Application of gynostemma pentaphyllum total saponins in preparation of medicine for resisting diabetic osteoporosis

Abstract

The invention discloses application of gynostemma pentaphyllum total saponins in preparation of a medicine for resisting diabetic osteoporosis, and belongs to the technical field of traditional Chinese medicines. The invention provides an application of gynostemma pentaphyllum total saponins in preparation of a medicine for resisting diabetic osteoporosis. The gynostemma pentaphyllum total saponins can promote the expression of ossification related genes. And the ossification related gene is at least one of ALP (Alkaline Phosphate Protein), OPG (Osteotropin Growth) or beta-catenin genes. The administration concentration of the gynostemma pentaphyllum total saponins is 25 to 50 [mu] g / mL. The Gynostemma pentaphyllum total saponins (GPMS) disclosed by the invention can be used for remarkably improving the diabetic osteoporosis symptom of an alloxan-induced zebra fish model. The dosage of the gynostemma pentaphyllum total saponins (GPMS) in the medicine for resisting diabetic osteoporosis is relatively low, and the toxicity is relatively low in an effective dosage range.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of total saponins of Gynostemma pentaphyllum in preparing medicines for treating diabetic osteoporosis. Background Art

[0002] Diabetes and osteoporosis are chronic diseases of glucose and bone metabolism, respectively, and share many contributing factors, including aging and genetic susceptibility, unhealthy lifestyle, diet, and physical activity, so the two conditions often coexist and influence each other.

[0003] Osteoporosis can be divided into primary osteoporosis and secondary osteoporosis. Primary osteoporosis is mostly caused by aging, especially in postmenopausal women. It is mainly due to the autocrine reduction of age-related sex hormones, which leads to the activation of osteoclasts and the reduction of bone marrow mesenchymal stem cell ossification, which bidirectionally breaks the osteoblast-osteoclast balance in the body, and finally causes osteoporosis. For the treatment of primary osteoporosis, estrogen, calcitonin, and bisphosphonates can achieve good therapeutic effects. Diabetes is a systemic metabolic disease, often accompanied by insulin deficiency, decreased insulin sensitivity, endocrine disorders, and endocrine dysfunction. Diabetic osteoporosis is a typical secondary osteoporosis, which is mainly caused by the adverse symptoms of diabetes such as high blood sugar, inflammation, oxidative stress, bone marrow obesity, changes in collagen properties, and sarcopenia, which change the microenvironment in the body, affect the osteoblast-osteoclast balance from multiple dimensions, and are closely related to the disruption of calcium and other mineral metabolism. It is easy to cause calcium and phosphorus metabolism disorders in the body, leading to reduced bone density and abnormal bone metabolism. The treatment needs of diabetic osteoporosis are higher than those of primary osteoporosis. In addition to improving bone metabolism to restore the osteoblast-osteoclast balance, it is still necessary to consider the improvement of the diabetic microenvironment in the body, especially since most drugs for treating diabetes have the side effect of aggravating osteoporosis.

[0004] Zebrafish is an ideal animal model for pharmacological research. Zebrafish is a vertebrate with highly conserved signal transduction pathways for human diseases, and it maintains a high degree of consistency with human genes. Most drug trials based on zebrafish models are consistent with the results of human trials. The mechanism of zebrafish skeletal development is similar to that of higher vertebrate skeletal ossification. When zebrafish are at the 3-9dpf stage, most of the skull is formed by endochondral ossification. The body length is 3-4mm, and it can survive in 50μL of liquid. The modeling is sensitive and suitable for high-throughput screening in the form of microplates. The zebrafish spine begins to gradually ossify and develop in segments from head to tail at 7dpf. It is known that compared with the skull, the zebrafish spine ossification mechanism is quite different from the endochondral ossification of the mammalian spine. At present, many studies have established a zebrafish model of diabetic osteoporosis by using alloxan, and observed its skull development to explore the improvement effect of drugs on diabetic osteoporosis.

