CN118021856A - Herba Selaginellae extract for treating osteosarcoma and its preparation method - Google Patents
Herba Selaginellae extract for treating osteosarcoma and its preparation method Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/11—Pteridophyta or Filicophyta (ferns)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
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Abstract
The invention relates to a selaginella extract for treating osteosarcoma and a preparation method thereof. The selaginella extract prepared by the invention can inhibit the expression level of MMP2 and MMP9 in osteosarcoma cells, further effectively inhibit the proliferation, migration and other abilities of the osteosarcoma cells, and promote the apoptosis of the osteosarcoma cells. Further molecular docking analysis shows that the selaginella extract has good connection with p53 and can be well embedded into a binding site; the space-filling model shows that two hydrogen bonds and five hydrophobic interactions are formed between the selaginella extract and the amino acid residues, indicating a potential interaction relationship that may exist between the selaginella extract and p53, thereby affecting osteosarcoma cells through the p53 pathway. In general, the invention defines the therapeutic activity of the selaginella extract on osteosarcoma, and particularly researches the related action mechanism thereof, provides a new active substance for treating osteosarcoma, and provides a theoretical basis and a new direction for the development of the subsequent osteosarcoma drugs.
Description
Technical Field
The invention belongs to the technical field of biology, and in particular relates to a selaginella extract for treating osteosarcoma and a preparation method thereof.
Background
Osteosarcoma is one of the most common primary malignant tumors in bone tumors, is well developed at the diaphyseal end of the limb long bone of children and teenagers, especially at the distal femur, proximal tibia and proximal humerus, has high malignant degree, is easy to develop early lung metastasis and has extremely poor prognosis effect. Osteosarcoma was treated by surgery in more than 70 years of the last century, however the 5-year survival rate of patients was not higher than 20% after surgery. With advances in surgical technology and the use of neoadjuvant chemotherapy, while neoadjuvant chemotherapy has increased the 5-year survival rate of patients to about 60% -70%, some patients still fail chemotherapy due to lung metastasis. Therefore, there is an urgent need for a safe, effective and low-toxic side effect drug in osteosarcoma treatment, especially a nontoxic or low-toxic antitumor drug derived from natural substances, which has important clinical significance.
The Chinese medicine can play a role in the development of tumor generation, such as inhibiting cell proliferation and metastasis, promoting apoptosis, improving the sensitivity of the body to chemotherapeutic drugs, reducing the side effect of chemotherapy and reversing multi-drug resistance. The Chinese medicinal herba Selaginellae is a plant of the genus herba Selaginellae of the class of ferns, has about 700 species, is widely distributed worldwide, has about 60-70 species in China, and is firstly collected in Shennong's herbal channel, pottery Hongdalian's herbal channel collection and injection's cloud: "Jinzhuan dao", on the clay of the cluster stone, the fine leaves are like cypress, the roll-bending shape is like chicken feet, and the color is green and yellow. For this purpose, the place near the stone with sand is removed. The distribution, chemical components and pharmacological activity of two varieties of selaginella and Selaginella pulvinata recorded in the pharmacopoeia of the people's republic of China are important representatives in selaginella plants, and in recent years, research on the pharmacological activity of the selaginella plants attracts high importance to researchers, and modern pharmacological research shows that the selaginella plants have pharmacological activities such as anti-tumor, anti-oxidation, anti-inflammatory, antibacterial and antiviral, antiallergic, anti-osteoporosis, blood sugar reducing and blood vessel expanding, and the action and mechanism of the selaginella in osteosarcoma are freshly reported.
Osteosarcoma is one of the most common primary malignant tumors, and early lung metastasis is likely to occur, with extremely poor prognosis. The current standard of treatment for osteosarcoma is surgery plus chemotherapy, but the effect of chemotherapy is affected by the large side effects of chemotherapeutic drugs and drug resistance of tumor genes. Therefore, a safe and effective medicament with small toxic and side effects is sought in the osteosarcoma treatment, and the medicament has important clinical significance.
