CN110934944A - Method for extracting total saponins of Trillium japonicum and pharmaceutical application - Google Patents
Method for extracting total saponins of Trillium japonicum and pharmaceutical application Download PDFInfo
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Abstract
The invention provides an extraction method of total saponins of Trillium japonicum and pharmaceutical application thereof. The results show that the total saponins of the trillium fortunei can effectively inhibit the proliferation of the colon cancer CT26 cells, and the IC of the total saponins50It was 9.28. mu.g/mL. Western blotting results show that the total saponins of the Trillium wilfordii can reduce the expression of Ras, Akt, Erk proteins and phosphorylation proteins thereof in a concentration-dependent manner. In vivo experiments show that compared with a model group, the Trillium wilfordii total saponin shows better inhibition rates of high, medium and low, which are respectively 51.22%, 34.88% and 25.02%, and shows a certain concentration dependence trend. HE staining shows that the total saponins of Trillium fortunei can promote apoptosis of colon cancer cells, wherein large tumor cell necrosis can be seen in high-dose groups. Western blotting results show that the total saponins of the Trillium arvense can reduce the expression level of Ras protein in a concentration-dependent manner. The total saponins of the Trillium arvense can effectively inhibit the proliferation of the colon cancer CT26 cells, and the mechanism is that the expression of Ras, Akt and Erk proteins is reduced through a Ras signal path.
Description
Technical Field
The invention relates to an extraction method of total saponins of trillium fortunei, in particular to application of total saponins of trillium fortunei in preparation of a medicine for treating colon cancer. Belongs to the field of biological pharmacy.
Background
Colon cancer is one of the most common malignant tumors in the digestive system, and seriously threatens human health. Chemotherapy is still one of the main means for treating colon cancer, but the clinical application of the chemotherapy is greatly limited due to the serious side effect and the multi-drug resistance of the chemotherapy drugs. Therefore, the search for a drug which can efficiently kill tumor cells and has small toxic and side effects is a hotspot in the research and development of new anti-tumor drugs at present.
Trillium tschnoskii Maxim is one of four famous medicines in the Tujia, has sweet and warm taste, has the effects of relieving pain and swelling, stopping bleeding and detoxifying, soothing nerves and activating blood and the like, and is mainly used for treating lung carbuncle and sore, furuncle and pyogenic infections, headache and dizziness, neurasthenia and the like. Researches show that the Trillium wilfordii extract and saponin have obvious inhibiting effect on colon cancer, lung cancer, cervical cancer and the like. RAS is one of the most known oncogenes, is involved in important physiological processes such as cell growth, proliferation, development and differentiation regulation, and plays an important role in malignant transformation of cells. Continued activation of RAS proteins can lead to rapid cell proliferation and hyperactivity of anti-apoptotic signals, thereby leading to tumorigenesis and progression. Hans Raskov et al found that the occurrence of colon cancer was highly correlated with mutations in K-ras, APC and P53 genes.
Disclosure of Invention
Aiming at the technical problems, the invention adopts an MTT method, an RT-PCR method and a Western blotting method to observe the effects of the total saponins of the Trillium age on the proliferation of colon cancer CT26 cells and the inhibition of subcutaneous tumors of nude mice and discuss the action mechanism of the total saponins of the Trillium age.
A method for extracting total saponins of Trillium japonicum Thunb comprises the following steps: drying and crushing Trillium arvense beads, soaking the Trillium arvense beads in ethanol for several hours, performing reflux extraction, concentrating an extracting solution under reduced pressure, and performing freeze drying to obtain a total Trillium arvense extract;
dissolving the Trillium wilfordii total extract in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, concentrating under reduced pressure, freeze drying to obtain extract, separating with macroporous adsorbent resin, eluting with methanol, concentrating, and drying to obtain Trillium wilfordii total saponin.
The invention also provides an application of the trillium total saponin obtained by extraction in preparation of a medicine for treating colon cancer.
As a preferred scheme, the invention applies the total saponins of trillium fortunei extracted in the preparation of the medicine for inhibiting the proliferation of the colon cancer CT26 cells. In particular to application of the total saponins of the Trillium aizoon obtained by extraction in preparing medicines for reducing expression of Ras, Akt, Erk proteins and phosphorylation proteins thereof.
