CN105061533B - Hexa methoxy flavanone rhamnopyranosyl rhamnoside and its application - Google Patents

Abstract

The invention belongs to the technical field of medicine, and relates to hexamethoxydihydroflavone-rhamnosyl-rhamnoside and an application thereof. The present invention extracts and separates the jiuli xiang through a solvent, then uses a silica gel column to separate, further separates by semi-preparative chromatography, concentrates and freeze-dries to obtain the finished product hexamethoxydihydroflavone-rhamnosyl-rhamnoside. Through the evaluation of anti-tumor activity, the present invention finds that hexamethoxyflavanone-rhamnosyl-rhamnoside has little cytotoxicity; it can adhere to colon cancer cell HT-29 and Fn (nectin), and to HUVECs cells It has a significant inhibitory effect on adhesion; it has a significant inhibitory effect on the migration ability (scratch healing test) and invasion ability of HT-29; it has a significant inhibitory effect on B16-F10 lung metastasis in mice. Therefore, hexamethoxyflavanone-rhamnosyl-rhamnoside has the activity of inhibiting tumor metastasis, can be used to prepare anticancer drugs, and has good development and application prospects.

Description

Hexamethoxyflavanone-rhamnosyl-rhamnoside and its application

technical field

The invention belongs to the technical field of medicine, and in particular relates to a hexamethoxydihydroflavone-rhamnosyl-rhamnoside and an application thereof.

Background technique

Tumor metastasis is a particularly complex process involving multiple steps and factors. This whole process is affected by many factors, such as the tumor itself and the host environment. The scariest aspect of cancer is metastasis, the spread of cells from a primary tumor to distant organs, where they continue to grow without interruption. Currently, despite significant improvements in diagnosis, surgical techniques, patient care, and local and systemic adjuvant therapy, patients often die from cancer metastasis. Circulating tumor cells (CTCs) remaining in "sub-healthy people" after primary tumor surgery are the fatal source of future tumor metastasis.

Primary tumors are mostly organ-localized diseases, but in many patients after surgery, CTCs in their bodies will spread to other tissues and organs through the bloodstream to form metastasis, which is the main cause of death in cancer patients. CTCs can exist in a dormant or low-activity state for a long time in circulatory systems such as bone marrow and lymphatic system. Because of its dormancy (or low activity), chemotherapy drugs are not sensitive to it; and because its content in sub-healthy people after surgery is very low, chemotherapy drugs do not work on it. Compared with normal blood cells, CTCs have antigenic properties due to the presence of abundant biomarkers on their surface, such as SialyLewis, epithelial cell adhesion molecule (EpCAM), rabbit anti-human proliferating cell nuclear antigen monoclonal antibody (Ki-67) , human epidermal growth factor receptor (HER2), CD44+ and folate receptor (Folate Receptor), etc. These substances are not expressed or lowly expressed on the surface of normal cells, but they can be highly expressed on the surface of tumor cells.

Surgery can remove early-stage tumors with a success rate of over 95%. However, once tumor metastasis occurs, there is no way to remove the metastatic tumors in the whole body by surgery. At this time, the use of "anticancer drugs" is also ineffective; chemotherapy drugs cannot prevent tumor metastasis. Although "anticancer drugs" have shown some effects, they still have not significantly reduced cancer mortality, because the number of circulating tumor cells in the patient's body is drastically reduced after surgery and most of them are in a quiescent state, while chemotherapy drugs only Rapidly growing and dividing tumor cells work, but the use of chemotherapy drugs severely hits the patient's own immune system, tipping the patient's own balance along the unfavorable side. Therefore, we believe that if we can drug-intervene the adhesion of CTCs to endothelial cells, which is an important source step of tumor metastasis, or even comprehensively control several key sites, it is possible to effectively prevent CTCs-related tumor recurrence after surgery. transfer. Therefore, the development of low-toxic drugs to prevent tumor metastasis after surgery has become an urgent task to be solved.