Disclosure of Invention
Aiming at the defects in the prior art, the selaginella extract which can act on osteosarcoma is obtained by extracting and purifying selaginella by adopting a specific method, and can effectively inhibit activities such as proliferation, migration and the like of osteosarcoma and induce apoptosis of osteosarcoma cells; meanwhile, the influence of the selaginella extract on apoptosis and metastasis of osteosarcoma cells and the influence of the selaginella extract on the expression level of key factors in a p53 signal path are researched, the inhibition effect of the selaginella extract on the apoptosis and metastasis of osteosarcoma cells and the mechanism thereof are clarified, and an experimental basis is provided for developing clinical therapeutic drugs for osteosarcoma metastasis.
In order to solve the technical problems, the invention is realized by the following technical scheme.
The first aspect of the invention provides an application of selaginella extract in preparing a medicament for treating osteosarcoma, wherein the selaginella extract is prepared by the following method:
(1) Pulverizing herba Selaginellae, and soaking in organic solvent;
(2) Absorbing the leaching solution by macroporous adsorption resin and then carrying out gradient elution; collecting eluent, concentrating and separating out yellow solid;
(3) Dissolving the yellow solid obtained in the step (2) in methanol by heating, and filtering to obtain a supernatant;
(4) Concentrating the supernatant in the step (3) to separate out a solid substance, and drying the solid substance in vacuum to obtain the solid substance.
Preferably, the organic solvent in step (1) is selected from ethanol; most preferably, the organic solvent is selected from ethanol at a concentration of 75%.
Preferably, in the step (1), the mass-to-volume ratio of the selaginella to the organic solvent is 1:1-50; most preferably, the mass-to-volume ratio of the selaginella to the organic solvent is 1:20.
It is to be understood that, unless otherwise specified, the term "mass to volume ratio" in the context of the present invention is to be understood in a manner conventional in the art, i.e. the ratio of the mass of solid matter (in g) to the volume of liquid (in mL), for example when the "mass to volume ratio of selaginella to organic solvent is 1: at 30", if the addition amount of selaginella is 10g, the addition amount of the organic solvent is 300mL.
Preferably, the soaking time in the step (1) is 5-15h, and the soaking times are 1-3 times; most preferably, the soaking time is 10 hours, and the soaking times are 2 times.
Preferably, the macroporous adsorbent resin in step (2) is selected from NKA-9 type resins.
Preferably, the gradient elution in step (2) is specifically: sequentially eluting with 20%,40%,60%,80% and 90% ethanol.
Preferably, the temperature of the vacuum drying in the step (4) is 30-50 ℃ and the time is 10-15h; most preferably, the vacuum drying is carried out at a temperature of 40 ℃ for a period of 12 hours.
The second aspect of the invention provides a pharmaceutical composition for treating osteosarcoma, comprising herba Selaginellae extract and pharmaceutically acceptable adjuvants; the herba Selaginellae extract is prepared by the following method:
(1) Pulverizing herba Selaginellae, and soaking in organic solvent;
(2) Absorbing the leaching solution by macroporous adsorption resin and then carrying out gradient elution; collecting eluent, concentrating and separating out yellow solid;
(3) Dissolving the yellow solid obtained in the step (2) in methanol by heating, and filtering to obtain a supernatant;
(4) Concentrating the supernatant in the step (3) to separate out a solid substance, and drying the solid substance in vacuum to obtain the solid substance.
Preferably, the pharmaceutically acceptable auxiliary materials are selected from one or more of filling agents, disintegrating agents, binding agents, lubricants, bacteriostats, chelating agents, antioxidants, flavoring agents, coloring agents and solvents.
Preferably, the organic solvent in step (1) is selected from ethanol; most preferably, the organic solvent is selected from ethanol at a concentration of 75%.
Preferably, in the step (1), the mass-to-volume ratio of the selaginella to the organic solvent is 1:1-50; most preferably, the mass-to-volume ratio of the selaginella to the organic solvent is 1:20.
It is to be understood that, unless otherwise specified, the term "mass to volume ratio" in the context of the present invention is to be understood in a manner conventional in the art, i.e. the ratio of the mass of solid matter (in g) to the volume of liquid (in mL), for example when the "mass to volume ratio of selaginella to organic solvent is 1: at 30", if the addition amount of selaginella is 10g, the addition amount of the organic solvent is 300mL.
