CN112220812A - Fresh elm leaf water extract powder, its preparation, anti-tumor metastasis effect and application - Google Patents
Fresh elm leaf water extract powder, its preparation, anti-tumor metastasis effect and application Download PDFInfo
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- CN112220812A CN112220812A CN201910632742.9A CN201910632742A CN112220812A CN 112220812 A CN112220812 A CN 112220812A CN 201910632742 A CN201910632742 A CN 201910632742A CN 112220812 A CN112220812 A CN 112220812A
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- acryloyloxy
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- quinic acid
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Abstract
The invention discloses a fresh elm leaf aqueous extract powder, a preparation method and an anti-tumor metastasis effect thereof. The fresh elm leaf aqueous extract powder prepared by the method contains 17 quinic acid compounds and 14 relaxin compounds. Experiments prove that the fresh elm leaf water extract powder can effectively inhibit tumor lung metastasis, so the invention discloses the application of the fresh elm leaf water extract powder in preparing anti-tumor metastasis medicaments. The invention provides an effective technical means for treating and preventing tumor metastasis.
Description
Technical Field
The invention relates to a method for preparing fresh elm leaf water extract powder, and relates to application of the fresh elm leaf water extract powder prepared by the method in preparing anti-tumor metastasis medicaments. The invention belongs to the field of biological medicine.
Background
Malignant tumors are characterized by metastasis. Tumor cell infiltration, migration and proliferation play an important role in the process of human tumor development and transformation to malignancy. Especially in the process of tumor metastasis, tumor cell infiltration, migration and proliferation play an important role. Tumor metastasis seriously affects the patient's expectations for health and life. When malignant tumors grow, metastases often appear. Some patients develop micrometastases in the early stages of malignancy. This condition renders conventional treatment methods no longer effective. The cause of death in more than 90% of cases of malignancy-associated death is tumor metastasis. Most prostate cancer patients also die from metastases. Lung cancer is one of the most aggressive human cancers for malignancies. For example, only 10% -15% of patients with advanced lung cancer typically survive 5 years. This difficult situation has not improved significantly over the last 30 years. In many clinical cases, lung cancer has metastasized to surrounding tissues before it is diagnosed. Tumor metastasis, especially tumor metastasis to the lung, is the greatest risk of death for tumor patients. In the first diagnosed colon cancer patients, a proportion of liver metastases occurs of about 25%. The proportion of liver metastases in the advanced stage of colon cancer patients is 40-50%. Those patients with liver metastases had an average survival rate of 9% after 5 years, which was reduced to 6-12 months without treatment. The proportion of leptomeningeal metastases in breast cancer patients is 12-35%. Among the many metastatic pathways, lymphatic vessels are the most common pathway. The incidence rate exceeds 60 percent. To date, there are no drugs against tumor metastasis for clinical use. Thus, the development of a drug for preventing tumor metastasis without toxic side effects is receiving more and more attention. Despite the great efforts that have been made, no substantial progress has been made.
Elm leaves are picked in summer and autumn and dried in the sun or eaten fresh. For example, fresh and tender elm leaves are picked, washed clean and cut into pieces, and then the pieces can be mixed with flour to make elm leaf cakes and dumpling fillings. The tender leaves of the elm which just germinate are quickly boiled in boiling water and are cold and dressed with sauce are good dishes. Elm leaves are considered by folks to treat symptoms such as neurasthenia, insomnia and dreaminess. The folk also thinks that the dried elm leaves, three to five coins, are ground into powder, mixed into paste by white spirit, and directly applied to the injured part to stop pain and stop bleeding. The ointment has good therapeutic effect on fracture, and can relieve pain and promote bone regeneration. The folk further thinks that the elm leaves can induce diuresis to alleviate edema, and relieve dysuria and body edema.
The inventor conducts long-term research on chemical components and treatment effect of the fresh elm leaf water extract powder. The inventor carries out mass spectrum analysis on chemical components of fresh elm leaf water extract powder, and finds 31 unreported components which mainly comprise cinnamoyl quinic acid and polysubstituted relaxin. Although the biological activity of a single component of the fresh elm leaf aqueous extract powder has been reported, the biological activity of the full-component combination is unknown. Through repeated research, the inventor finds that the combination of the 31 components can prevent tumor metastasis. Compared with all the known inventions of elm leaves, the invention of the fresh elm leaves aqueous extract powder which can prevent tumor metastasis reaches a brand new height. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
The invention aims to provide a method for preparing fresh elm leaf water extract powder and the fresh elm leaf water extract powder prepared by the method. Experiments prove that the fresh elm leaf aqueous extract powder prepared by the invention has the function of resisting tumor metastasis. In order to achieve the purpose, the invention adopts the technical means of four aspects.
