CN107095888B

CN107095888B - Tung flower leaf n-butyl alcohol extract, preparation method thereof and application thereof in treating colon cancer

Info

Publication number: CN107095888B
Application number: CN201710375858.XA
Authority: CN
Inventors: 罗花; 邓家刚; 侯小涛; 郝二伟; 杜正彩; 易湘西; 谢滟; 谭德超; 冯小慧
Original assignee: Guangxi University of Chinese Medicine
Current assignee: Guangxi University of Chinese Medicine
Priority date: 2017-05-24
Filing date: 2017-05-24
Publication date: 2020-11-27
Anticipated expiration: 2037-05-24
Also published as: CN107095888A

Abstract

The invention discloses a tung tree leaf n-butyl alcohol extract and a preparation method thereof, which comprises the steps of adding tung tree leaves into ethanol for soaking to obtain a tung tree leaf ethanol total extract, volatilizing the ethanol in the ethanol total extract, adding petroleum ether for extraction to obtain a lower-layer water phase, adding ethyl acetate into the lower-layer water phase for extraction, adding n-butyl alcohol for continuous extraction after obtaining the lower-layer water phase, obtaining an upper-layer n-butyl alcohol part extract, and drying in a water bath to obtain the tung tree leaf n-butyl alcohol extract. The research of in vitro molecular biology experiments shows that the n-butyl alcohol extract of the tung tree leaves has the function of proliferation inhibition on HT-29, SW480 and DLD-1 cells of human colon cancer, can obviously inhibit the cloning formation of HT-29, SW480 and DLD-1 cells of human colon cancer, and mainly induces the apoptosis of cancer cells by regulating related proteins in a plurality of apoptosis signal paths. Therefore, the n-butyl alcohol extract of the tung tree leaves has potential application prospect in the aspect of preparing medicaments for treating colon cancer, and can be used for preparing medicaments for resisting colon cancer.

Description

Tung flower leaf n-butyl alcohol extract, preparation method thereof and application thereof in treating colon cancer

Technical Field

The invention belongs to the technical field of tung tree, and particularly relates to a tung tree leaf n-butanol extract, and preparation and application thereof in treating colon cancer.

Background

The Aegiceras corniculatum, also known as black olive, red gourd, black peduncle and the like, is a common mangrove plant belonging to the genus Ceramia of the family Myrsinaceae, and is mainly distributed in the islands of Guangxi, Guangdong, Fujian and Hainan in China, and also distributed in India, the peninsula, the Philippines and Australia. Tung flower tree is a traditional Chinese medicine, and bark and leaves of the Tung flower tree are commonly used for treating diseases such as asthma, diabetes, rheumatism and the like. Modern pharmacological research finds that the tung tree extract has the pharmacological effects of cytotoxicity, antifungal, antidiarrheal, antioxidation, liver protection and the like. Although the derivatives obtained from petroleum ether parts of stems and twigs of tung flowers have been reported to have the effect of inhibiting liver tumors at present, the research on the anticancer of n-butyl alcohol part extracts of tung flower leaves has not been reported yet. The chemical components contained in different parts of plants and different solvent extracts thereof are different, such as bamboo shavings and bamboo leaves, wherein the bamboo shavings are derived from the middle layer of the stalk of the gramineous plant caulis bambusae, the bamboo leaves are derived from the leaves of the gramineous plant lophatherum gracile, and the studies of the plum, the pangolin and the like show that the chemical components contained in the bamboo shavings and the bamboo leaves are different.

Colon cancer is a malignant lesion of colon mucosal epithelium under the action of various carcinogenic factors such as environment or heredity, is one of common malignant tumors, and the morbidity and mortality of the colon cancer are located at the 3 rd position in the whole malignant tumor. Statistics data published by the cancer center in China in 2017 show that five cancers such as colon cancer and the like are used as main causes of tumor death and account for about 70% of all tumor death cases. The incidence of colon cancer in China is on a rising trend in both cities and rural areas. The normalized mortality rate for colon cancer increased by 11.2% compared to thirty years ago. Based on the increasing population base of China, a huge colon cancer patient group is bound to become a heavy burden of the whole public health service.