Preferably, the soaking time in the step (1) is 5-15h, and the soaking times are 1-3 times; most preferably, the soaking time is 10 hours, and the soaking times are 2 times.
Preferably, the macroporous adsorbent resin in step (2) is selected from NKA-9 type resins.
Preferably, the gradient elution in step (2) is specifically: sequentially eluting with 20%,40%,60%,80% and 90% ethanol.
Preferably, the temperature of the heated dissolution in step (3) is 35-50 ℃; most preferably, the temperature of the heated dissolution is 40 ℃.
Preferably, the temperature of the vacuum drying in the step (4) is 30-50 ℃ and the time is 10-15h; most preferably, the vacuum drying is carried out at a temperature of 40 ℃ for a period of 12 hours.
In a third aspect, the present invention provides a method for preparing a selaginella extract for treating osteosarcoma, comprising the steps of:
(1) Pulverizing herba Selaginellae, and soaking in organic solvent;
(2) Absorbing the leaching solution by macroporous adsorption resin and then carrying out gradient elution; collecting eluent, concentrating and separating out yellow solid;
(3) Dissolving the yellow solid obtained in the step (2) in methanol by heating, and filtering to obtain a supernatant;
(4) Concentrating the supernatant in the step (3) to separate out a solid substance, and drying the solid substance in vacuum to obtain the solid substance.
Preferably, the organic solvent in step (1) is selected from ethanol; most preferably, the organic solvent is selected from ethanol at a concentration of 75%.
Preferably, in the step (1), the mass-to-volume ratio of the selaginella to the organic solvent is 1:1-50; most preferably, the mass-to-volume ratio of the selaginella to the organic solvent is 1:20.
It is to be understood that, unless otherwise specified, the term "mass to volume ratio" in the context of the present invention is to be understood in a manner conventional in the art, i.e. the ratio of the mass of solid matter (in g) to the volume of liquid (in mL), for example when the "mass to volume ratio of selaginella to organic solvent is 1: at 30", if the addition amount of selaginella is 10g, the addition amount of the organic solvent is 300mL.
Preferably, the soaking time in the step (1) is 5-15h, and the soaking times are 1-3 times; most preferably, the soaking time is 10 hours, and the soaking times are 2 times.
Preferably, the macroporous adsorbent resin in step (2) is selected from NKA-9 type resins.
Preferably, the gradient elution in step (2) is specifically: sequentially eluting with 20%,40%,60%,80% and 90% ethanol.
Preferably, the temperature of the vacuum drying in the step (4) is 30-50 ℃ and the time is 10-15h; most preferably, the vacuum drying is carried out at a temperature of 40 ℃ for a period of 12 hours.
The effect of the Chinese herba selaginellae extract in osteosarcoma is freshly reported, and in the invention, by adopting a specific method to extract active ingredients from herba selaginellae, subsequent researches find that the proliferation, migration and other capacities of osteosarcoma cells are obviously inhibited after the herba selaginellae extract is treated, and the herba selaginellae extract can obviously inhibit the apoptosis of osteosarcoma cells. Matrix Metalloproteinases (MMPs) are key mediators of cell matrix degradation and promotion of cell migration, playing an important role in the development and progression of a variety of neoplastic diseases. According to analysis, the herba selaginellae extract can effectively inhibit the expression level of MMP2 and MMP9 in osteosarcoma cells, and further inhibit the functional activities of the osteosarcoma cells. Further molecular docking analysis shows that the selaginella extract has good connection with p53 and can be well embedded into a binding site; the space-filling model shows that two hydrogen bonds are formed between the herba Selaginellae extract and amino acid residues (ASN 200 and THR 231), and five hydrophobic interactions are formed between amino acid residues (PRO 222, GLU224, THR230, ILE232 and VAL 197), indicating potential interactions that may exist between the herba Selaginellae extract and p53, thereby affecting osteosarcoma cells through the p53 pathway. In general, the invention defines the therapeutic activity of the selaginella extract on osteosarcoma, and particularly researches the related action mechanism thereof, provides a new active substance for treating osteosarcoma, and provides a theoretical basis and a new direction for the development of the subsequent osteosarcoma drugs.