The technical means of the first aspect is to provide a fresh elm leaf aqueous extract powder, wherein the fresh elm leaf aqueous extract powder is determined to contain 17 quinic acid compounds and 14 deuterodermine compounds by analyzing the mass spectrum total ion current spectrum and 31 components of the corresponding mass spectrum peak of the fresh elm leaf aqueous extract powder, wherein the 17 quinic acid compounds are 3-O- [ E-2, 6-dimethoxy-3- (4-carboxyl n-butylcarbonyloxy) -4- (2-carboxyl ethylcarbonyloxy) -5- (2-hydroxyl ethylcarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2,3, 6-tri (carboxyl n-acryloyloxy) -4- (hydroxyethyl n-acryloyloxy) -5- (5-hydroxyl n-pentylcarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2-hydroxyacetoxy-3, 4, 5-tris (carboxyacetoxy) -6-hydroxymethyl-n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2-hydroxyacetoxy-3, 5-dimethylolacetoxy-4, 6-dihydroxy-n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2-carboxyacetoxy-3, 5-bis (hydroxymethylacetoxy) -4-carboxymethyl-n-acryloyloxy-6-hydroxycinnamoyl ] quinic acid, 3-O- [ E-2, 6-dihydroxy-3, 5-bis (4-carboxy-1, 3-butadienoyloxy) -4-carboxymethacryloyloxycinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (carboxyethyl acetoxy) -3, 4-dihydroxy-5-carboxy n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2-carboxyacetoxy-3, 5-bis (hydroxymethyl acetoxy) -4-carboxymethyl n-acryloyloxy-6-methoxycinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (4-carboxy-1-butenoyloxy) -3, 5-bis (3-hydroxy n-propionyloxy) -4-hydroxy n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2-carboxyethyl acetoxy-3, 5-bis (hydroxy n-acryloyloxy) -4- (3-carboxy-1-propenoyloxy) -6-hydroxy n-acryloyloxy -methoxycinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (3-hydroxy-n-propionyloxy) -3, 5-bis (carboxy-n-acryloyloxy) -4-hydroxy-n-propionyloxy-cinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (carboxy-n-acryloyloxy) -3-carboxy-n-propionyloxy-4-hydroxy-n-propionyloxy-5-hydroxy-n-valeryloxy-cinnamoyl ] quinic acid, 3-O- [2, 6-bis (1-hydroxy-1-acryloyloxy) -3- (6-hydroxy-1-n-hexylenecarbonyloxy) -4- (7-hydroxy-n-heptoyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (carboxy-n-acryloyloxy) -4-methoxy-5- (5-hydroxy-1-n-pentenylcarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2-methoxy-3, 5-bis (hydroxy-n-acryloyloxy) -4- (3-hydroxy-1-propenylcarbonyloxy) -6- (4-hydroxy-n-butylcarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (3-hydroxy-n-propionyloxy) -3- (4-hydroxy-1-n-butenylcarbonyloxy) -4- (4-hydroxy-n-butylcarbonyloxy) cinnamoyl ] quinic acid and 3-O- [ E-2, 6-bis (6-hydroxy-n-hexylcarbonyloxy) -4-formyloxy-5- (6- Hydroxy-1-n-hexylenecarbonyloxy) cinnamoyl ] quinic acid; the 14 kinds of kadsurin compounds are 3-O-glucoside-5, 7-dimethyl-2 ' -methoxy-3 ', 4 ' -bis (3-hydroxy n-propionyloxy) -5 ', 6 ' -bis (3-hydroxy n-propionyloxy) kadsurin, 3-O-glucoside-5, 7-dimethyl-2 ' -hydroxy-4 ' - (3-hydroxy n-propionyloxy) -6 ' - (3-carboxy n-propionyloxy) kadsurin, 3-O-glucoside-5, 7-dimethyl-2 ' - (5-hydroxy-1, 3-pentadiene carbonyloxy) -4 ' - (3-hydroxy-1-n-propenylcarbonyloxy) -5 ' - (3-hydroxy n-propenyloxy) kadsurin, 3-O-glucoside-5, 7-dimethyl-2 ', 3' -dihydroxy-4 '- (5-hydroxy-n-pentanoyloxy) -5' - (4-hydroxy-n-butanoyloxy) -6 '-hydroxymethoxylespedezin, 3-O-glucoside-2, 6-dimethyl-2' - (4-hydroxy-1-n-butenylcarbonyloxy) -4 '- (3-hydroxy-1-n-propenylcarbonyloxy) -5' - (5-hydroxy-1-n-pentenylcarbonyloxy) lespedezin, 3-O-glucoside-5, 7-dimethyl-2 '- (4-hydroxy-1-n-butenylcarbonyloxy) -4' - (3-hydroxy-n-propenoyloxy) -5 '- (4' - (3-hydroxy-n-propenoyloxy) -hydroxy n-butylcarbonyloxy) lacustrin, 3-O-glucoside-5, 7-dimethyl-2 ', 6' -dihydroxy-3 '- (4-hydroxy-1, 3-butadiencarbonyloxy) -4' - (5-hydroxy-1, 3-pentadienylcarbonyloxy) lacustrin, 3-O-glucoside-2 '-methoxy-3', 6 '-bis (4-hydroxy-n-butyryloxy) -3' -hydroxy-n-acryloyloxy-4 '- (3-hydroxy-n-propionyloxy) lacustrin, 3-O-glucoside-2' -hydroxy-3 ', 5' -diformyloxy-4 '-hydroxy-n-propionyloxy-6' - (3-hydroxy-1-n-acryloyloxy) lacustrin, 3-O-glucoside-2 '-hydroxy-4' - (3-hydroxy-n-propylcarbonyloxy) -5 '-hydroxy-n-propionyloxy relaxin, 3-O-glucoside-2' -methoxy-4 '- (6-hydroxy-1, 3-n-hexyldienoyloxy) -5' - (4-carboxy-1, 3-n-butyldienoyloxy) -6 '- (5-carboxy-1, 3-n-pentadienoyloxy) relaxin, 3-O-glucoside-5, 7-dimethyl-2' -methoxy-4 '- (6-hydroxy-n-hexylcarbonyloxy) -5' - (5-hydroxy-n-pentylcarbonyloxy) relaxin, 3-O-glucoside-5, 7-dimethyl-2 ', 3' -dimethoxy-4 '- (6-hydroxy-n-hexylcarbonyloxy) -5' - (5-hydroxy-n-pentylcarbonyloxy) -6 '-hydroxymethoxylansopirin and 3-O-glucoside-5, 7-dimethyl-2', 3 ', 6' -trimethoxy-4 '- (6-hydroxy-n-hexylcarbonyloxy) -5' - (5-hydroxy-n-pentylcarbonyloxy) lansopirin.