Murraya exotica (L.) is a plant of the genus Murraya in the family Rutaceae. Like most natural plants, the chemical components in Murraya are also very complex, mainly flavonoids, coumarins and alkaloids.

Among them, flavonoids have been proved by a large number of experiments to have anti-metastasis activity of various tumors, including cancers of the thyroid, colon, oral cavity, cervix, lung and liver. For example: quercetin can inhibit the migration and invasion of breast cancer cell MDA-MB-231. Li Chunhua from Southern Medical University found that apigenin can inhibit the growth and metastasis of colorectal cancer. The activation of hepatocyte growth factor (HGF) and its receptor c-Met plays an important role in the invasion and metastasis of various tumor cells, and in the study of liver cancer HepG2 through cell adhesion and Transwell migration and invasion assay found that nobiletin can significantly inhibit the ability of adhesion, invasion and migration induced by HGF.

After conducting a series of related studies, we found an effective method for extracting and separating a single compound of jiulixiang flavonoid glycosides that may have anti-tumor metastasis, and completed the present invention accordingly.

Contents of the invention

The object of the present invention is to provide the compound hexamethoxyflavanone-rhamnosyl-rhamnoside and its application.

Hexamethoxyflavanone-rhamnosyl-rhamnoside, its structural formula is:

.

The preparation method of the hexamethoxyflavanone-rhamnosyl-rhamnoside comprises the following steps:

(1) Take 4.2 Kg of dried Gurixiang medicinal material (twigs) and pulverize it, and reflux extract it in 5 L of 75% ethanol for 3 times, each time for 3 hours. Filter and combine the filtrates;

(2) Concentrate the filtrate obtained in (1) under reduced pressure to obtain a syrupy total extract. The total extract was ultrasonically suspended in water, the suspension was extracted with dichloromethane, the extraction volume ratio was 1:1, and extracted 4 times, and the solvent part was concentrated under reduced pressure to obtain the crude extract of Murata japonica;

(3) The crude extract was separated by gradient chromatography on a silica gel column with ethyl acetate-petroleum ether, the fraction eluted with ethyl acetate was collected, and the solvent was removed by rotary evaporation to obtain the flavonoid component of Jiulixiang with a yield of 0.05-0.1 %;

(4) The components in (3) were separated by semi-preparative high performance liquid chromatography to obtain the monomeric extract of jiulixiang flavonoid glycosides; after identification, the structure was: hexamethoxydihydroflavone-rhamnosyl-rhamnosyl glycosides.

The preparative liquid phase separation conditions in step (4) are: chromatographic column: Agela Venusil XBP C18; flow rate: 1-4mL/min; column temperature: 35°C; detection fluorescence excitation wavelength 352 nm, emission wavelength 458nm; mobile phase is Methanol and pure water mixture, the volume ratio of methanol and pure water is 9:11, isocratic elution.

Application of the hexamethoxyflavanone-rhamnosyl-rhamnoside in drugs for preventing tumor metastasis.

The advantages of the present invention are: the monomeric compound of hexamethoxydihydroflavone-rhamnosyl-rhamnoside has the following advantages at low toxic concentrations: 1. Obvious anti-tumor cells and matrix component Fn (fibronectin) Adhesion and its adhesion to endothelial cells HUVECs; 2. Obvious anti-tumor cell migration and invasion ability; 3. Promote tumor cell apoptosis.

Description of drawings

Fig. 1 UV-Vis absorption spectrum of crude extract of flavonoid glycosides of Jiulixiang.

Fig. 2 The fluorescence emission spectrum of the crude extract of flavonoid glucosides of Jiulixiang.

Figure 3 is the semi-preparative HPLC chromatogram of the flavonoid components of Jiulixiang, and the interpolation is the HPLC analysis chart of the prepared monomer.

Fig. 4 Mass spectrum of hexamethoxyflavanone-rhamnosyl-rhamnoside.