Drawings
FIG. 1 is a schematic diagram showing the effect of herba Selaginellae extract on migration of osteosarcoma cells.
FIG. 2 is a schematic diagram showing the quantitative analysis result of the effect of herba Selaginellae extract on osteosarcoma cell migration.
FIG. 3 is a schematic representation of the effect of Selaginella tamariscina extract on Saos2 apoptosis.
FIG. 4 is a schematic diagram showing the effect of herba Selaginellae extract on apoptosis of U2OS cells.
FIG. 5 is a schematic representation of the effect of Selaginella tamariscina extract on mitochondrial membrane potential in Saos2 cells.
FIG. 6 is a schematic diagram showing the effect of herba Selaginellae extract on mitochondrial membrane potential in U2OS cells.
FIG. 7 is a graph showing the effect of herba Selaginellae extract on Cyto-c protein expression level in osteosarcoma cells.
FIG. 8 is a graph showing the effect of herba Selaginellae extract on the levels of MMP2 and MMP9 protein expression in Saos2 cells.
FIG. 9 is a graph showing the effect of herba Selaginellae extract on the levels of MMP2 and MMP9 protein expression in U2OS cells.
FIG. 10 is a graph showing the potential linkage between Selaginella extract and p53 using molecular docking analysis.
FIG. 11 is a graph showing the effect of p53 inhibitor treatment of Saos2 cells on inhibition activity of Selaginella tamariscina extract.
FIG. 12 is a graph showing the effect of p53 inhibitor on inhibition activity of Selaginella tamariscina extract after treatment of U2OS cells.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention more clear and clear, the present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Cell lines including Saos2, U2OS, etc. listed in the context of the present invention were all cultured according to conventional methods, without specific explanation. All cell lines were identified by short tandem repeat analysis by the chinese collection of typical cultures (martial arts) and verified for mycoplasma contamination using the PCR detection kit (Shanghai Biothrive Sci) while being stored in liquid nitrogen and used for subsequent experiments. All of the reagents used in the present invention are commercially available. The experimental methods used in the present invention, such as cell culture, cell migration experiments, apoptosis experiments, western blot, etc., are all conventional methods and techniques in the art.
Representative results of selection from the biological experimental replicates are presented in the context figures, and data are presented as mean±sd and mean±sem as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GRAPHPAD PRISM 8.0.0 or SPSS22.0 software. And comparing the average value difference of two or more groups by adopting conventional medical statistical methods such as t-test, chi-square test, analysis of variance and the like. p < 0.05 was considered a significant difference.
Example 1
First, the preparation of selaginella extract is carried out, comprising the following steps:
(1) Pulverizing herba Selaginellae, soaking in 75% ethanol twice for 10 hr each time; wherein the mass volume ratio of the selaginella tamariscina to the 75% ethanol is 1:20.
(2) Collecting and combining the leaching solutions of the two times, adsorbing by adopting NKA-9 type macroporous adsorption resin, and then carrying out gradient elution by adopting ethanol with the concentration of 20%,40%,60%,80% and 90% in sequence; the eluent was collected and concentrated to precipitate a yellow solid.
(3) Dissolving the yellow solid obtained in the step (2) in methanol at the temperature of 40 ℃ and filtering to obtain supernatant.
(4) Concentrating the supernatant in the step (3) to separate out a solid substance, and vacuum drying the solid substance in a vacuum drying oven at 40 ℃ for 12 hours to obtain the solid substance, wherein the purity of the solid substance is more than 98% through High Performance Liquid Chromatography (HPLC).
Taking the selaginella extract prepared by the method for performing cell scratch experiments, and specifically comprises the following steps:
(1) Saos2 and U2OS cells in the logarithmic growth phase were digested, cell counts of 2×10 5 cells/mL were seeded in 6-well plates, and when cell densities were 95% or more, a scratch assay was performed, and serum starvation was performed for 4h prior to the assay.
(2) Streaking with a 10 μl pipette tip vertical well plate to form a narrow streak, washing the washed cells with 2mL PBS 3 times; dividing the cells into an experimental group and a Control group, wherein different concentrations (Saos 2 cells: 75. Mu.M and 150. Mu.M; U2OS cells: 100. Mu.M and 200. Mu.M) of herba Selaginellae extract are added to the experimental group, and an equal volume of solvent is added to the Control group (Control group, 0. Mu.M); cultivation was continued in a CO 2 incubator (37 ℃,5% CO 2) for 24h.