The second technical means is to provide a method for preparing the fresh elm leaf water extract powder, which can be prepared by the steps of cleaning fresh elm leaves with tap water, heating in distilled water at 50-100 ℃ for 0.5-4 hours, cooling to room temperature, filtering, washing filter cakes with distilled water for 3 times, and concentrating the combined filtrate under reduced pressure to obtain the powder, namely the fresh elm leaf water extract powder.
The third technical means is to provide a preferable method for preparing the fresh elm leaf water extract powder, which is characterized in that fresh elm leaves are taken and washed by tap water, heated for 2 hours in distilled water at 50-80 ℃ and stirred at 220rpm/min, cooled to room temperature, filtered, filter cakes are washed by distilled water for 3 times, and the combined filtrate is decompressed and concentrated to obtain the powder, namely the fresh elm leaf water extract powder.
The technical means of the fourth aspect is to provide the anti-tumor metastasis effect of the fresh elm leaf water extract powder.
Drawings
FIG. 1 shows UPLC-mass spectrum of total ion current of fresh folium Ulmi Pumilae extract powder.
FIG. 2.3-O- [ E-2, 6-dimethoxy-3- (4-carboxy-n-butylcarbonyloxy) -4- (2-carboxyethylcarbonyloxy) -5- (2-hydroxyethylcarbonyloxy) cinnamoyl ] quinic acid and cleavage products.
FIG. 3.3-O- [ E-2,3, 6-tris (carboxy n-acryloyloxy) -4- (hydroxyethyl n-acryloyloxy) -5- (5-hydroxy n-pentylcarbonyloxy) cinnamoyl ] quinic acid and cleavage products.
FIG. 4.3-O- [ E-2-hydroxyacetoxy-3, 4, 5-tris (carboxyacetoxy) -6-hydroxymethyl-n-acryloyloxy cinnamoyl ] quinic acid and cleavage products.
FIG. 5.3-O- [ E-2-Hydroxyacetoxy-3, 5-dimethylol acetoxy-4, 6-dihydroxyn-acryloyloxy cinnamoyl ] quinic acid and cleavage products.
FIG. 6.3-O- [ E-2-carboxyacetoxy-3, 5-bis (hydroxymethylacetoxy) -4-carboxymethyln-acryloyloxy-6-hydroxycinnamoyl ] quinic acid and cleavage products.
FIG. 7 is a schematic representation of 3-O- [ E-2, 6-dihydroxy-3, 5-bis (4-carboxy-1, 3-butadienecarbonyloxy) -4-carboxymethacryloxycinnamoyl ] quinic acid and its cleavage products.
FIG. 8 is a schematic representation of 3-O- [ E-2, 6-bis (carboxyethylacetoxy) -3, 4-dihydroxy-5-carboxy n-acryloyloxy cinnamoyl ] quinic acid and its cleavage products.
FIG. 9. 3-O- [ E-2-carboxyacetoxy-3, 5-bis (hydroxymethylacetoxy) -4-carboxymethyln-acryloyloxy-6-methoxycinnamoyl ] quinic acid and cleavage products.
FIG. 10.3-O- [ E-2, 6-bis (4-carboxy-1-butenylcarbonyloxy) -3, 5-bis (3-hydroxy-n-propenylcarbonyloxy) -4-hydroxy-n-propenoyloxy cinnamoyl ] quinic acid and cleavage products.
FIG. 11.3-O- [ E-2-carboxymethylacetoxy-3, 5-bis (hydroxy-n-propionyloxy) -4- (3-carboxy-1-propenylcarbonyloxy) -6-methoxycinnamoyl ] quinic acid and cleavage products.
FIG. 12.3-O- [ E-2, 6-bis (3-hydroxy-n-propylcarbonyloxy) -3, 5-bis (carboxy-n-acryloyloxy) -4-hydroxy-n-propylcarbonyloxy-cinnamoyl ] quinic acid and cleavage products.
FIG. 13.3-O- [2, 6-bis (1-hydroxy-1-propenecarbonyloxy) -3- (6-hydroxy-1-n-hexenecarbonyloxy) -4- (7-hydroxy-n-heptylcarbonyloxy) cinnamoyl ] quinic acid and cleavage products.
FIG. 14.3-O- [ E-2, 6-bis (carboxy n-propionyloxy) -3-carboxy n-propionyloxy-4-hydroxy n-propionyloxy-5-hydroxy n-valeryloxy cinnamoyl ] quinic acid and cleavage products.
FIG. 15.3-O- [ E-2, 6-bis (carboxy n-acryloyloxy) -4-methoxy-5- (5-hydroxy-1-pentenylcarbonyloxy) cinnamoyl ] quinic acid and cleavage products.