[0005] Gynostemmapentaphyllum (Thunb.) Makino is the dried aerial part of the plant of the genus Gynostemma of the Cucurbitaceae family. It is known as the "herb of immortality" and "southern ginseng" in the folk. As a traditional Chinese medicine, Gynostemma was first recorded in the Ming Dynasty's "Compendium of Materia Medica for Famine Relief" and was later included in the "Compendium of Materia Medica". The book records that Gynostemma is cold in nature and bitter in taste, and has the effects of "curing insect bites, cooling blood and detoxifying, and promoting urination". Modern pharmacological studies have shown that Gynostemma can lower blood sugar, blood lipids, and blood pressure, and has the effects of preventing aging, enhancing immunity, inhibiting tumors, preventing atherosclerosis, and reducing cardiovascular and cerebrovascular diseases. Summary of the invention

[0006] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide the use of total saponins of Gynostemma pentaphyllum in the preparation of drugs for resisting diabetic osteoporosis.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides the use of total saponins of Gynostemma pentaphyllum in the preparation of a drug for resisting diabetic osteoporosis.

[0009] The application, the total saponins of Gynostemma pentaphyllum can promote the expression of genes related to osteogenesis.

[0010] In the application, the osteogenesis-related gene is at least one of ALP, OPG or β-catenin gene.

[0011] The application is characterized in that the total saponins of Gynostemma pentaphyllum contain escin XVII, ginsenoside Re, ginsenoside Rg1, escin XLVI, glycyrrhizic acid, ginsenoside F1, ginsenoside Rh1 and notoginsenoside Fe.

[0012] The application is characterized in that the total saponins of Gynostemma pentaphyllum are eluates obtained by eluting the crude extract of total saponins of Gynostemma pentaphyllum with 45% to 55% gradient ethanol, wherein the concentration of total saponins in the eluate is 70.39% to 75.09%.

[0013] In the application, the administration concentration of the total saponins of Gynostemma pentaphyllum is 25-50 μg / mL; preferably, the administration concentration of the total saponins of Gynostemma pentaphyllum is 50 μg / mL.

[0014] In a second aspect, the present invention provides a drug for preventing diabetic osteoporosis, comprising total saponins of Gynostemma pentaphyllum and pharmaceutically acceptable excipients.

[0015] The drug for preventing diabetic osteoporosis is in the form of tablets, granules, pills, capsules, powders, oral liquids or injections.

[0016] In the drug for preventing diabetic osteoporosis, the dosage concentration of the total saponins of Gynostemma pentaphyllum is 25-50 μg / mL.

[0017] In the drug for preventing diabetic osteoporosis, the dosage concentration of the total saponins of Gynostemma pentaphyllum is 50 μg / mL.

[0018] The total saponin of Gynostemma pentaphyllum is an alcohol extract of Gynostemma pentaphyllum, which is obtained by purifying the alcohol extract of Gynostemma pentaphyllum. The purification refers to extracting the Gynostemma pentaphyllum water extract with organic reagents, separating and purifying with macroporous resins, concentrating under reduced pressure, freeze drying and other operations.

[0019] The preparation method of total saponins of Gynostemma pentaphyllum comprises the following steps:

[0020] (1) Drying Gynostemmapentaphyllum (Thunb.) Makino at 40-60° C. for 18-24 hours, crushing and sieving through a 40-60 mesh sieve to obtain a coarse powder of Gynostemmapentaphyllum saponin;

[0021] (2) extracting the crude powder of Gynostemma pentaphyllum by ultrasonic-enzymatic extraction method, with the solvent being 75% ethanol; performing ultrasonic extraction twice, enzymatic hydrolysis once, combining the two extracts, filtering, combining, concentrating under reduced pressure at 40-60° C., and drying to constant weight to obtain an alcohol extract of Gynostemma pentaphyllum;

[0022] (3) dissolving the alcohol extract of Gynostemma pentaphyllum in water to prepare an alcohol extract solution of Gynostemma pentaphyllum, extracting with equal volumes of petroleum ether, ethyl acetate and n-butanol in sequence, retaining the n-butanol phase extract, concentrating under reduced pressure at 40-60° C., and freeze-drying to obtain a crude extract of total saponins of Gynostemma pentaphyllum;

[0023] (4) The crude extract of total saponin of Gynostemmapentaphyllum Makino was dissolved with an appropriate amount of deionized water, loaded onto the pretreated D101 resin, and then eluted with water, 30%, 50%, 70% and 90% gradient ethanol in sequence, and each component was collected and freeze-dried to obtain each gradient ethanol eluate. Among them, the 50% ethanol gradient eluate was identified as the total saponin of Gynostemmapentaphyllum Makino (GPMS).

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention found that Gynostemma pentaphyllum total saponins (GPMS) significantly improved the symptoms of diabetic osteoporosis in the alloxan-induced zebrafish model.

