further analyzed the OPG / RANKL ratio in the serum samples of mice in different groups, and the results showed ( Figure 7E), The ratio of OPG / RANKL in the OVX group was significantly lower than that in the Sham group (P<0.001). After drug treatment, compared with the OVX group, the OPG / RANKL ratio in the OVX+AS group was significantly increased, and the difference was statistically significant (P<0.01). After CP-1 intervention, the OPG / RANKL ratio increased significantly with the increase of CP-1 administration concentration, showing a certain dose-dependence. Among them, the high-dose CP-1 group (120mg / kg) had the most significant effect on increasing the OPG / RANKL ratio (P<0.0001), approaching the normal level (Sham group). The smaller the OPG / RANKL value, the more severe the bone loss. In the OVX mouse model, the expression of RANKL increased while the expression of OPG decreased, breaking the balance between OPG and RANKL, resulting in osteoclast activation, increased bone resorption and decreased bone density, ultimately leading to osteoporosis. Our study found that the CP-1 compound can effectively inhibit the content of RANKL, increase the OPG / RANKL ratio, and improve the bone degradation of OVX mice through the OPG / RANK / RANKL axis.

[0125] There are many specific application ways of the present invention, and the above description is only the preferred embodiment of the present invention. It should be noted that the above embodiments are only used to illustrate the present invention, and do not limit the protection scope of the present invention. For those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements can be made, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A compound having a triterpenoid saponin structure, characterized in that: The structure of the compound is shown in the following formula (I) or (II): Among them, R1 represents a glucosyl group or a methyl group; R2 represents a methyl group, a hydroxymethyl group, a glucosyl group or a glucose disaccharide group; R3 represents a glucosyl group or a hydroxymethyl group; R4 represents a hydrogen or a hydroxyl group; and R5 represents a hydroxyl group or a glucose trisaccharide group.

2. The compound having a triterpene saponin structure according to claim 1, characterized in that The structures of the compounds are shown in the following formulas CP-1 to CP-8:

3. A method for preparing a compound having a triterpene saponin structure according to any one of claims 1 to 2, characterized in that: The following steps are involved: A. Dry and crush the whole herb of Pimpinella foenum-leaf, add 50-90% by volume ethanol for extraction, and concentrate the obtained extract under reduced pressure until there is no alcohol taste to obtain an extract; B. After diluting the extract with water, extracting with petroleum ether, ethyl acetate and n-butanol in sequence, recovering the solvent under reduced pressure to obtain a petroleum ether extract, an ethyl acetate extract and a n-butanol extract, respectively; C. Remove the pigment from the n-butanol extract, apply it to a 100-200 mesh normal phase silica gel column, and perform gradient elution with a mixed solution of dichloromethane and methanol. After the eluate is detected by thin layer chromatography, collect the eluted fractions containing the triterpenoid saponin structures represented by formula (I) and (II); D. The eluted fraction containing the triterpenoid saponin structure represented by formula (I) or (II) is further eluted and subdivided to obtain compounds of the triterpenoid saponin structure represented by formula (I) or (II).

4. The method for preparing a compound having a triterpene saponin structure according to claim 3, characterized in that: In step A, the extraction is performed 2-5 times, the extraction temperature is 80-90° C., and the extraction time for each time is 1-3 hours.

5. The method for preparing a compound having a triterpene saponin structure according to claim 3, characterized in that: In step B, the extract is diluted with 2-6 times of water; 2-6 times amount of petroleum ether, ethyl acetate and n-butanol are respectively used for extraction, and the number of times of extraction with each solvent is 2-4 times.

6. The method for preparing a compound having a triterpene saponin structure according to claim 3, characterized in that: In step C, in the mixed solution of dichloromethane and methanol used for the gradient elution, the volume ratios of dichloromethane and methanol are 1:0, 100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1 and 1:1, respectively.

7. The method for preparing a compound having a triterpene saponin structure according to claim 3, characterized in that: In step D, the specific step of eluting and subdividing is: using a methanol-water gradient elution to subdivide the eluted fractions containing the triterpenoid saponin structures represented by formula (I) and (II), so as to obtain eluted subdivided fractions containing the triterpenoid saponin structures represented by formula (I) or (II); In the methanol-water solution used for the gradient elution, the volume ratios of methanol to water are 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, respectively.

8. The method for preparing a compound having a triterpene saponin structure according to claim 3 or 7, characterized in that: The method further comprises the step of purifying the compounds with triterpenoid saponin structures shown in formula (I) or (II) obtained in step D by preparative high performance liquid chromatography to obtain compounds with triterpenoid saponin structures shown in formula (I) or (II).

9. The method for preparing a compound having a triterpene saponin structure according to claim 3 or 7, characterized in that: The purification is eluted with methanol-water solution; the methanol-water solution uses a methanol volume concentration of 50-80% and a volume of 8-12L.

10. Use of the compound with a triterpene saponin structure according to any one of claims 1 to 2 in the preparation of a medicine or health product for promoting osteoblast proliferation and differentiation or treating osteoporosis.

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