FIG. 16.3-O- [ E-2-methoxy-3, 5-bis (hydroxy-n-acryloyloxy) -4- (3-hydroxy-1-propenylcarbonyloxy) -6- (4-hydroxy-n-butylcarbonyloxy) cinnamoyl ] quinic acid and cleavage products.
FIG. 17.3-O- [ E-2, 6-bis (3-hydroxy-n-propylcarbonyloxy) -3- (4-hydroxy-1-n-butenylcarbonyloxy) -4- (4-hydroxy-n-butylcarbonyloxy) cinnamoyl ] quinic acid and cleavage products.
FIG. 18.3-O- [ E-2, 6-bis (6-hydroxy-n-hexylcarbonyloxy) -4-formyloxy-5- (6-hydroxy-1-n-hexylenecarbonyloxy) cinnamoyl ] quinic acid and cleavage products.
FIG. 19.3-O-glucoside-5, 7-dimethyl-2 ' -methoxy-3 ', 4 ' -bis (3-hydroxy-n-propionyloxy) -5 ', 6 ' -bis (3-hydroxy-n-propionyloxy) relaxin and cleavage products.
FIG. 20.3-O-glucoside-5, 7-dimethyl-2 ' -hydroxy-4 ' - (3-hydroxy-n-propylcarbonyloxy) -6 ' - (3-carboxy-n-propylcarbonyloxy) relaxin and cleavage products.
FIG. 21.3-O-glucoside-5, 7-dimethyl-2 ' - (5-hydroxy-1, 3-pentadiene carbonyloxy) -4 ' - (3-hydroxy-1-n-propenylcarbonyloxy) -5 ' - (3-hydroxy-n-propenoyloxy) relaxin and cleavage products.
FIG. 22.3-O-glucoside-5, 7-dimethyl-2 ', 3 ' -dihydroxy-4 ' - (5-hydroxy-n-valeryloxy) -5 ' - (4-hydroxy-n-butyryloxy) -6 ' -hydroxymethoxylansciin and cleavage products.
FIG. 23.3-O-glucoside-2, 6-dimethyl-2 ' - (4-hydroxy-1-n-butenylcarbonyloxy) -4 ' - (3-hydroxy-1-n-propenylcarbonyloxy) -5 ' - (5-
Hydroxy-1-n-pentenylcarbonyloxy) relaxin and cleavage products.
FIG. 24.3-O-glucoside-5, 7-dimethyl-2 ' - (4-hydroxy-1-n-butenylcarbonyloxy) -4 ' - (3-hydroxy-n-acryloyloxy) -5 ' - (4-hydroxy-n-butylcarbonyloxy) relaxin and cleavage products.
FIG. 25.3-O-glucoside-5, 7-dimethyl-2 ', 6' -dihydroxy-3 '- (4-hydroxy-1, 3-butadienecarbonyloxy) -4' - (5-hydroxy-1, 3-pentadienecarbonyloxy) relaxin and cleavage products.
FIG. 26.3-O-glucoside-2 ' -methoxy-3 ', 6 ' -bis (4-hydroxy-n-butyryloxy) -3 ' -hydroxy-n-acryloyloxy-4 ' - (3-hydroxy-n-propionyloxy) relaxin and cleavage products.
FIG. 27.3-O-glucoside-2 ' -hydroxy-3 ', 5 ' -diformyloxy-4 ' -hydroxy-propionyloxy-6 ' - (3-hydroxy-1-n-propenoyloxy) relaxin and cleavage products.
FIG. 28.3-O-glucoside-2 ' -hydroxy-4 ' - (3-hydroxy-n-propylcarbonyloxy) -5 ' -hydroxy-n-propionyloxy-lospermin and cleavage products.
FIG. 29.3-O-glucoside-2 '-methoxy-4' - (6-hydroxy-1, 3-n-hexyldienoyloxy) -5 '- (4-carboxy-1, 3-n-butyldienoyloxy) -6' - (5-carboxy-1, 3-n-pentadienoyloxy) relaxin and cleavage products.
FIG. 30.3-O-glucoside-5, 7-dimethyl-2 ' -methoxy-4 ' - (6-hydroxy-n-hexylcarbonyloxy) -5 ' - (5-hydroxy-n-pentylcarbonyloxy) relaxin and cleavage products.
FIG. 31.3-O-glucoside-5, 7-dimethyl-2 ', 3 ' -dimethoxy-4 ' - (6-hydroxy-n-hexylcarbonyloxy) -5 ' - (5-hydroxy-n-pentylcarbonyloxy) -6 ' -hydroxymethoxyrelaxin and cleavage products.
FIG. 32.3-O-glucoside-5, 7-dimethyl-2 ', 3 ', 6 ' -trimethoxy-4 ' - (6-hydroxy-n-hexylcarbonyloxy) -5 ' - (5-hydroxy-n-pentylcarbonyloxy) relaxin and cleavage products.
Detailed Description
To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.
Example 1 preparation of an aqueous extract powder of fresh elm leaves
Cleaning fresh folium Ulmi Pumilae with tap water, heating 300g in 900mL distilled water at 60-90 deg.C for 2 hr while stirring (220 rpm/min). Cooling to room temperature, filtering, and concentrating under reduced pressure to obtain 36g fresh folium Ulmi Pumilae water extract powder.