On the basis of the technical scheme, the using amount of the trillium total saponin is 5mg/kg-15 mg/kg.
The in vitro cell experiment of the invention shows that the Trillium repens total saponin has the inhibiting effect on the proliferation of colon cancer CT26 cells cultured in vitro, IC50The value was 9.28. mu.g/mL, indicating that it is an active ingredient against colon cancer. In vivo experiments show that the trillium fortunei total saponins effectively inhibit the growth of subcutaneous tumors of nude mice, and the trillium concentrations of the trillium fortunei total saponins also show better inhibition rates, which are respectively 51.22%, 34.88% and 25.02%, and show concentration dependence trend. The result shows that the total saponins of the Trillium fortunei have the effect of inhibiting the growth of tumors in vivo and are in line with in vitro cell experiments.
The colon cancer generation is related to the continuous activation of RAS protein, the dose-dependent effect of the colon cancer is further verified by the results of in vivo experimental tumor tissue protein level, and the high, medium and low dose groups of the Trillium arvense total saponin can obviously reduce the expression of RAS gene on the mRNA level and are dose-dependent. Therefore, the total saponins of the Trillium arvense have the correlation of inhibiting the growth of tumors and the expression of RAS genes or the downstream channels thereof. Then, in an in vitro experiment, the expression levels of Ras, Akt and Erk proteins and phosphorylated proteins thereof in cells are detected by using a Western blotting method to further confirm the action mechanism, and the expression levels of the Ras, Akt and Erk proteins are reduced in a concentration dependence manner, and the Akt and Erk are reduced in a time dependence manner. The immunohistochemical result also indicates that the total saponins of the Trillium aizoon of high and medium dose in vivo can promote the apoptosis of colon cancer cells.
In conclusion, the total saponins of the Trillium fortunei have antitumor activity in vivo and in vitro, and the mechanism is to interfere the expression of Ras, Akt and Erk proteins through a Ras signal channel. The method is expected to provide a new idea for drug development.
Drawings
FIG. 1 shows RT-PCR method for detecting the effect of total saponins of Trillium arvense on RAS gene expression in colon carcinoma tissues, compared with model group, P < 0.01; n is 8.
FIG. 2 shows the effect of total saponins of Trillium ramosum on the expression of Akt, p-Akt, Erk and p-Erk proteins of CT-26 cells of colon cancer detected by Western blotting method.
FIG. 3 is a gray scale analysis statistical chart of the influence of the total saponins of Trillium arvense on the expression of the protein Akt, P-Akt, Erk and P-Erk of the colon cancer CT-26 cells, a is the time-dependent influence of the total saponins of Trillium arvense on the expression of the protein Akt of the colon cancer CT-26 cells, b is the time-dependent influence of the total saponins of Trillium arvense on the expression of the protein P-Akt of the colon cancer CT-26 cells, c is the time-dependent influence of the total saponins of Trillium arvense on the expression of the protein Erk of the colon cancer CT-26 cells, d is the time-dependent influence of the total saponins of Trillium arvense on the expression of the protein P-Erk of the; n is 3.
FIG. 4 shows the effect of total saponins of Trillium ramosum on the concentration dependence of Ras, Akt, Erk and their expression of phosphorylated proteins of CT26 cells of colon cancer detected by Western blotting method.
FIG. 5 is a gray scale analysis statistical chart of the dependency influence of the total saponins of Trillium japonicum on the Ras, Akt and Erk of colon cancer CT26 cells and the expression concentration of the phosphorylation protein thereof, wherein a is the dependency influence of the total saponins of Trillium japonicum on the Ras protein expression concentration of colon cancer CT26 cells, b is the dependency influence of the total saponins of Trillium japonicum on the p-Ras concentration of colon cancer CT26 cells, c is the dependency influence of the total saponins of Trillium japonicum on the Akt concentration of colon cancer CT26 cells, d is the dependency influence of the total saponins of Trillium japonicum on the p-Akt concentration of colon cancer CT26 cells, e is the dependency influence of the total saponins of Trillium japonicum on the Erk concentration of colon cancer CT26 cells, and f is the dependency influence of the total saponins of colon cancer CT26 cells; p <0.05, P < 0.01; n is 3.
FIG. 6 shows the HE staining results of Trillium aizoon total glycosides in tumor tissues, wherein a is a model group, b is a high dose group, c is a medium dose group, and d is a low dose group.