Because malignant tumor is hidden, the early diagnosis rate of colon cancer is only 11% -15%, more than 80% of patients are diagnosed in middle and late stages, and the five-year survival rate of advanced colon cancer is lower than 20%. At present, the method for treating colon cancer by western medicine mainly comprises the schemes of surgery, radiotherapy, chemotherapy, combination treatment of the three, and the like, but has large toxic and side effects and is easy to cause tumor variation and drug resistance. Therefore, the search for new, safe and effective therapeutic drugs from Chinese medicine is an important research direction for treating colon cancer at present.

Disclosure of Invention

The invention aims to provide a tung tree leaf n-butanol extract, a preparation method thereof and application thereof in treating colon cancer, so as to expand the application range of the tung tree and open up a new way for treating cancer.

In order to solve the technical problems, the invention adopts the following technical scheme:

application of n-butanol extract of Tung flower leaf in preparing medicine for treating colon cancer is provided.

The medicine is capsule, tablet, pill, granule, paste, mixture or suspension, and is prepared by adding normal butanol fraction of folium Tung Hua as active ingredient into conventional adjuvants according to conventional process.

The colon cancer is derived from human colon cancer cells HT-29, SW480 and DLD-1.

The preparation method of the n-butyl alcohol extract of the tung tree leaves comprises the steps of adding the tung tree leaves into ethanol for soaking to obtain a tung tree leaf ethanol total extract, volatilizing the ethanol in the ethanol total extract, adding petroleum ether for extraction to obtain a lower-layer water phase, adding ethyl acetate into the lower-layer water phase, adding n-butyl alcohol for continuous extraction to obtain an upper-layer n-butyl alcohol part extract, and drying in a water bath to obtain the tung tree leaf n-butyl alcohol extract.

The preparation method of the normal butanol extract of the tung tree leaves comprises the following steps: sun drying and pulverizing the tung tree leaves into coarse powder, adding 25 times of 95% ethanol, and soaking for 7 days; concentrating under reduced pressure to obtain total ethanol extract of the leaf of the tung tree, dissolving the total ethanol extract with pure water to obtain suspension, volatilizing until no ethanol smell exists, adding petroleum ether for extraction to obtain a lower-layer water phase, adding ethyl acetate into the lower-layer water phase for continuous extraction, adding n-butanol into the obtained lower-layer water phase for continuous extraction to obtain an upper-layer n-butanol part extract, and drying the upper-layer n-butanol part extract in a water bath to obtain the n-butanol extract of the leaf of the tung tree.

The n-butyl alcohol extract of the tung tree leaves obtained by the preparation method.

In order to fully develop tung tree resources, the inventor establishes a preparation method of tung tree leaf n-butyl alcohol extract, which comprises the steps of adding tung tree leaves into ethanol for soaking to obtain tung tree leaf ethanol total extract, volatilizing the ethanol in the ethanol total extract, adding petroleum ether for extraction to obtain a lower-layer water phase, adding ethyl acetate into the lower-layer water phase, adding n-butyl alcohol for continuous extraction to obtain an upper-layer n-butyl alcohol part extract, and drying in a water bath to obtain the tung tree leaf n-butyl alcohol extract. The research of in vitro molecular biology experiments shows that the n-butyl alcohol extract of the tung tree leaves has the function of proliferation inhibition on HT-29, SW480 and DLD-1 cells of human colon cancer, can obviously inhibit the cloning formation of HT-29, SW480 and DLD-1 cells of human colon cancer, and mainly induces the apoptosis of cancer cells by regulating related proteins in a plurality of apoptosis signal paths. Therefore, the n-butyl alcohol extract of the tung tree leaves has potential application prospect in the aspect of preparing medicaments for treating colon cancer, and can be used for preparing medicaments for resisting colon cancer. The deep development of the n-butyl alcohol extract of the tung tree leaves can enlarge the application range of the tung tree and open up a new way for treating cancers.

Drawings

FIG. 1 is a graph showing the results of the inhibitory effect of n-butanol extract of Tung flower leaves on the proliferation of colon cancer cells.