Example 2 determination of chromatography and Mass Spectrometry ion flow Spectroscopy of fresh Ulmus pumila leaf aqueous extract powder
2-1 preparation of sample solution (10mg/mL)
26.7mg of fresh elm leaf aqueous extract powder was weighed and dissolved in 2.67mL of ultrapure water. The resulting solution was sonicated for 1 minute, followed by centrifugation at 13000r/min for 10 minutes. The supernatant was taken, filtered through a 0.22 μm filter and placed in a sample vial for chromatographic and mass spectrometric determination.
2-2. chromatographic conditions
Chromatographic conditions
Chromatographic column Waters, Acquity
A HSS T3 column (2.1 × 100mm i.d.,1.7 μm);
the sample injection volume is 2 mu L;
190-400nm of PDA detector;
mobile phase water (0.1% formic acid), acetonitrile;
the mobile phase washed the column according to the gradient of table 1.
TABLE 1 mobile phase gradiometer
2-3 measuring chromatogram
UPLC chromatograms of fresh elm leaf aqueous extract powder were measured and recorded according to the above chromatographic assay conditions (see fig. 1).
2-4. conditions for measuring ion flow spectrum and mass spectrum
Electrospray ionization modes are Positive (PI) and Negative (NI) modes. The ion mode parameters comprise that the capillary tube voltage is 1000V, the desolvation air flow rate is 800L/h, the temperature is 450 ℃, the source temperature is 120 ℃, the taper hole air flow rate is 50L/h, the spraying air pressure is 6bar, the fragmentation voltage is 20-45V, the sampling cone voltage is 6V, the acquisition mode is an MSE continuum resolution mode, the data acquisition range of the ratio (m/z) of the mass number of the charged particles to the charge number is 100-1500, the Trap fragmentation voltage of the low-energy channel is 6V, the gradient voltage of the Trap fragmentation voltage of the high-energy channel is 20-60V, LE (leucine enkephalin) is selected as a mass lock to acquire m/z, and the range is 100-1500.
2-5, recording ion flow spectrum and mass spectrum
The ion flow spectrum of the fresh elm leaf aqueous extract powder was determined and recorded according to the above conditions (see fig. 1).
Example 3 specifying the Structure of 31 Components in an aqueous extract powder of fresh Ulmus pumila leaves
The UPLC chromatogram of example 2 was coupled to a mass spectrum and the UPLC-mass spectrum of the fresh elm leaf aqueous extract powder was determined. The mass spectrometry conditions are two modes of electrospray ionization, positive and negative ions. The ion mode parameters comprise that the capillary tube voltage is 1000V, the desolvation air flow rate is 800L/h, the temperature is 450 ℃, the source temperature is 120 ℃, the taper hole air flow rate is 50L/h, the spraying air pressure is 6bar, the fragmentation voltage is 20-45V, the sampling cone voltage is 6V, the acquisition mode is an MSE continuum resolution mode, the data acquisition range of the ratio (m/z) of the mass number of the charged particles to the charge number is 100-1500, the Trap fragmentation voltage of the low-energy channel is 6V, the gradient voltage of the Trap fragmentation voltage of the high-energy channel is 20-60V, LE (leucine enkephalin) is selected as a mass lock to acquire m/z, and the range is 100-1500. 31 independent peaks were separated within 140 minutes. The peaks (in order of the peaks from left to right of the total ion current spectrum) are assigned according to the mass fragmentation law in Table 2. These peaks (peaks numbered sequentially from left to right for the total ion flux spectrum) were assigned to the corresponding components of table 2 according to the mass spectrometry fragmentation law. The cleavage products of these 31 components under mass spectrometric conditions are shown in FIGS. 2-32.
Table 2 shows the retention time of the peak-corresponding component, the mass number of the negative ion, the structure and the name of the negative ion in the total ion current spectrum
Example 4 evaluation of antitumor cell migration Effect of fresh Ulmus pumila leaf aqueous extract powder
1) RGDS is prepared into a solution with the concentration of 100 mu M by using a DMEM culture medium, and the fresh elm leaf water extract powder is prepared into a solution with the concentration of 10mg/mL by using the DMEM culture medium.
2) The cells were a549 (adenocarcinoma human alveolar basal epithelial cells) and 95D (human highly metastatic lung adenocarcinoma cells).
3) Experimental methods
After A549 cells are digested, washed 3 times by serum-free DMEM medium, counted and prepared into cell suspension with the density of 2 multiplied by 106one/mL. mu.L of cell suspension was added to each well, and 25. mu.L of fresh elm leaf aqueous extract powder (final concentration of 0.039mg/mL) or RGDS solution (final concentration of 20. mu.M) was added simultaneously. Blank plus 25 μ L DMEM medium. Add 600. mu.L serum-free DMEM medium to the lower chamber at 37 ℃ and 5% CO2The culture was carried out in an incubator for 6 hours. After the 95D cells are digested, washed for 3 times by serum-free DMEM medium, counted and prepared into cell suspension with the density of 2 x 106one/mL. mu.L of cell suspension was added to each well, and 25. mu.L of fresh elm leaf aqueous extract powder (final concentration of 0.156mg/mL) or RGDS solution (final concentration of 20. mu.M) was added at the same time. Blank plus 25 μ L DMEM medium. Add 600. mu.L serum-free DMEM medium to the lower chamber at 37 ℃ and 5% CO2The culture was carried out in an incubator for 6 hours.