Figure 5A Schematic diagram of the hexamethoxyflavinone ion.

Fig. 5B Schematic diagram of fragmentation ions of hexamethoxyflavanone-rhamnoside.

Fig. 6 C-NMR spectrum of hexamethoxyflavanone-rhamnosyl-rhamnoside.

Fig. 7 Proton nuclear magnetic resonance spectrum of hexamethoxyflavanone-rhamnosyl-rhamnoside.

Fig. 8 Effect of hexamethoxyflavanone-rhamnosyl-rhamnoside on the viability of colon cancer cells HT-29.

Figure 9 The effect of hexamethoxyflavanone-rhamnosyl-rhamnoside on the adhesion between colon cancer cell HT-29 and Fn (fibronectin) was detected by MTT method (**p<0.01).

Figure 10 Effects of hexamethoxyflavanone-rhamnosyl-rhamnoside (1, 10, 30 μg/mL) on the adhesion rate of colon cancer cells HT-29 and HUVECs cells (**p<0.01) .

Fig. 11 Effect of hexamethoxydihydroflavone-rhamnosyl-rhamnoside on migration ability of colon cancer cell HT-29 (scratch healing test) (*p<0.05, **p<0.01).

Fig. 12 Effects of hexamethoxyflavanone-rhamnosyl-rhamnoside (1, 10, 30 μg/mL) on the invasion ability of colon cancer cell HT-29 (**p<0.01).

Figure 13 Typical photographs (A) of the effect of hexamethoxyflavanone-rhamnosyl-rhamnoside on B16-F10 lung metastasis in C57BL/6 mice and the lung tissue morphology. Histogram of the effect of hexamethoxyflavin-rhamnosyl-rhamnoside on B16-F10 transfer in mice (B).

detailed description

The invention provides the research of the flavonoid extract and the separation and purification method of the new monomer compound. The research and the method include the following steps. The pre-separation process is:

(1) Take 4.2 Kg of dried Jiulixiang medicinal material (twigs) and pulverize, heat and boil under reflux in 5 L of 75% ethanol, and extract 3 times, 3 hours each time. Filter and combine the filtrates;

(2) Concentrate the filtrate obtained in (1) under reduced pressure to obtain a syrupy total extract. The total extract was ultrasonically suspended in water, the suspension was extracted with dichloromethane, the extraction volume ratio was 1:1, and extracted 4 times, and the solvent part was concentrated under reduced pressure to obtain the crude extract of Murata japonica;

(3) The crude extract was separated by gradient chromatography on a silica gel column with ethyl acetate-petroleum ether, and the eluted part of ethyl acetate was collected, and the solvent was removed by rotary evaporation to obtain the flavonoid component of Jiulixiang with a yield of about 0.09% ;

(4) The components in (3) were separated by semi-preparative high-performance liquid chromatography to obtain the monomeric extract of flavonoid glycosides of Jiulixiang; identified by mass spectrometry, the structure was: hexamethoxydihydroflavone-rhamnosyl-ratamine Lee Glycoside.

The preparation liquid phase separation conditions in the step (4) are: chromatographic column: Agela Venusil XBP C18; flow rate: 1-4 ml/min; column temperature: 35°C; detection fluorescence excitation wavelength 352 nm, emission wavelength 458 nm , isocratic elution, the mobile phase is a mixture of methanol and pure water, the volume ratio of methanol and pure water is 9:11. The injection volume was 200 μL, and the 20 mg/mL sample was prepared with methanol solvent.

The operating conditions of the mass spectrometer in the step (4) are, ion source: atmospheric pressure electrospray ion source (ESI); scan mode: full scan in positive ion mode; scan range: 150-800 m/z ; spray voltage (spray voltage): 4.0 kV; sheath gas flow rate: 35 Arb; auxiliary gas flow rate (aux gas flow rate): 5 Arb; ion transfer tube temperature (capillary temperature): 350 ° C; tube diameter voltage (tube lens ): 60.00 V.