FIG. 7 shows the effect of total glycosides of Trillium japonicum on Ras protein expression level detected by Western blotting method.
FIG. 8 is a statistical plot of the intensity analysis of the effect of Trillium stolonifera on Ras protein expression levels by tumor tissues with P <0.05 and P < 0.01; n is 3.
Detailed Description
Example 1
The invention relates to a medicament and a reagent, wherein the traditional Chinese medicine of trillium age is picked in Shennong forest district and is identified as the trillium age through the three-level laboratory of traditional Chinese medicine pharmacology of the university of Sanxia.
The apparatus used in the present invention comprises: EYELA model N-1100 rotary evaporator: shanghai Ailang instruments, Inc.; an electronic balance: shanghai balance instrumentation works; thermo full-wavelength microplate reader: thermo Electron corporation; CO type MCD1752An incubator: SANYO, Japan; eppendorf centrifuge 5804R type low temperature high speed centrifuge: eppendorf, Germany; an ultra-clean bench: suzhou Antai air technologies, Inc.; micro instantaneous centrifuge: BIO-BAD Bio Inc., USA; immunoblotter, gel imager was purchased from Bio-Rad.
Tumor mouse CT26 cells from Shanghai Koehley Biotechnology Ltd
BALB/c-nu female mice were bred in the animal experiment center of the university of three gorges
Preparation of total saponins of Trillium repens L.Diels is prepared by drying Trillium repens L.Diels in a constant temperature oven at 48 ℃, crushing by a traditional Chinese medicine crusher, sieving by a 60-mesh sieve, soaking for several hours by ethanol, reflux extracting, repeating for many times, concentrating the extracting solution under reduced pressure, and freeze drying to obtain the total extract of Trillium repens L.Diels. Dissolving in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, concentrating under reduced pressure, and freeze drying to obtain extracts. Separating the n-butanol extract with macroporous adsorbent resin, eluting with 60% methanol, concentrating, and drying to obtain total saponins of Trillium vulgare L.
Establishment of a transplanted tumor mouse model BALB/c-nu female mice the experiment was started one week after adaptive rearing at the animal experiment center of the university of three gorges (average body weight 22 g). Taking colon cancer CT26 cells in logarithmic growth phase, and trypan blue dye exclusion to show live cells>95%, adjusting the cell concentration to 4X 106Cell suspension was prepared, and solid tumors were formed by subcutaneous inoculation of 0.2mL in the right axilla. The size of the tumor to be detected is about 100mm3Then, tumor-bearing mice were randomly divided into 5 groups, namely a model group, a 5-FU group, a high dose group, a medium dose group and a low dose group, wherein each group contains 8 mice. The model group is administrated with normal saline for intragastric administration (0.2mL), the 5-FU group is administrated by intraperitoneal injection with 5-FU (10mg/kg), and the low, medium and high dose groups are respectively administrated with Trillium japonicum total saponin aqueous solution (5mg/kg, 10mg/kg, 15 mg/kg). The administration was 1 time per day for 9 days.
Calculation of tumor inhibition rates body weights were measured every two days, and the signs and changes in body weight of tumor-bearing mice in the administration group and the control group were observed and a body weight change chart was drawn. On day 9, tumor bearing mice were sacrificed, tumor tissue was stripped and weighed. Tumor inhibition rate (tumor inhibition rate ═ average tumor body weight in control group-average tumor body weight in administration group)/average tumor body weight in control group x 100%) was calculated.
Activity of Trillium arvense total saponins on CT26 in vitro proliferation inhibition Colon cancer CT26 cells in logarithmic growth phase were cultured at 5 × 103The cells were inoculated in 96-well plates and placed in 5% CO2And culturing for 24 hours in a constant temperature incubator at 37 ℃. 100 mul of culture solution containing total extracts of Trillium wilfordii (0, 0.5, 1, 2, 4, 8, 16mg/L) with different concentrations are respectively added, only culture solution containing 0.54 thousandth of DMSO is added to a solvent control group, and 4 parallel multiple wells are arranged in each well. Continuously culturing cellsAnd (5) cultivating for 24 hours. And then taking out the culture plate, adding 20 mu L of MTT solution of 5g/L into each hole, continuously incubating for 4h, discarding the remaining incubation solution, adding 150uL of DMSO into each hole, placing in a shaking table, shaking for 10min, fully dissolving the crystals, and detecting the absorbance value of each hole at 570nm on an enzyme-labeling instrument. The cell proliferation rate (%) was calculated as (a test group-a control group)/a control group × 100%.