After swabbing the matrigel and cells in the upper chamber with a cotton swab, the cells were fixed with 4% paraformaldehyde for 30 min. The fixative was aspirated, washed 3 times with PBS, and stained with 0.1% crystal violet stain for 30 min. The staining solution was aspirated and washed 3 times with PBS.
The cells were observed in approximately 9 fields, photographed and counted. The experimental data were statistically analyzed by t-test and variance analysis, and the number of migrated cells was expressed as mean ± SD.
4) The results are shown in tables 3 and 4. As can be seen from Table 3, the fresh elm leaf aqueous extract powder can effectively inhibit the migration of A549 cells at the concentration of 0.039mg/mL, and has no significant difference from the RGDS activity for inhibiting the migration of A549 cells. As can be seen from Table 4, the fresh elm leaf aqueous extract powder can effectively inhibit 95D cell migration at a concentration of 0.156mg/mL, and is significantly stronger than the inhibitory activity of RGDS on 95D cell migration.
TABLE 3 migration inhibitory Activity of fresh Ulmus pumila leaf aqueous extract powder on A549 cells
TABLE 4 fresh elm leaves aqueous extract powder inhibits 95D cell migration activity
Example 5 evaluation of antitumor metastasis Effect of fresh Ulmus pumila leaf aqueous extract powder
1) Dissolving fresh elm leaf water extract powder with physiological saline, dissolving RGDS with physiological saline as positive control, and using physiological saline as negative control.
2) The oral dosage of the fresh elm leaf water extract powder is 50 mg/kg/day, the oral dosage of the normal saline is 0.2mL/20g, and the oral dosage of RGDS is 20 mu mol/kg/day. Two weeks after tumor inoculation, tumors with a diameter of about 4-5mm were observed in the mouse axilla, and 10 consecutive doses were administered for 10 days for 10 times.
3) The experimental animals were C57BL/6 male mice (clean grade), weighing 20. + -.2 g, 10 mice per group.
4) Lewis mouse lung carcinoma cells (LLC) LLC cells were purchased from ATCC, adherent cells, DMEM medium.
5) LLC cells were digested and diluted to 2X 10 with physiological saline7Number of viable cells per mL, placental blue (Tryanblue) dye exclusion assay>95 percent. Taking a C57BL/6 male mouse, male, left-hand fixed mouse, sterilizing the right anterior limb axillary skin of the mouse with 75% ethanol, and holding a 1mL sterile syringe with the right hand to inject 0.2mL of tumor cell suspension into the axillary part of the mouse subcutaneously (the number of the tumor cells is about 2 multiplied by 10)6/mL). Tumors with the diameter of about 1.5-2 cm can grow out 15 days after the mice are inoculated, and the tumors are used as standby tumor sources.
6) Taking a Lewis lung cancer tumor-bearing mouse which is inoculated for 15 days, dislocating the cervical vertebra, soaking and sterilizing the mouse by using 75 percent ethanol for 10min, carrying out inactive peeling on solid tumors in a super clean bench, selecting tumor tissues which grow well, washing floating blood, shearing the tumor tissues in a watch glass, putting the sheared tumor tissues into a glass tissue homogenizer, adding 4 ℃ precooled physiological saline, grinding the mixture into cell suspension, and filtering the cell suspension twice by using a 200-mesh nylon net to prepare single cell suspension. Dilution of the cell suspension to 2X 107cell/mL, staining with Trypan blue, and counting viable cell number>95 percent. The qualified cell suspension was inoculated 0.2mL per mouse to the right axilla of a C57BL/6 male mouse and observed. Tumors of about 4-5mm in diameter were observed in the mice axilla 12 days after inoculation, and tumor volumes were measured and divided into groups on average according to tumor volume, and administration was started. The fresh elm leaf water extract powder is orally taken by mice with 50mg/kg fresh elm leaf water extract powder every day, the normal saline group mice orally take 0.2mL/20g normal saline every day, and the RGDS group mice orally take 20 mu mol/kg RGDS every day. The oral administration is continued for 10 days.
7) Weighing the weight of the mouse on the eleventh day, anesthetizing with ether, removing the cervical vertebra, killing the mouse, fixing the right axillary tumor growth part of the mouse with forceps, cutting the skin, exposing the tumor, carrying out blunt dissection, and weighing; the lungs of the mice were removed and the number of tumor nodules metastasized in the tumor lungs was recorded. The experimental data are measured by t-test and variance analysis, and mean + -SD g represents the tumor weight in situ, and mean + -SD represents the number of metastatic nodules. As can be seen from Table 5, the fresh elm leaf aqueous extract powder was as effective in inhibiting lung tumor metastasis in mice at 50 mg/kg/day oral dose as 20 μmol/kg/day RGDS. Therefore, the invention has obvious technical effect.