[0026] (2) The present invention found that Gynostemma pentaphyllum total saponins (GPMS) can significantly increase the expression of ALP, OPG, and β-catenin mRNA in the alloxan-induced zebrafish model.

[0027] (3) The dosage of GPMS in the drug for treating diabetic osteoporosis of the present invention is relatively low, and the toxicity is relatively low within the effective dosage range. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 HPLC spectra of different batches of total saponins of Gynostemma pentaphyllum (GPMS) samples obtained in Example 2;

[0029] Figure 2 The LC-MS spectrum of the total saponins of Gynostemma pentaphyllum (GPMS) obtained in Example 2 and its analysis results, wherein A is the LC-MS spectrum of GPMS, B is the secondary mass spectrum of the saponin component aesculoside XVII identified in the detection and its comparison with the mzVault database, C is the secondary mass spectrum of the saponin component ginsenoside Re identified in the detection and its comparison with the mzVault database, D is the secondary mass spectrum of the saponin component ginsenoside Rg1 identified in the detection and its comparison with the mzVault database, E is the secondary mass spectrum of the saponin component aesculoside XLVI identified in the detection and its comparison with the mzVault database, F is the secondary mass spectrum of the saponin component glycyrrhizic acid identified in the detection and its comparison with the mz Vault database, G is the secondary mass spectrum of the saponin component ginsenoside F1 identified in the detection and its comparison with the mz Vault database. Vault database, H is the secondary mass spectrum of the saponin component 20(R)-ginsenoside Rh1 identified in the test and its comparison with the mzVault database, I is the secondary mass spectrum of the saponin component Panax notoginsenoside Fe identified in the test and its comparison with the mzVault database;

[0030] Figure 3 This is a graph showing the toxicity of total gynostemma saponins (GPMS) obtained in Example 2 to zebrafish without a diabetic background;

[0031] Figure 4 This is a graph showing the toxicity of total gynostemma pentaphyllum saponins (GPMS) obtained in Example 2 on zebrafish with a diabetic background;

[0032] Figure 5 The figure is a result diagram of the effect of total saponins of Gynostemma pentaphyllum (GPMS) obtained in Example 2 on diabetic osteoporosis in the zebrafish model induced by alloxan, wherein A is the zebrafish alizarin red staining result, B is the semi-quantitative analysis of the staining area in the alizarin red staining result, and C is the semi-quantitative analysis of the optical density in the alizarin red staining result;

[0033] Figure 6 This is a graph showing the effect of GPMS obtained in Example 2 on the expression of ALP, OPG, and β-catenin mRNA in the zebrafish model induced by alloxan, wherein A is the expression result of ALP, B is the expression result of OPG, and C is the expression result of β-catenin. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. In the following examples, AB wild-type zebrafish eggs were purchased from the Central Laboratory of Southern Medical University; Gynostemma pentaphyllum raw materials were purchased from Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd. (National Medicine Standard Z44021756).

[0035] Example 1: Preparation of a crude extract of total saponins from Gynostemma pentaphyllum

[0036] (1) Grinding Gynostemma pentaphyllum and passing it through a 40-mesh sieve to obtain a coarse powder of Gynostemma pentaphyllum medicinal material;

[0037] (2) Accurately weigh 5 g of Gynostemma pentaphyllum powder, with a solid-liquid ratio of 1 g:15 mL, sonicate twice with 75% ethanol at an ultrasonic power of 80 W for 30 min each time, filter, and set aside the filtrate;

[0038] (3) adding 3% pectinase to the residue and placing it in a 50°C constant temperature water bath for enzymolysis for 1 hour. After the enzymolysis is completed, heating it in a 90°C water bath for 5 minutes to inactivate it. Filtering it while hot, combining the filtrate, and freeze-drying it to obtain the Gynostemma pentaphyllum ethanol extract;

[0039] (4) The ethanol extract was fully dissolved in an appropriate amount of water, and then extracted with an equal volume of petroleum ether, ethyl acetate, and n-butanol for three times, each time for 30 minutes. The n-butanol phase extract was concentrated under reduced pressure and freeze-dried to obtain the crude extract of total saponins of Gynostemma pentaphyllum.