FIG. 2 is a graph of the experimental results of plate cloning formation of the n-butanol extract of tung tree leaves of the present invention on the effect of human colon cancer cell activity.

FIG. 3 is a bar graph of the results of the effect of n-butanol extract of Tung flower leaves on human colon cancer cell activity.

FIG. 4 is a flow cytometry image of the results of the n-butanol extract from Tung flower leaves of the present invention inducing apoptosis of colon cancer cells (HT-29, SW 480).

FIG. 5 is a bar graph of the results of the n-butanol extract of Tung flower leaves of the present invention inducing apoptosis of colon cancer cells human colon cancer cells (HT-29, SW 480).

FIG. 6 is a flow cytometry image of the result of the n-butanol extract of the tung tree leaves of the present invention inducing the apoptosis of colon cancer cells (DLD-1).

FIG. 7 is a bar graph of the results of the n-butanol extract of Tung flower leaves of the present invention inducing apoptosis of colon cancer cells human colon cancer cells (DLD-1).

FIG. 8 is a graph showing the results of the n-butanol extract from Tung-flower leaves of the present invention regulating the expression of apoptosis signaling pathway proteins.

Detailed Description

Preparation of n-butyl alcohol part extract (NACL) of tung tree leaves

1. The medicinal materials are as follows: the tung tree leaves are collected in Guangxi city-defense harbors and are originally identified as the leaves of the tung tree of the ceratitis of the family Jinniu, family Ceramia, by the ocean research center of northern gulf of Guangxi.

2. Extraction and separation: sun drying the tung tree leaves, pulverizing into coarse powder (2500g), adding 25 times (62.5L) of 95% ethanol, and soaking for 7 days; concentrating under reduced pressure to obtain total ethanol extract of the leaf of the tung tree, dissolving the total ethanol extract with pure water to obtain suspension, volatilizing until no ethanol smell exists, adding petroleum ether for extraction to obtain a lower-layer water phase, adding ethyl acetate into the lower-layer water phase for continuous extraction, adding n-butanol into the obtained lower-layer water phase for continuous extraction to obtain an upper-layer n-butanol part extract, and drying the upper-layer n-butanol part extract in a water bath to obtain the n-butanol extract of the leaf of the tung tree.

Pharmacodynamic study of n-butyl alcohol part extract of tung tree leaves

1. Experimental Material

The cell lines, human colon cancer cells HT-29, SW480 and DLD-1, are provided by cell banks of Chinese academy of sciences.

Chinese medicinal material tung tree leaf

Main reagents and consumables

Main agent-

Apoptosis kit (Pharmingen FITC Annexin V Apoptosis DetectionKit 1): RayBiotech, USA;

cell proliferation assay kit (MTS): promega corporation, usa;

crystal violet staining solution: biyuntian, China;

chloroform, isopropanol, absolute ethyl alcohol, chloral hydrate and the like are domestic analytical pure reagents.

Main consumables (food) -X

25cm²Cell culture flasks: BD corporation, usa;

6cm cell culture dish, 10cm cell culture dish: BD corporation, usa;

cell 6-well plate, 24-well plate, 96-well plate: BD corporation, usa;

15ml centrifuge tube, 50 ml centrifuge tube: BD corporation, usa;

freezing and storing the tubes: BD corporation, usa;

a pipette: american corn corporation;

gun head box, EP pipe, PCR pipe: AXYGEN corporation, usa;

main Instrument-

Electric pipettor, adjustable pipettor, low-temperature high-speed centrifuge: manufactured by Eppendorf, Germany;

cell culture incubator, biological safety cabinet, liquid nitrogen tank: thermo scientific, usa;

-80 ℃ bio ultra-low temperature refrigerator: thermo scientific, usa;

superclean bench: suzhou jinjing corporation;

37 ℃ bacteria incubator, oven: china Jinghong corporation;

inverted fluorescence microscopy: OlyMPUS, Japan;

palm centrifuge: thermo Fisher Scientific, USA;

autoclave, -20 ℃ bio-refrigerator, 4 ℃ bio-refrigerator: china Haier corporation;

an ice crushing and making machine: sanyo corporation, Japan;

a computer: china Association company;

nanodrop 2000 spectrophotometer: thermo Scientific, usa;

electromagnetic stirrer, enzyme linked immunosorbent assay: thermo Scientific, usa.