The extraction method of the active ingredient of embodiment 1 Murata japonica

Take 4.2 Kg of dried Gurixiang medicinal materials (twigs) and crush them, and reflux extract them in 5L of 75% ethanol. Each batch of medicinal materials is extracted 3 times for 3 hours each time. Combine and reflux the extracts, concentrate the ethanol under reduced pressure to obtain a syrupy total extract. The total extract was ultrasonically suspended in water, the suspension was extracted with dichloromethane, the extraction ratio was 1:1 (v:v), extracted 4 times, combined and concentrated under reduced pressure, the obtained crude extract was placed on a silica gel column, distilled with ethyl acetate -Petroleum ether was separated by gradient chromatography, the fraction eluted with ethyl acetate was collected, and the solvent was removed by rotary evaporation to obtain the target component with a yield of about 0.09%. The extract was dissolved in an equal amount of methanol, and its ultraviolet absorption spectrum (Figure 1) and fluorescence spectrum (Figure 2) were measured under a UV-visible absorption spectrometer and a fluorescence spectrometer.

Example 2 Semi-preparative chromatography

The conditions are, instrument: Waters2695; detector: Waters2475 FLR; chromatographic column: Agela Venusil XBPC18 (10 mm×250 mm, 5 μm); flow rate: 3mL/min; column temperature: 35°C; detection wavelength: λex=352 nm, λem=458nm; isocratic elution and the ratio of methanol and pure water is 9:11. Inject 200 μl of 20 mg/mL sample, and collect the components within 23-27 minutes, as shown in Figure 3.

Embodiment 3 Nuclear Magnetic Resonance Spectrum Determines the Monomer Compound Structure

We used ESI-MS, ¹³ C-NMR and ¹ H-NMR to characterize the structure of the target compound, and assigned its hydrogen and carbon atoms. The deduced product structure is hexamethoxyflavanone-o-[rhamnopyranosyl-(1→4)-rhamnopyranoside], the molecular formula is C ₃₃ H ₄₄ O ₁₇ , and the relative molecular weight for 712.26. The chemical structural formula is:

.

As shown in Figure 4, the ^ion peak signal of flavonoid aglycone can be seen in the ESI-MS figure; ⁺ : 427.14; dihydroflavone is easy to ring-open and convert to isomer chalcone, and then cracked to obtain a series of fragment molecular ion peaks, including m/z=374.12, 362.32, 347.09, 331.11 ion peaks.

As shown in Figure 6, ¹³ C-NMR gives 33 carbon signal peaks; (C-5''), 92.06(C-2''), 90.76(C-1''), 78.94(C-3'''), 76.83(C-3''), 60.84(C-2 '''), 60.47(C-5'''), is the signal of ten carbon atoms on rhamnose, δ 26.05, δ 25.09, is the signal of carbon atom of methyl group on rhamnose. There are also 15 aromatic carbon signals belonging to the flavone core, ¹³ C-NMR δ=188.6(C-4), 162.03(C-3'), 161.01(C-5'), 158.81(C-5), 157.73 (C-7), 156.01(C-9), 155.51(C-6), 154.18(C-4'), 153.39(C-1'), 139.46(C-8), 137.8(C-10), 135.2 (C-6'), 130.5 (C-2'), 72.39 (C-2), 45.31 (C-3). Among them, δ=188.6 is the characteristic signal of flavonoid C-4, which is inferred to be dihydroflavone according to the C2-C3 system, and δ=56.40, 56.44, 56.61, 56.74, 56.80 are the carbon signals of the methoxy group on the flavone. The molecular structural formula is as follows:

.