(3) Photographs were taken to record cell migration for 0h, 24h, and data processing was performed using ImageJ software.
The results are shown in FIG. 1, and the results show that the scratch closure was rapid within 24 hours in the control group without treatment with Selaginella tamariscina extract. After the treatment of the selaginella extract, the scratch closing speed of Saos2 and U2OS cells is obviously reduced, and the difference has statistical significance. The quantitative analysis of the migration distance of the cells revealed that Saos2 and U2OS cells after treatment with Selaginella tamariscina extract had significantly shorter migration distance than the Control group (p < 0.01, vs Control group) (see FIG. 2). From this, it was confirmed that the selaginella extract was effective in inhibiting the migration ability of osteosarcoma cells, and that the inhibition activity exhibited a dose-dependence.
Subsequently, the influence of the selaginella extract on osteosarcoma cell apoptosis is examined, and the specific steps are as follows:
(1) Saos2 and U2OS cells in logarithmic growth phase were taken, digested and counted, appropriate cell densities were selected according to doubling time of each cell, inoculated into 96-well plates (3 replicates), and herba Selaginellae extract (Saos 2 cells: 0. Mu.M, 75. Mu.M and 150. Mu.M; U2OS cells: 0. Mu.M, 100. Mu.M and 200. Mu.M) was added at different concentrations.
(2) Culturing in a 37 ℃ incubator, collecting cells in a 10mL centrifuge tube after 48 hours, centrifuging at 1000r/min for 5min with the number of cells per sample being 5X 10 6/mL, and discarding the culture solution.
(3) Washing with PBS buffer for 1 time, and centrifuging at 1000r/min for 5min.
(4) The cells were resuspended in 100. Mu.L of labelling buffer and incubated at room temperature for 15min in the absence of light.
(5) Centrifugation at 1000r/min for 5min and washing the pelleted cells with PBS 1 time.
(6) Mu.L of Annexin V-FITC was added and incubated at 4℃for 30min, protected from light and from time to time.
(7) 200. Mu.L of the labeling buffer was added and the mixture was detected by a flow cytometer.
The results are shown in FIGS. 3-4. The results show that after the treatment by using the selaginella extract, the proliferation capacity of Saos2 and U2OS cells is obviously reduced, the growth of osteosarcoma cells can be obviously inhibited and the apoptosis of the cells can be induced, so that the apoptosis rate of the Saos2 and U2OS cells is obviously increased, and the difference has statistical significance.
The decrease in mitochondrial membrane potential (Δψm) is a hallmark event in the early phase of apoptosis. The transition of JC-1 from red fluorescence to green fluorescence can easily detect a decrease in cell membrane potential. Meanwhile, the transition of JC-1 from red fluorescence to green fluorescence can be used as an early detection index of apoptosis. Mitochondrial membrane potential assays performed after Saos2 and U2OS cells were treated with different concentrations of selaginella extract found that the selaginella extract was able to significantly promote the red to green transition of JC-1 fluorescence in osteosarcoma cells (see fig. 5-6), and the ability to promote the transition exhibited a dose dependency, further demonstrating that the selaginella extract could induce an increase in cytochrome c (Cyto-c) in osteosarcoma cells (see fig. 7) and activate the mitochondrially mediated apoptosis pathway.
Example 2
In the previous examples, it was confirmed that the herba Selaginellae extract can effectively inhibit proliferation and migration activities of osteosarcoma cells and induce apoptosis, and on the basis of this, the action mechanism of herba Selaginellae extract was further studied. Matrix Metalloproteinases (MMPs) are key mediators of cell matrix degradation and promotion of cell migration, play an important role in the development of various tumor diseases, and for this purpose, the influence of herba Selaginellae extract on MMPs is studied first, and the specific steps are as follows:
(1) Taking Saos2 and U2OS cells in logarithmic growth phase, discarding original culture medium, adding PBS, cleaning for 2 times, adding culture medium containing herba Selaginellae extract at different concentrations (Saos 2 cells: 0. Mu.M, 75. Mu.M and 150. Mu.M; U2OS cells: 0. Mu.M, 100. Mu.M and 200. Mu.M), and processing.