TABLE 5 antitumor metastasis activity of fresh elm leaf aqueous extract powder
a) P <0.01 to saline, p >0.05 to RGDS; b) p >0.05 to normal saline; n-10
Example 6 evaluation of the effect of fresh elm leaf water extract powder on the tumor metastasis mouse serum MMP-2 and MMP-9 tumor invasion and metastasis is an important biological characteristic of malignant tumor, and is a series of results related to the interaction of multiple steps of tumor cell adhesion, matrix degradation, tumor cell migration and the like. The degradation of Extracellular Matrix (EMC) is an important link of tumor metastasis, and Matrix Metalloproteinase (MMP) in enzymes participating in EMC degradation is closely related to tumor invasion and metastasis. MMPs play a key role in tumor invasion and metastasis by promoting tumor cell invasion to surrounding tissues through degradation of the extracellular matrix. MMP-2, also known as gelatinase A, is a type IV collagenase with a molecular weight of 72 kDa. MMP-9, also known as gelatinase B, is a zinc ion dependent endo-dicer with a molecular weight of 92 kDa. MMP-2 and MMP-9 are directly involved in tumor metastasis mainly by degrading collagen type IV, a main component of basement membrane. Research shows that the high MMP-9 level in the blood of squamous carcinoma patients at the head, neck and esophagus parts is related to tumor distant metastasis and shortened survival period. In order to investigate the relation between the anti-tumor metastasis activity of the fresh elm leaf water extract powder and MMP-2 and MMP-9, the invention uses an enzyme linked immunosorbent assay (Elisa) to determine the contents of MMP-2 and MMP-9 in the blood of a treated mouse. The results are shown in tables 6 and 7. The data in Table 6 show that the fresh elm leaf aqueous extract powder not only significantly reduced serum MMP-2 levels in tumor metastasis mice at an oral dose of 50 mg/kg/day, but also had activity at the same level as RGDS at an oral dose of 20. mu. mol/kg/day. The data in Table 7 show that the fresh elm leaf aqueous extract powder not only significantly reduced serum MMP-9 content in tumor metastasis mice at an oral dose of 50 mg/kg/day, but also had activity at the same level as RGDS at an oral dose of 20. mu. mol/kg/day. This phenomenon indicates that the enhancement of MMP-2 and MMP-9 expression in the blood of mice with tumor metastasis is one of the mechanisms of inhibiting tumor lung metastasis by the fresh elm leaf water extract powder. Therefore, the invention has obvious technical effect.
TABLE 6 influence of powder of fresh folium Ulmi Pumilae water extract on serum MMP-2 content of tumor metastasis mice
| Therapeutic agents | Dosage form | Mean value of serum MMP-2. + -. SD ng/ |
| Physiological saline | ||
| 10 mL/kg/day | 486.20±86.12 | |
| RGDS | 20 mu mol/kg/day | 344.90±32.09 |
| Fresh elm leaf water extract powder | 50 mg/kg/day | 340.30±17.02a |
a) P <0.01 to saline, p >0.05 to RGDS; n is 6
TABLE 7 influence of fresh folium Ulmi Pumilae water extract powder on serum MMP-9 content of tumor metastasis mice
| Therapeutic agents | Dosage form | Mean value of serum MMP-9. + -. SD ng/ |
| Physiological saline | ||
| 10 mL/kg/day | 143.20±10.31 | |
| RGDS | 20 mu mol/kg/day | 90.40±17.79 |
| Fresh elm leaf water extract powder | 50 mg/kg/day | 97.01±15.21a |
a) P <0.01 to saline, p >0.05 to RGDS; n is 6.
Claims (5)
1. A fresh elm leaf aqueous extract powder is characterized by comprising 17 quinic acid compounds, wherein the 17 quinic acid compounds are 3-O- [ E-2, 6-dimethoxy-3- (4-carboxyl n-butylcarbonyloxy) -4- (2-carboxyethyl carbonyloxy) -5- (2-hydroxyethyl carbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2,3, 6-tri (carboxyl n-acryloyloxy) -4- (hydroxyethyl n-acryloyloxy) -5- (5-hydroxyl n-pentylcarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2-hydroxyacetoxy-3, 4, 5-tri (carboxyacetoxy) -6-hydroxymethyl n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2-hydroxyacetoxy-3, 5-dimethylolacetoxy-4, 6-dihydroxy n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2-carboxyacetoxy-3, 5-bis (hydroxymethylacetoxy) -4-carboxymethyl n-acryloyloxy-6-hydroxycinnamoyl ] quinic acid, 3-O- [ E-2, 6-dihydroxy-3, 5-bis (4-carboxy-1, 3-butadienylcarbonyloxy) -4-carboxymethyl acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (carboxyethylacetoxy) -3, 4-dihydroxy-5-carboxy n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2-carboxyacetoxy-3, 5-bis (hydroxymethylacetoxy) -4-carboxymethyln-acryloyloxy-6-methoxycinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (4-carboxy-1-butenylcarbonyloxy) -3, 5-bis (3-hydroxy-n-acryloyloxy) -4-hydroxy-n-acryloyloxy-cinnamoyl ] quinic acid, 3-O- [ E-2-carboxymethylacetoxy-3, 5-bis (hydroxy-n-acryloyloxy) -4- (3-carboxy-1-acryloyloxy) -6-methoxycinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (3-hydroxy-n-acryloyloxy) -3, 5-bis (carboxy-n-acryloyloxy) -4-hydroxy-n-acryloyloxy cinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (carboxy-n-acryloyloxy) -3-carboxy-n-acryloyloxy-4-hydroxy-n-propionyloxy-5-hydroxy-n-valeryloxy cinnamoyl ] quinic acid, 3-O- [2, 6-bis (1-hydroxy-1-acryloyloxy) -3- (6-hydroxy-1-n-hexylenecarbonyloxy) -4- (7-hydroxy-n-heptylcarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (carboxy-n-acryloyloxy) -4-methoxy-5- (5-hydroxy-1-n-pentylenecarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2-methoxy-3, 5-bis (hydroxy-n-acryloyloxy) -4- (3-hydroxy-1-propenylcarbonyloxy) -6- (4-hydroxy-n-butylcarbonyloxy) cinnamoyl ] quinic acid, 3-O- [ E-2, 6-bis (3-hydroxy-n-propylcarbonyloxy) -3- (4-hydroxy-1-n-butenylcarbonyloxy) -4- (4-hydroxy-n-butylcarbonyloxy) cinnamoyl ] quinic acid and 3-O- [ E-2, 6-bis (6-hydroxy-n-hexylcarbonyloxy) -4-formyloxy-5- (6-hydroxy-1-n-hexenylcarbonyloxy) cinnamoyl ] quinic acid.