RT-PCR detection of RAS gene expression level of tumor body tissue cells 150mg of tumor tissue is weighed and placed in a dry glass homogenizer, 1mL of Trizol is added, the complete grinding is carried out, RNA is extracted, the RNA is inverted into cDNA by using a reverse transcription kit, and then the expression level of RAS is detected by using an RT-PCR method.
Pathological section HE staining tumor-bearing mice are weighed and killed, tumor body tissues are stripped, 1cm multiplied by 1cm tissues are cut and fixed in 10 percent formaldehyde solution, the sections are embedded by normal paraffin, HE staining is carried out, and histopathological changes are observed under a mirror.
Western blot determination of Ras, p-Ras, Akt, p-Akt, Erk, p-Erk expression in colon cancer cells and tumor tissue cell RAS protein expression the cells of control group and Trillium procumbens total saponin groups (5, 10 and 15mg/L) were collected by cell scraping, RIPA cell lysate was added to extract the proteins, a Bio-Rad microplate reader was used for protein quantification, polyacrylamide gel electrophoresis was performed after protein denaturation, PVDF membrane was sealed with 5% skim milk at room temperature for 2h, rabbit anti-Ras (1: 1000), p-Ras (1: 1000), Akt (1: 1000), p-Akt (1: 1000), Erk (1: 1000), p-Erk (1: 1000) and rabbit anti- β -actin (1: 1000) were added at room temperature overnight, after TBST membrane washing, corresponding markers diluted at 1: 3000 were added, secondary antibodies, after TBHRP, TBST membrane washing, chemiluminescence was performed, 0.1g, tumor tissue was taken and added to the same level of protein, and protein was incubated in a homogenizer with 10 ml, and extracted with the same protein and homogenized by a method.
Statistical processing SPSS 13.0 statistical software is adopted for data statistics, and quantitative data are calculated as mean plus or minus standard deviation
It is shown that the differences were compared using one-way anova, with P <0.05 being statistically significant.
Tumor inhibition rate
After one week of administration, except for the model and the negative control group, the mice in the other groups showed messy fur, no longer smooth and bright color, slow movement and slow weight increase, which probably indicates that 5-FU and the total saponins of Trillium japonicum also have some toxic and side effects on the mice in the process of killing tumor cells. The tumor inhibition rate of the 5-FU group is 65.15% at most, and the high, medium and low concentrations of the Trillium arvense total saponins also show good inhibition rates which are 51.22%, 34.88% and 25.02% respectively, and show a certain concentration dependence trend (see Table 1). The results show that the total saponins of Trillium arvense have an inhibitory effect on the growth of tumors in vivo.
Table 1 influence of total saponins of trillium fortunei on tumor weight in mice (n ═ 8)
Proliferation inhibiting effect of Trillium arvense total saponins on colon cancer CT-26 cells
The trillium total saponin has the effect of inhibiting the proliferation of CT26 cell cultured in vitro, and the half inhibitory concentration is 9.278 mug/ml, and the result is shown in Table 2.
TABLE 2 proliferation inhibitory effect of Trillium arvense total saponins on colon cancer CT-26 cells (n ═ 3)
Influence of Trillium japonicum total saponin on RAS gene expression of tumor tissue
The results are shown in figure 1, and the high, medium and low dose groups of the total saponins of the Trillium arvense can obviously reduce the expression of RAS genes and are dose-dependent. Therefore, it is presumed that the aged grass total saponins have a correlation in inhibiting the growth of tumors and in inhibiting RAS gene expression or its downstream channels.
Influence of Trillium arvense total saponins on expression of Akt, p-Akt, Erk and p-Erk proteins in colon cancer CT-26 cells
FIGS. 2 and 3 show that the expression of Akt, Erk and phosphorylated proteins thereof can be reduced in a time-dependent manner in three time groups of the total saponins of Trillium vulgare L. It is speculated that the total saponins of the aged grass are related to inhibiting the growth of tumors and Ras signal pathways.