[0040] Example 2: Preparation of Gynostemma pentaphyllum saponins (GPMS)

[0041] The macroporous adsorption resin is pretreated by soaking it in distilled water for 12 to 24 hours, then soaking it in a hydrochloric acid solution with a mass fraction of 3 to 5% for 0.5 to 3 hours, washing it with distilled water until it is neutral, and then soaking it in a sodium hydroxide solution with a mass fraction of 3 to 5% for 0.5 to 3 hours, washing it with distilled water until it is neutral, thereby obtaining a pretreated macroporous adsorption resin.

[0042] The crude extract of total saponins of Gynostemma pentaphyllum was loaded onto the pre-treated D101 macroporous adsorption resin. Then, water, 30%, 50%, 70% and 90% gradient ethanol were used for elution in sequence, and each component was collected and freeze-dried to obtain each gradient ethanol eluate. Among them, the 50% gradient ethanol eluate was identified as total saponins of Gynostemma pentaphyllum (GPMS).

[0043] Example 3: Determination of total saponin content in each gradient ethanol eluate by vanillin-glacial acetic acid-perchloric acid colorimetric method (VPAC method)

[0044] (1) Drawing of standard curve:

[0045] Weigh 6 mg of ginsenoside Rb1 standard dried to constant weight accurately, dissolve in anhydrous ethanol, transfer to a 10 mL volumetric flask, make up to volume with anhydrous ethanol, and accurately pipette 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the solution into 10 mL stoppered test tubes, evaporate the reagents in an oven, add 0.2 mL of 5% vanillin-glacial acetic acid solution (prepared and used immediately) and 0.8 mL of perchloric acid in sequence, and mix well. Heat in a 60°C water bath for 20 min, cool in an ice water bath for 3 min, add 5 mL of glacial acetic acid, and shake well. The corresponding reagent is used as a blank, and the absorbance value is measured at a wavelength of 540 nm.

[0046] (2) Sample testing:

[0047] Weigh 6 mg of each gradient ethanol eluate sample that has been dried to constant weight, dissolve it with a small amount of DMSO, transfer it to a 10 mL volumetric flask, and dilute it to 0.6 mg / mL with anhydrous ethanol. Pipette 1 mL of the sample solution, dilute it to 5 mL with anhydrous ethanol, and accurately pipette 1 mL of the sample solution into a 10 mL stoppered test tube. According to the method of drawing the standard curve, measure the absorbance at 540 nm, and calculate the content of Gynostemma pentaphyllum saponins under different extraction conditions according to the standard curve.

[0048] Saponin content in sample (%) = saponin content calculated from standard curve (mg) × dilution factor / sampling amount × 100%

[0049] Result analysis:

[0050] The total saponin content of each gradient ethanol eluate prepared in Example 2 was detected as shown in Table 1. The results showed that the total saponin content in the 50% ethanol gradient eluate was 72.74±2.35%, and the recovery rate was 59.22%. Compared with the same period last year, the total saponin content and yield of the 50% ethanol gradient eluate were the highest, so the 50% ethanol eluate was identified as the total saponins of Gynostemma pentaphyllum (GPMS) prepared in Example 2.

[0051] Table 1 Total saponin concentration of each gradient ethanol eluate

[0052]

[0053] Example 4: High Performance Liquid Chromatography (HPLC) Analysis of Gynostemma Pentaphyllum Saponins (GPMS)

[0054] (1) Preparation of test solution:

[0055] Accurately weigh 1 mg of Gynostemma pentaphyllum total saponins (GPMS) sample, add 1 mL of methanol solution for ultrasonic-assisted dissolution, shake well, and filter through a 0.22 μm filter membrane to obtain a 1 mg / mL test solution.

[0056] (2) Chromatographic conditions:

[0057] YMC C18 chromatographic column, column temperature 25°C, mobile phase acetonitrile (A)-water (B), elution gradient as shown in Table 2, flow rate 1.0 mL / min, detection wavelength 203 nm, injection volume 30 μL. Elution was performed according to the gradient elution conditions shown in Table 2.

[0058] Table 2 HPLC analysis gradient elution conditions

[0059]

[0060] Result analysis:

[0061] like Figure 1 As shown, each batch of GPMS has two main peaks, with retention times around 17.3min and 19.6min, respectively, and several small peaks. The results show that the material basis of the Gynostemma pentaphyllum total saponins (GPMS) sample extracted in Example 2 is stable.