(2) After 24h incubation, the original medium was discarded, washed 2 times with PBS, and then cell lysate (containing PMSF) was added and lysed on ice for 30min.
(3) Scraping the cells with the cells, transferring the cells into a sterilizing centrifuge tube, centrifuging at 12000rpm for 10min at 4 ℃, and taking the supernatant to obtain a cell lysate for extracting cell total proteins. Each histone concentration (Thermo) was measured using BCA protein quantification. Diluting each histone to an equal concentration by using a cell lysate according to a volume ratio of 4:1 plus 5 Xloading buffer, denatured at 98℃for 5min.
(4) Extracting 20 mug of total protein, performing 10% SDS-PAGE gel electrophoresis (100V, 90 min), transferring the membrane to a PVDF membrane at a constant current of 300mA for 100 min; blocking for 2h at room temperature by a shaker with 5% BSA, incubating the primary antibody (1:1000) at 4 ℃ overnight (CST), washing with TBST for 3 times, each time for 5min, and incubating for 1h at normal temperature after diluting the secondary antibody (1:20000) by blocking solution; TBST was washed 3 times, 7min each, and the ECL luminescence kit (Santa Cruz) was exposed in an exposure apparatus, and the net optical density values of the strips were analyzed using a gel Image processing system (Image-Pro Plus 6.0) with reference GAPDH as a control.
The experimental results are shown in FIGS. 8-9. The results show that compared with a control group, the expression of MMP2 and MMP9 in Saos2 and U2OS cells is obviously reduced after the herba selaginellae extract is treated, and the herba selaginis extract prepared by the invention can effectively inhibit the expression level of MMP2 and MMP9 in osteosarcoma cells.
Molecular docking analysis of herba Selaginellae extract revealed that herba Selaginellae extract has good connection with p53 and can be well embedded into binding site. According to the Dock6 protocol analysis in the silver cloud platform (https:// closed. Yinfotek. Com /), there is a high affinity of-64.52 kcal/mol and hydrogen bonds are formed (see FIG. 10). The space-filling model shows that two hydrogen bonds are formed between the herba Selaginellae extract and amino acid residues (ASN 200 and THR 231), and five hydrophobic interactions are formed between amino acid residues (PRO 222, GLU224, THR230, ILE232 and VAL 197), indicating potential interactions that may exist between the herba Selaginellae extract and p53, thereby affecting osteosarcoma cells through the p53 pathway.
To further confirm the above results of inhibition of osteosarcoma proliferation by the p 53-mediated mitochondrial apoptosis pathway of Selaginella tamariscina extract, saos2 and U2OS cells were pretreated with PFT- α (p 53 transcriptional activity inhibitor) or PFT- μ (p 53 mitochondrial translocation inhibitor) for 2 hours, respectively, before treatment with Selaginella tamariscina extract, followed by treatment of the cells with Selaginella tamariscina extract at different concentrations (5 μM,10 μM) to examine changes in cell viability, as shown in FIG. 11-12 (panel 1-panel 9 represents Selaginella tamariscina extract, PFT- α (5 μM), selaginella tamariscina extract+PFT- α (5 μM), PFT- α (10 μM), PFT- μ (5 μM), selaginella tamariscina extract+PFT- μ (10 μM), and Selaginella tamariscina extract+PFT- μ (10 μM), respectively. The results show that pretreatment of Saos2 and U2OS cells with 5-10. Mu.M PFT-alpha or 2.5-5. Mu.M PFT-mu.M can significantly increase the activity of osteosarcoma cells, reversing the inhibition of cell viability by herba Selaginellae extract. The above results indicate that apoptosis induced by selaginella extract is involved in p 53-mediated transcription-dependent and independent pathways.