As shown in Figure 7 and Table 1, according to the ¹ H-NMR chemical shift, peak shape and integrated area, the structure of the target compound was further confirmed. ¹ H-NMR (400 MHz, DMSO) δ=1.14(3H) and δ=1.24(3H) are typical chemical shifts caused by protons of rhamnose methyl groups. δ 3.6-4.0 has a typical proton signal on the methoxy group, and it is divided into six groups, indicating that there are six methoxy groups on the flavonoid aglycone. Among them, δ=0 ppm is the peak of the reference substance tetramethoxysilane (TMS), δ=2.5 ppm is the peak of deuterated dimethyl sulfoxide, and δ=3.4 ppm is the peak of water of deuterated dimethyl sulfoxide.

Table 1 H NMR analysis of HMFRR

Embodiment 4MTT method detects the inhibitory effect of target drug on colon cancer cell HT-29 proliferation

Method: select cells in logarithmic growth phase and in good condition, digest with trypsin, count with a hemocytometer, and dilute the medium to prepare a cell suspension of 8×10 ⁴ ~1×10 ⁵ cells/mL, and then in 96-well Add 100 µL of diluent to each well of the plate, and incubate in a 37°C, 5% CO ₂ incubator for 24 h; the target drug is dissolved in DMSO and sterilized by filtration through a 0.22 μm filter membrane. Dilute the primary mother solution with DMSO into the secondary mother solution with different concentration gradients, and then dilute the secondary mother solution 100 times with the medium to obtain the final concentration of the drug. The concentration of DMSO in the solution was 1 wt%; the old medium was removed, and 100 µL of medium containing different concentrations of the target drug was added to each well, and a blank control group and a solvent control group were set up. Continue to incubate for 24 h; remove the drug-containing medium, add 100 µL of serum-free and phenol red-free RPMI 1640 medium and 10 µL of 5 mg/mL MTT solution to each well, and continue to incubate for 4 h in the incubator; suck off the supernatant of each well and remove Add 100 µL of DMSO, shake well for 20 min, fully dissolve, measure the OD value of each well with a microplate reader at a wavelength of 570 nm, and calculate the cell survival rate and the IC50 value of drug action. Survival rate (%)=(light absorption value of experimental group-light absorption value of solvent control group)/(light absorption value of blank control group-light absorption value of solvent control group)×100%.

As shown in Figure 8, the activity of HT-29 decreased with the increase of the concentration of the target drug. It can be known that the target drug inhibits the proliferation of tumor cells. The activity of HT-29 cells in the concentration of 60 μg/mL was above 80%, indicating that the cytotoxicity of the target drug was low.

Example 5 MTT method to detect the effect of target drugs on the adhesion of colon cancer cell HT-29 and matrix component Fn (fibronectin)

Method: Coating matrix membrane: Dilute the Fn solution with serum-free culture medium to 10 μg/mL working solution, add 100 μL of coating solution to each well of a 96-well plate, and incubate at 37 °C, 5 % CO ₂ (V/ V) Continue culturing in the incubator for 24 h; BSA sealing: suck out the working solution in the culture plate, add 100 μL of 1 wt% BSA solution to each well of the well to be tested, and place in 37 °C, 5% CO ₂ (V/V) After blocking in the incubator for 1 h, wash with sterile PBS solution 3 times, and discard the PBS thoroughly; inoculate cells: take HT-29 cells in the logarithmic growth phase, digest and adjust the cell concentration to 2×10 ⁵ /ml, Mix with the target drug so that the final concentrations are 1, 10, and 30 μg/mL, and add 100 µL to each well of the 96-well culture plate that has been coated with Fn, and set up a blank control group and a solvent control group. Set up 6 replicate wells in each group and culture in a conventional incubator for 1 h; MTT colorimetric assay: carefully aspirate and discard the culture medium, wash with PBS three times to remove non-adherent cells, add 10 µL 100 µL of 5 mg/mL MTT serum-free and phenol red-free culture solution, continue to incubate for 4 h, discard the supernatant, add 100 µL DMSO to each well, shake well for 20 min, and use a microplate reader to detect the concentration at 570 nm after dissolution. OD value. Relative adhesion rate (%) = OD value of medication group/OD value of blank control group × 100%.