[0062] Example 5: Liquid chromatography-mass spectrometry (LC-MS) analysis of total saponins of Gynostemma pentaphyllum (GPMS)

[0063] (1) Preparation of test solution:

[0064] Take an appropriate amount of Gynostemma pentaphyllum total saponins (GPMS) sample, add an appropriate amount of methanol solution for ultrasonic-assisted dissolution, centrifuge at 15000g for 10 min, take the supernatant, and obtain the test solution.

[0065] (2) Mobile phase conditions:

[0066] The mobile phase conditions were the same as those in Example 4.

[0067] (3) Mass spectrometry conditions:

[0068] Mass spectrometry conditions: ion source: ESI; scanning range: m / z 100-1200; heating module temperature: 200°C; CDL temperature: 200°C; nebulizer gas flow rate: 1.5 L / min; drying gas pressure: 10 8 kpa; IT vacuum degree: 1.8×10 -2 pa; TOF vacuum degree: 2.2×10 -4 Pa; ion source voltage negative ion mode: -3.5kV; detector voltage: 1.6kV.

[0069] Result analysis:

[0070] The total saponins (GPMS) sample obtained in Example 2 contained 57 potential components, including 8 saponin components, including 2 gypenosamine saponin compounds, 5 Araliaceae saponin compounds, and 1 glycyrrhizin saponin compound. The potential compounds obtained by LC-MS analysis are listed in Table 3 below, and the LC-MS spectrum is shown in Table 3. Figure 2 As shown in A, the secondary mass spectra of 8 saponins were compared with the database spectra. Figure 2 As shown in BI.

[0071] Table 3 UPLC / MS separation and identification of components in total saponins of Gynostemma pentaphyllum

[0072]

[0073]

[0074]

[0075] Example 6: Toxicity of Gynostemma pentaphyllum total saponins (GPMS) to background-free zebrafish

[0076] (1) Experimental animals

[0077] 3dpf zebrafish were selected as experimental subjects. Adult male and female AB zebrafish in the spawning period were scooped into the breeding tank, and a partition was inserted. After 10 hours of dark treatment, the middle partition was removed. After light stimulation, the male and female zebrafish mated, and the embryos were collected after 15 minutes. The embryos were transferred to a 90mm culture dish, added with embryo buffer and placed in a 28℃ constant temperature incubator for culture, and 3dpf AB zebrafish with normal development were selected for the experiment.

[0078] (2) Grouping and drug administration

[0079] Normal 3dpf zebrafish were randomly divided into 24-well plates for toxicity test. Embryonic culture water was used as blank control for toxicity test of zebrafish without diabetes background. Embryonic culture water was used as solvent for GPMS, and a series of gradient concentration groups were set up, including 5μg / mL, 25μg / mL, 100μg / mL, 200μg / mL, 400μg / mL, 800μg / mL, and 1600μg / mL. Three replicate wells were set up in each group, with 10 fish / well, and 2mL of corresponding solution was added. The solution was changed every 24h, and the death of zebrafish was observed at 24, 48, and 72hpf, and the survival rate was calculated.

[0080] Result analysis:

[0081] The total saponins (GPMS) of Gynostemma pentaphyllum obtained in Example 2 were tested in zebrafish without diabetic background. Figure 3 As shown, 24 hours after administration, the survival rate of the 800μg / mL GPMS solution group dropped to 80%±3.38, and the survival rate of the 1600μg / mL GPMS solution group dropped to 35%±3.03; 48 hours after administration, the survival rate of the 800μg / mL GPMS solution group continued to decrease to 75±4.97% and then remained unchanged, all zebrafish in the 1600μg / mL GPMS solution group died, and the other groups were normal and no deaths occurred.

[0082] Example 7: Toxicity of Gynostemma pentaphyllum saponins (GPMS) to patients with diabetes

[0083] (1) Experimental animals

[0084] Same as step (1) in Example 6.

[0085] (2) Grouping and drug administration

[0086] A zebrafish diabetes model was constructed by taking a 3dpf zebrafish with normal development and using alloxan (400mM) solution as a modeling agent. The embryonic fish culture water was used as a solvent, and the alloxan solution (400mM) was used as a control group administration reagent to conduct a toxicity test on zebrafish with a diabetic background. The remaining test conditions were the same as step (2) of Example 6.