In the invention, by adopting a specific method to extract active ingredients from selaginella tamariscina, subsequent researches show that after the selaginella tamariscina extract is treated, the proliferation, migration and other capacities of osteosarcoma cells are obviously inhibited, and the selaginella tamariscina extract can obviously inhibit apoptosis of osteosarcoma cells. Matrix Metalloproteinases (MMPs) are key mediators of cell matrix degradation and promotion of cell migration, playing an important role in the development and progression of a variety of neoplastic diseases. According to analysis, the herba selaginellae extract can effectively inhibit the expression level of MMP2 and MMP9 in osteosarcoma cells, and further inhibit the functional activities of the osteosarcoma cells. Further molecular docking analysis shows that the selaginella extract has good connection with p53 and can be well embedded into a binding site; the space-filling model shows that two hydrogen bonds are formed between the herba Selaginellae extract and amino acid residues (ASN 200 and THR 231), and five hydrophobic interactions are formed between amino acid residues (PRO 222, GLU224, THR230, ILE232 and VAL 197), indicating potential interactions that may exist between the herba Selaginellae extract and p53, thereby affecting osteosarcoma cells through the p53 pathway. In general, the invention defines the therapeutic activity of the selaginella extract on osteosarcoma, and particularly researches the related action mechanism thereof, provides a novel active substance for treating osteosarcoma, and provides a novel direction for the development of the subsequent osteosarcoma medicines.
The above detailed description describes the analysis method according to the present invention. It should be noted that the above description is only intended to help those skilled in the art to better understand the method and idea of the present invention, and is not intended to limit the related content. Those skilled in the art may make appropriate adjustments or modifications to the present invention without departing from the principle of the present invention, and such adjustments and modifications should also fall within the scope of the present invention.
Claims (10)
1. The application of the selaginella extract in preparing the medicines for treating osteosarcoma is characterized in that the selaginella extract is prepared by the following method:
(1) Pulverizing herba Selaginellae, and soaking in organic solvent;
(2) Absorbing the leaching solution by macroporous adsorption resin and then carrying out gradient elution; collecting eluent, concentrating and separating out yellow solid;
(3) Dissolving the yellow solid obtained in the step (2) in methanol by heating, and filtering to obtain a supernatant;
(4) Concentrating the supernatant in the step (3) to separate out a solid substance, and drying the solid substance in vacuum to obtain the solid substance.
2. The use according to claim 1, wherein the organic solvent in step (1) is selected from ethanol.
3. The use according to claim 1, wherein the mass-to-volume ratio of selaginella to organic solvent in step (1) is 1:1-50.
4. The use according to claim 1, wherein the soaking time in step (1) is 5-15 hours and the number of soaking times is 1-3.
5. The use according to claim 1, wherein the macroporous adsorbent resin in step (2) is selected from NKA-9 type resins.
6. The use according to claim 1, wherein the vacuum drying in step (4) is carried out at a temperature of 30-50 ℃ for a time of 10-15 hours.
7. The use according to claim 1, wherein the gradient elution in step (2) is in particular: sequentially eluting with 20%,40%,60%,80% and 90% ethanol.
8. A pharmaceutical composition for treating osteosarcoma, which is characterized by comprising selaginella extract and pharmaceutically acceptable auxiliary materials; the herba Selaginellae extract is prepared by the following method:
(1) Pulverizing herba Selaginellae, and soaking in organic solvent;
(2) Absorbing the leaching solution by macroporous adsorption resin and then carrying out gradient elution; collecting eluent, concentrating and separating out yellow solid;
(3) Dissolving the yellow solid obtained in the step (2) in methanol by heating, and filtering to obtain a supernatant;
(4) Concentrating the supernatant in the step (3) to separate out a solid substance, and drying the solid substance in vacuum to obtain the solid substance.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipients are selected from one or more of fillers, disintegrants, binders, lubricants, bacteriostats, chelating agents, antioxidants, flavoring agents, colorants, solvents.
10. A method for preparing selaginella extract for treating osteosarcoma, comprising the steps of:
(1) Pulverizing herba Selaginellae, and soaking in organic solvent;
(2) Absorbing the leaching solution by macroporous adsorption resin and then carrying out gradient elution; collecting eluent, concentrating and separating out yellow solid;
(3) Dissolving the yellow solid obtained in the step (2) in methanol by heating, and filtering to obtain a supernatant;
(4) Concentrating the supernatant in the step (3) to separate out a solid substance, and drying the solid substance in vacuum to obtain the solid substance.
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