As shown in Figure 9, within the concentration range of 30 μg/mL, the inhibitory rate of the adhesion of colon cancer cells HT-29 to Fn gradually increased with the increase of the concentration. Therefore, targeted drugs can block the binding and adhesion of extracellular matrix to integrins on the surface of cancer cells.

Example 6 Fluorescence microscope photography method to investigate the effect of target drugs on the adhesion of colon cancer cells HT-29 and HUVECs

Method: Take the umbilical cord of the newborn baby from a healthy puerpera in the hospital, quickly put it in a biological safety cabinet, and put it into a preheated PBS culture dish under aseptic conditions; find out the umbilical vein and wash it with preheated PBS More than 3 times, then pour into the preheated 0.1wt% type II collagenase solution, and then immerse it in a sterile beaker of double distilled water warmed in advance, digest it for 20-30min, and make the collagenase solution slowly flow into the container. In a 50 mL centrifuge tube containing 20 mL of M-199 medium containing 10 wt% standard fetal bovine serum; centrifuge, discard the supernatant, add M-199 medium and transfer to a cell culture bottle, place at 37 ℃, 5 % CO ₂ (V/V) culture in the incubator; after 4-5 hours of culture, change the medium and wash off the non-adherent cells; after the cells grow and fuse into a single layer of cells, discard the culture medium and wash with PBS for 2-3 Second, trypsinized and then subcultured. Human umbilical vein endothelial cells in logarithmic growth phase and in good condition were selected, digested with trypsin, and prepared into cell suspension. Inoculate 500 µL per well into a 24-well plate, culture in a 37°C, 5 % CO ₂ (V/V) incubator, and grow until the coverage reaches 80%-90%, then remove the old medium , washed once with sterile PBS solution, and then added 500 µL of medium containing stimulatory factor IL-1β at a concentration of 1 ng/mL to each well to increase its ability to adhere to cancer cells. A blank control group was also set up to culture Continue to incubate in the box for 4 h to activate the endothelial cells; during this period, the colon cancer cells HT-29 were digested with trypsin solution and made into a cell suspension. Centrifuge at 1000 rpm for 5 min, discard the supernatant, take 1 mL of PBS solution and pipette evenly, add 10 µL rhodamine-123 solution in the dark and mix well, stain at room temperature for 15 min in the dark; centrifuge at 1000 rpm for 5 min, discard After the supernatant, wash the cells twice with PBS, take 1 mL of phenol red-free RPMI 1640 medium, pipette the cell pellet evenly, count, and dilute to a cell density of ³ ×106/mL; the target drug is phenol red-free RPMI Dilute the 1640 medium to 1, 10, and 30 μg/mL respectively, remove the old medium, and add 450 µL of the medium containing the target drug and 50 µL of the diluted HT-29 cell suspension to each well of the 24-well plate , set a blank control group, continue to incubate in the incubator for 1 h, and operate in the dark during the whole process; slowly discard the drug-containing medium, gently rinse the cells with PBS twice to remove non-adherent cells, and add 500 µL to each well RPMI 1640 medium without phenol red, photographed with a fluorescent inverted microscope at 10 times. Select 10 fields of view for each well, count the average number of adherent cells, and calculate the relative adhesion rate according to the following formula.

As shown in Figure 10, compared with the fluorescence map of the control group, within the concentration range of 30 μg/mL, the number of HT-29 cells adhered to HUVECs gradually decreased with the increase of concentration, showing a dose-dependent decrease. With the increase of target drug concentration, the relative adhesion rate decreased gradually, and the higher the concentration, the smaller the relative adhesion rate. Therefore, the target drug can significantly inhibit the adhesion of tumor cells to HUVECs.