2. The powder of an aqueous extract of fresh elm leaves according to claim 1, characterized in that it further comprises 14 relaxin compounds, wherein the 14 relaxin compounds are 3-O-glucoside-5, 7-dimethyl-2 ' -methoxy-3 ', 4 ' -bis (3-hydroxy-n-propionyloxy) -5 ', 6 ' -bis (3-hydroxy-n-propionyloxy) relaxin, 3-O-glucoside-5, 7-dimethyl-2 ' -hydroxy-4 ' - (3-hydroxy-n-propionyloxy) -6 ' - (3-carboxy-n-propionyloxy) relaxin, 3-O-glucoside-5, 7-dimethyl-2 ' - (5-hydroxy-1, 3-pentadienecarbonyloxy) -4 '- (3-hydroxy-1-n-propenylcarbonyloxy) -5' - (3-hydroxy-n-propenoyloxy) melaleukin, 3-O-glucoside-5, 7-dimethyl-2 ', 3' -dihydroxy-4 '- (5-hydroxy-n-pentanoyloxy) -5' - (4-hydroxy-n-butanoyloxy) -6 '-hydroxymethoxymelaleukin, 3-O-glucoside-2, 6-dimethyl-2' - (4-hydroxy-1-n-butenylcarbonyloxy) -4 '- (3-hydroxy-1-n-propenylcarbonyloxy) -5' - (5-hydroxy-1-n-pentenylcarbonyloxy) melaleukin, 3-O-glucoside-5, 7-dimethyl-2 '- (4-hydroxy-1-n-butenylcarbonyloxy) -4' - (3-hydroxy-n-acryloyloxy) -5 '- (4-hydroxy-n-butylcarbonyloxy) relaxin, 3-O-glucoside-5, 7-dimethyl-2', 6 '-dihydroxy-3' - (4-hydroxy-1, 3-butadienylcarbonyloxy) -4 '- (5-hydroxy-1, 3-pentadienylcarbonyloxy) relaxin, 3-O-glucoside-2', 5 '-dihydroxy-3' -formyloxy-4 '-hydroxy-n-propionyloxy-6' - (3-hydroxy-n-propionyloxy) relaxin, 3-O-glucoside-2 '-hydroxy-3', 5 '-diformyloxy-4' -hydroxy-n-propionyloxy-6 '- (3-hydroxy-1-n-propenylcarbonyloxy) deuterocortin, 3-O-glucoside-2' -hydroxy-4 '- (3-hydroxy-n-propenylcarbonyloxy) -5' -hydroxy-n-propionyloxy deuterocortin, 3-O-glucoside-2 '-methoxy-4' - (6-hydroxy-1, 3-n-hexadienylcarbonyloxy) -5 '- (4-carboxy-1, 3-n-butadienylcarbonyloxy) -6' - (5-carboxy-1, 3-n-pentadienylcarbonyloxy) deuterocortin, 3-O-glucoside-5, 7-dimethyl-2 ' -methoxy-4 ' - (6-hydroxy-n-hexylcarbonyloxy) -5 ' - (5-hydroxy-n-pentylcarbonyloxy) relaxin, 3-O-glucoside-5, 7-dimethyl-2 ', 3' -dimethoxy-4 '- (6-hydroxy-n-hexylcarbonyloxy) -5' - (5-hydroxy-n-pentylcarbonyloxy) -6 '-hydroxymethoxylansopirin and 3-O-glucoside-5, 7-dimethyl-2', 3 ', 6' -trimethoxy-4 '- (6-hydroxy-n-hexylcarbonyloxy) -5' - (5-hydroxy-n-pentylcarbonyloxy) lansopirin.
3. A process for preparing the powder of the aqueous extract of fresh elm leaves according to claim 1 or 2, characterized in that fresh elm leaves are washed with tap water, heated in distilled water at 50-100 ℃ for 0.5-4 hours, cooled to room temperature, filtered, the filter cake is washed with distilled water 3 times, the combined filtrates are concentrated under reduced pressure to obtain the powder of the aqueous extract of fresh elm leaves.
4. The method of claim 3, wherein the fresh elm leaves are washed with tap water, heated in distilled water at 50-80 ℃ for 2 hours and stirred at 220rpm/min, cooled to room temperature, filtered, the filter cake is washed with distilled water 3 times, the combined filtrates are concentrated under reduced pressure to obtain powder, i.e. fresh elm leaves aqueous extract powder.
5. Use of the fresh elm leaf aqueous extract powder of claim 1 or 2 in the preparation of a medicament for the treatment of tumor metastasis.
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