FIGS. 4 and 5 show that the high, medium and low dose groups of the Trillium wilfordii total saponin can reduce the expression of Ras, Akt, Erk and phosphorylation protein thereof in a concentration-dependent manner. It is speculated that the total saponins of the aged grass are related to inhibiting the growth of tumors and Ras signal pathways.
Total glycosides of Trillium japonicum (L.) Merr tumor tissue HE staining result
As a result, it was found that most of the groups administered with 15mg/kg and 10mg/kg of total saponins from Trillium arvense exhibited dark nuclei, dense cytoplasm, increased eosinophilic staining (eosin staining), and an apoptotic state. It is shown that the total saponins of Trillium fortunei can promote the apoptosis of colon cancer cells in vivo (see FIG. 6). The tumor tissue treated by the medicine can show tumor cell necrosis in three doses, particularly the necrosis of large tumor cells in a medium-high dose group.
Western blot for detecting influence of trillium fortunei total saponins on RAS protein expression
The colon cancer is associated with the continuous activation of RAS protein, the dose-dependent effect of the tumor histoprotein level in the in vivo experiment is further verified (see results 7 and 8), and the high, medium and low dose groups of the total saponins of the Trillium aizoon can obviously reduce the expression of the RAS gene on the mRNA level and are dose-dependent. Therefore, it is speculated that the total saponins of the Trillium aizoon have relevance to inhibiting the expression of RAS genes or the downstream channels thereof. Then, expression levels of Ras, Akt, Erk proteins and phosphorylation proteins thereof in cells are detected by using a Westernblotting method in vitro experiments to further prove an action mechanism of the protein, and the expression levels of the Ras, Akt and Erk proteins are reduced in a concentration-dependent manner, and the Akt and Erk are reduced in a time-dependent manner. The immunohistochemical result also indicates that the total saponins of the Trillium aizoon of high and medium dose in vivo can promote the apoptosis of colon cancer cells.
Claims (5)
1. The method for extracting the total saponins of the trillium fortunei is characterized by comprising the following steps of: drying and crushing Trillium arvense beads, soaking the Trillium arvense beads in ethanol for several hours, performing reflux extraction, concentrating an extracting solution under reduced pressure, and performing freeze drying to obtain a total Trillium arvense extract;
dissolving the Trillium wilfordii total extract in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, concentrating under reduced pressure, freeze drying to obtain extract, separating with macroporous adsorbent resin, eluting with methanol, concentrating, and drying to obtain Trillium wilfordii total saponin.
2. Use of the total saponins extracted from Trillium procumbens according to claim 1 in the preparation of a medicament for treating colon cancer.
3. The use according to claim 2, wherein the use of the total saponins of Trillium arvense obtained by extraction in the preparation of a medicament for inhibiting proliferation of CT26 cells in colon cancer.
4. The use of claim 3, wherein the total saponins extracted from Trillium wilfordii are used for preparing a medicament for reducing the expression of Ras, Akt, Erk proteins and their phosphorylated proteins.
5. The use according to any one of claims 2 to 4, wherein the amount of Trillium repens total saponins is from 5mg/kg to 15 mg/kg.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113318176A (en) * | 2021-05-24 | 2021-08-31 | 三峡大学 | Application of Trillium fortunei total saponin in preparing medicine for treating arthritis |
| CN115887570A (en) * | 2021-09-30 | 2023-04-04 | 中国人民解放军军事科学院军事医学研究院 | Application of Trillium arvense extract in preparation of product for regeneration and repair of injured intestinal mucosa |
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2019
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| 周慧萍 等: "延龄草总皂苷抑制结肠癌细胞增殖作用及机制研究", 《华南国防医学杂志》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113318176A (en) * | 2021-05-24 | 2021-08-31 | 三峡大学 | Application of Trillium fortunei total saponin in preparing medicine for treating arthritis |
| CN115887570A (en) * | 2021-09-30 | 2023-04-04 | 中国人民解放军军事科学院军事医学研究院 | Application of Trillium arvense extract in preparation of product for regeneration and repair of injured intestinal mucosa |
| CN115887570B (en) * | 2021-09-30 | 2024-02-13 | 中国人民解放军军事科学院军事医学研究院 | Application of longhairy antenoron herb extract in preparation of products for regeneration and repair after intestinal mucosa injury |
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