Example 7 Cell Scratch Test Detects the Effect of the Target Drug on the Migration Ability of Colon Cancer Cell HT-29

Method: Draw three horizontal lines evenly on the back of the 24-well plate with a marker pen in the ultra-clean workbench. Digest cells with trypsin to prepare cell suspension, add 1 mL of cell suspension containing 3×10 ⁵ cells to each well, and incubate overnight in an incubator at 37 °C and 5 % CO ₂ (V/V); Use a 20 μL pipette tip to scratch perpendicularly to the horizontal line on the back of the 24-well plate; gently rinse with PBS 3 times to remove the scratched cells, target drug and containing 2wt

% fetal bovine serum-free medium was mixed so that the final concentrations were 1, 10, and 30 μg/mL, and 1 mL was added to each well, and each concentration was repeated for three wells, and then placed in the culture medium for 24 h under the same conditions. Stop the culture; at 0 h and 24 h, observe each well of the 24-well plate under the bright field of a fluorescent inverted microscope and take pictures. At least 3 fields of view are taken for each well. The migration distance of the control group was compared.

As shown in Figure 11, the cell scratch experiment proved that after 24 h of different concentrations of the target drug, as the drug concentration increased, the migration distance of the cells relative to the blank group gradually decreased. Within the concentration range of 30 μg/mL, as the concentration gradually increased, the mobility of the target drug-dosed group decreased gradually compared with the blank group (0 μg/mL), and showed a dose-dependent manner. Therefore, the target drug can significantly prevent the migration of colon cancer cell HT-29.

Example 8 Transwell assay to detect the effect of the target drug on the invasion ability of colon cancer cell HT-29

Method: Matrigel preparation: BD matrigel frozen at -80°C refrigerator overnight at 4°C until it becomes liquid; take 200 μL of serum-free medium, add 50 μL of matrigel at 4°C, mix well, add 50 μL to each upper chamber (4 chambers); incubate in an incubator (37°C, 5% CO ₂ (V/V)) for 4-5 h, take out the small chamber, suck out the culture medium, and invert to air dry; add containing 20% fetal bovine serum culture medium, 750 μL/well; digest well-growing HT-29 cells, wash 3 times with serum-free medium, count, make cell suspension concentration 1.5×10 ⁵ cells/mL, centrifuge Afterwards, suspend with 0.1wt% BSA serum-free phenol red medium, and add 200 μL cell suspension containing corresponding drug concentration to the chamber; culture in the incubator for 24 h under the same conditions; take out the Tanswell chamber, and discard the culture medium in the well , washed twice with PBS, fixed with methanol for 20 min; discarded methanol, air-dried the chamber properly, stained with 0.1% crystal violet for 30 min at room temperature, washed twice with PBS, and gently wiped off the stain on the chamber with a wet cotton swab. Surface cells: 5 randomly selected fields of view were counted under the microscope, and the number of cells that had invaded was calculated.

As shown in Figure 12, the Transwell invasion test proved that the concentration was within the range of 30 μg/mL. As the concentration increased, the number of cells passing through the matrigel chamber in the target drug-dosed group gradually decreased, showing a dose-dependent manner. The number of cells passed through in the blank group (0 μg/mL) was 106, and when the concentration was 1, 10 and 30 μg/mL, the number of cells passed through was 86, 58 and 28, respectively. Therefore, the target drug can significantly reduce the invasion ability of colon cancer cell HT-29.

Embodiment 9 Animal model detects the effect of target drug on tumor metastasis in vivo

Method: 6-8 weeks old healthy female C57BL/6 mice were taken, with an average weight of 20 g each, and randomly divided into 2 groups with 6 mice in each group, marked as the blank group and the drug-dosed group; 100 mg/kg, the intragastric solvent was carboxymethylcellulose sodium (CMCNa), for four consecutive days, and the blank group was intragastrically administered the same volume of CMCNa under the same conditions; B16-F10 cells were digested with trypsin, each The mice were injected with ⁵ ×104 B16-F10 cells in the tail vein respectively; the mice in the drug-dosed group and the blank group were fed normally under the same conditions, and were intragastrically administered at a dose of 100 mg/kg in the drug-added group every day. Continuously administered for 31 days, under the same conditions, the same volume of CMCNa was administered to the blank group; the mice were killed, and the metastasis of B16-F10 melanoma in the lungs of the mice was observed by dissection, the weight of the lung tissue and the number of metastatic nodules were recorded, and the metastatic tissue for slice observation.