[0087] Result analysis:

[0088] The total saponins (GPMS) of Gynostemma pentaphyllum obtained in Example 2 were tested in zebrafish with a diabetic background, such as Figure 4 As shown, 24 hours after administration, the survival rate of the 800μg / mL GPMS solution group dropped to 50±3.19%, and the survival rate of the 1600μg / mL GPMS solution group was only 20±6.41%; 48 hours after administration, the 400μg / mL GPMS solution group began to die, and the survival rate dropped to 70±4.61%, and almost all zebrafish in the 800μg / mL GPMS solution group died, with a survival rate of only 5±4.51%, while all zebrafish in the 1600μg / mL GPMS solution group died; 72 hours after administration, the survival rate of the 200μg / mL GPMS solution group dropped to 90±6.43%, the survival rate of the 400μg / mL GPMS solution group continued to decrease to 60±1.51%, and all zebrafish in the 800μg / mL GPMS solution group died.

[0089] Based on the experimental results of Example 6, it is shown that the total saponins of Gynostemma pentaphyllum (GPMS) obtained in Example 2 have greater toxic side effects on diabetic zebrafish, that is, compared with zebrafish without a diabetic background, zebrafish with diabetes have lower tolerance to GPMS.

[0090] Example 8: Effect of Gynostemma pentaphyllum total saponins (GPMS) on alloxan-induced diabetic osteoporosis in zebrafish model

[0091] (1) Experimental animals

[0092] Same as step (1) in Example 6.

[0093] (2) Grouping and dosing

[0094] The experiment set up a blank group (Blank group, blank fish water), a model group (Model group, 400mM alloxan solution), and a GPMS gradient concentration group (25-50-100-200-400μg / mL). Each group was set up with 2 replicate wells, 10 fish / well. Normal 3dpf zebrafish were randomly divided into 12-well plates, and 5mL of the corresponding solution was added to each well. Except for the blank group, the corresponding dosing solution and modeling reagent were alternately replaced every 12h in the other groups until the zebrafish developed to 9dpf.

[0095] (3) Alizarin red staining

[0096] The zebrafish that had been given the drug were fixed with 4% paraformaldehyde for 1 hour, the paraformaldehyde was removed and rinsed with 100mM Tris-MgCl2 for 10 minutes. The rinse solution was removed and a freshly prepared 30% H2O2 and 0.5% KOH mixture was added to bleach and remove the pigment until the pigment of the eyes and body of the fry faded. The bleach solution was removed and 5% glycerol / 0.1% KOH was added to rinse the zebrafish fry several times, 10 minutes each time, until there were no bubbles. After the rinse solution was removed, 0.02% alizarin red dye solution was added and shaken for 1 hour. After the dye solution was aspirated, it was washed once with 50% glycerol and 0.1% KOH and soaked. The stained zebrafish were photographed using a stereo fluorescence microscope, and the corresponding alizarin red staining area and optical density were calculated using Image-J software. The blank group result was taken as 100%, and the other groups were normalized.

[0097] Result analysis:

[0098] After the total saponins of Gynostemma pentaphyllum (GPMS) obtained in Example 2 were administered to zebrafish at 9 dpf, it was clearly observed that the mineralized areas of the head bones of each group of zebrafish at 9 dpf were different. Figure 5 As shown in the figure, compared with the blank group, the mineralized area of ​​the zebrafish head bones in the model group after alloxan treatment was relatively small in the parasphenoid bone, notochord, and operculum, and the head was stained lighter. Compared with the model group, the mineralized area of ​​the zebrafish in the GPMS administration group in the parasphenoid bone, operculum, and notochord was significantly increased, the mineralized bones were more obvious, and the staining was darker, indicating that the bone density in this area increased ( Figure 5 A). Observation and semi-quantitative analysis of the zebrafish bone staining area in each group showed that different concentrations of GPMS had an effect on the mineralization area of ​​the zebrafish parasphenoid bone, operculum and notochord ( Figure 5 B) and the staining optical density showed significant differences ( Figure 5 C). When GPMS concentration was 100 μg / mL or above, the effect of improving diabetic osteoporosis was significantly reduced.

[0099] Example 9: Effect of Gynostemma pentaphyllum total saponins (GPMS) on the expression of ALP, OPG and β-catenin mRNA in alloxan-induced zebrafish model

[0100] (1) Experimental animals

[0101] Same as step (1) in Example 6.