The experimental results are shown in Figure 13. It can be seen that the tumor volume and number of nodules of melanoma metastasized to the lungs were significantly reduced compared with the blank control group. Lung histomorphology can be observed that the morphology of the drug-added group did not form obvious micro-metastases, and the tumor tissue was loose; while the blank group formed obvious micro-metastases, and the tumor tissue was denser. Compared with the blank group, the addition of drugs Tumor tissue showed significant tumor cell necrosis and apoptosis. Compared with the blank control group, the lung weight of the drug-added group after tumor metastasis was reduced. The average lung weight of the blank group was 0.25 g, and the average lung weight of the drug-added group was only 0.18 g. Compared with the blank group, there was a very significant difference (p< 0.01); and the number of nodules of lung tumor metastasis was also significantly reduced compared with the blank group, the average number of nodules of lung tumor metastasis in the blank group was 3, while the average number of nodules of lung tumor metastasis in the treatment group was 2 . Therefore, the target drug can significantly inhibit the metastasis of melanoma in mice.

In summary, the hexamethoxyflavanone-rhamnosyl-rhamnoside of the present invention has the following advantages at low toxic concentrations: 1. Obvious anti-tumor cell adhesion to matrix component Fn (fibronectin) And its adhesion to endothelial cells HUVECs; 2. Obvious anti-tumor cell migration and invasion ability; 3. Promote tumor cell apoptosis. Therefore, hexamethoxyflavanone-rhamnosyl-rhamnoside can be used as a drug for the prevention of tumor metastasis, which provides a scientific basis for the development and utilization of natural plant resources.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (3)

1. the extraction method of hexamethoxyflavanone-rhamnosyl-rhamnoside, is characterized in that: Including the following steps: (1) Grind 4.2 Kg of the dried Murata medicinal material, heat and reflux extraction in 5 L of 75% ethanol for 3 times, each time for 3 hours, filter, and combine the filtrates; (2) Concentrate the filtrate obtained in (1) under reduced pressure to obtain a syrupy total extract, which is ultrasonically suspended in water, and the suspension is extracted with dichloromethane. The extraction volume ratio is 1:1, and the extraction is performed 4 times. Concentrate the solvent part under pressure to obtain the crude extract of Murata japonica; (3) The crude extract was separated by gradient chromatography on a silica gel column with ethyl acetate-petroleum ether, the fraction eluted with ethyl acetate was collected, and the solvent was removed by rotary evaporation to obtain the flavonoid component of Jiulixiang with a yield of 0.05-0.1 %; (4) The components in (3) were separated by semi-preparative high performance liquid chromatography to obtain the monomeric extract of jiulixiang flavonoid glycosides; after identification, the structure was: hexamethoxydihydroflavone-rhamnosyl-rhamnosyl Glycoside; The chemical structural formula is as follows: . 2. The method for extracting hexamethoxyflavanone-rhamnosyl-rhamnoside according to claim 1, characterized in that: the preparation liquid phase separation condition in step (4) is, chromatographic column: Agela Venusil XBP C18; flow rate: 1-4 mL/min; column temperature: 35°C; detection fluorescence excitation wavelength 352 nm, emission wavelength 458nm; mobile phase is a mixture of methanol and pure water, the volume ratio of methanol and pure water is 9:11, Isocratic elution. 3. The application of the hexamethoxyflavanone-rhamnosyl-rhamnoside prepared by the preparation method as claimed in claim 1 in the preparation of a drug for preventing tumor metastasis.