[0102] (2) Grouping and dosing

[0103] The experiment set up a blank group (Blank group, blank fish water), a model group (Model group, alloxan solution, 400mM), a positive control group (Positive group, etidronate disodium solution, 80nM), and a GPMS gradient concentration group (25-50-100-200μg / mL). Two parallel groups were set up in each group, with 50 fish / dish. Normal 3dpf zebrafish were randomly divided into culture dishes (100mm×100mm), and 10mL of the corresponding solution was added to each dish. Except for the blank group, the corresponding dosing solution and modeling reagent were alternately replaced every 12h in the other groups until the zebrafish developed to 9dpf.

[0104] (3) RT-qPCR experiment

[0105] After the last administration, zebrafish were anesthetized in an ice bath and placed in centrifuge tubes, with 50 zebrafish embryos in each tube. Total RNA from each zebrafish group was extracted using Trizol reagent and NanoDrop TM One / One C The concentration and purity of RNA were determined by a micro-UV-Vis spectrophotometer, and then reverse transcribed into cDNA. The cDNA was amplified using the RT-qPCR system to detect the expression levels of OPG, CLP, and β-catenin in each group. Application 2 -ΔΔCt The relative expression of the gene was calculated by using the β-actin as an internal reference. The Roche FastStart Universal SYBR Green Master (Rox) kit was used for RT-qPCR amplification (pre-denaturation at 95°C for 7 min; denaturation at 95°C for 10 s, annealing at 60°C for 32 s, 40 cycles; melting curve at 95°C for 5 s, 65°C for 15 s, and 95°C for 1 s, once). The primer sequences are shown in Table 4 below.

[0106] Table 4 Primer sequences used in RT-PCR reactions

[0107]

[0108]

[0109] Result analysis:

[0110] After administration to 9 dpf, the total saponins of Gynostemma pentaphyllum (GPMS) obtained in Example 2 were Figure 6As shown in the results, GPMS has a significant effect on the expression of osteogenic genes such as ALP, OPG, and β-catenin in the diabetic zebrafish model. Among them, GPMS can significantly increase the expression of ALP, OPG, and β-catenin genes at a concentration of 25-50μg / mL, and the best effect is achieved at 50μg / mL. At a dosage of 100-200μg / mL, the effect of GPMS on the expression of ALP, OPG, and β-catenin genes is significantly weakened.

[0111] Based on the experimental results of Example 9, it is shown that the GPMS obtained in Example 2 has a significant improvement effect on diabetic osteoporosis in zebrafish, and the effect is concentration-related, with the best effect at a concentration of 25-50 μg / mL, and the effect decreases when the administration concentration is higher than 100 μg / mL.

Claims

1. Application of total saponins of Gynostemma pentaphyllum in the preparation of drugs for treating diabetic osteoporosis.

2. The use according to claim 1, It is characterized in that The total saponins of Gynostemma pentaphyllum can promote the expression of genes related to osteogenesis.

3. The use according to claim 2, It is characterized in that The osteogenesis-related gene is at least one of ALP, OPG or β-catenin gene.

4. The use according to claim 1, It is characterized in that The total saponins of Gynostemma pentaphyllum include esculin XVII, ginsenoside Re, ginsenoside Rg1, esculin XLVI, glycyrrhizic acid, ginsenoside F1, ginsenoside Rh1 and notoginsenoside Fe.

5. The use according to claim 1, It is characterized in that The total saponins of Gynostemma pentaphyllum are eluates obtained by eluting the crude extract of total saponins of Gynostemma pentaphyllum with 45% to 55% gradient ethanol, wherein the concentration of total saponins in the eluate is 70.39% to 75.09%.

6. The use according to claim 1, It is characterized in that The dosage concentration of the total saponins of Gynostemma pentaphyllum is 25-50 μg / mL; Preferably, the administration concentration of total saponins of Gynostemma pentaphyllum is 50 μg / mL.

7. A drug for treating diabetic osteoporosis, It is characterized in that The invention comprises total saponins of Gynostemma pentaphyllum and pharmaceutically acceptable excipients.

8. The drug for treating diabetic osteoporosis according to claim 7, It is characterized in that The dosage form of the drug is selected from one of tablets, granules, pills, capsules, powders, oral liquids or injections.

9. The drug for treating diabetic osteoporosis according to claim 7, It is characterized in that The administration concentration of the total saponins of Gynostemma pentaphyllum is 25-50 μg / mL.

10. The drug for treating diabetic osteoporosis according to claim 7, It is characterized in that The administration concentration of the total saponins of Gynostemma pentaphyllum is 50 μg / mL.

Images