2. Experimental methods

2.1 cell proliferation assay (MTS)

(1) When the cells are in the logarithmic growth phase, the cell culture medium is sucked away by a vacuum pump, the DPBS is added to clean the cells, and 0 is added after the DPBS is discarded.25% pancreatin, when most of the cells can be separated from the culture bottle, adding a complete culture medium containing 10% fetal calf serum to terminate digestion, sucking the culture solution by a pipette to blow the cells off the wall of the culture bottle, transferring the cells to a 15ml centrifuge tube, placing the cells in a centrifuge for 1000 rpm, centrifuging for 3 minutes, sucking the supernatant by a vacuum pump, adding 5ml of complete culture medium to carry out resuspension and mixing, counting the cells, resuspending, adjusting the density of the cells to be 2 x 10⁴One per ml, 100 μ l of cell suspension was added per well in a 96-well plate, i.e. 2000 cells per well, 3 replicates per set, 5 dosing gradient sets (25, 37.5, 50, 75, 100 μ g/ml), and culture control, DMSO control. Standing at 37 deg.C for 5% CO₂Carrying out conventional culture in a constant-temperature incubator.

(2) Preparing a drug mother solution to be tested: dissolving 0.2g of the drug to be detected in 2ml DMSO to prepare a mother solution of 100mg/ml, dissolving the extract with ultrasound in an ultrasonic instrument, filtering with a 0.22 μm nylon filter sieve, subpackaging in an EP tube, and storing at-20 deg.C.

(3) After the cells are cultured and adhered to the wall, n-butanol part extracts (25, 37.5, 50, 75, 100 mu g/ml) of the tung tree leaves with different concentration gradients are respectively added into each row, and the control group is added with the equal volume of complete culture medium containing DMSO with the corresponding concentration and the control group containing only the complete culture medium. The culture was continued for 24, 48, 72 h.

(4) After 24, 48 and 72 hours of incubation, 20. mu.l of completely thawed Cell Titer 96Aqueous One Solution Reagent was added to each well with a discharge gun, and the cells were returned to the incubator for further incubation for 2 hours, with absorbance values read on a microplate reader at 490nm wavelength. And (3) taking the hole without the cell and only containing the culture medium as a blank hole, making a table, and drawing an MTS result graph with the ordinate of cell activity and the abscissa of drug concentration. And calculating the cell growth inhibition rate by using software, wherein the formula is as follows:

(5) the above experiment was repeated 3 times, statistically analyzed by SPSS 21 test, and blankedCompared with the group, P is less than 0.01, and the difference is significant; and the IC was determined by plotting GraphPad Prism 5 as a line graph₅₀。

2.2 Single cell clonogenic experiments

(1) Taking cells in logarithmic phase for conventional digestion, resuspending the cells, repeatedly blowing and beating the cells to fully disperse the cells to the greatest extent, enabling the dispersity to reach more than 95%, counting the cells, adjusting the cell concentration to be 200/ml, inoculating 2ml of the cells into a 6-well plate per well, and culturing overnight.

(2) The next day, two drug concentrations were set at 12.5 and 25. mu.g/ml, respectively, and a blank (complete medium) was set.

(3) After 4 days of drug action, the culture medium was discarded and the same drug was administered again.

(4) When the two cell masses were combined, the culture medium was aspirated off by a vacuum pump, washed twice with DPBS buffer, and fixed with 4% paraformaldehyde for 15 min.

(5) Paraformaldehyde is discarded and the sample is stained with crystal violet staining solution for 35 min.

(6) The crystal violet was recovered, washed twice with DPBS buffer, the DPBS buffer was aspirated away, and the six well plate was scanned with a scanner.

(7) The results of the experiment repeated three times are analyzed by SPSS 21 test statistics, compared with a blank group, P is less than 0.01, the difference is significant, and GraphPad Ptism 5 is used as a histogram;

2.3 cell scratch test

(1) The horizontal lines are drawn on the back of the 6-hole plate by a marker with the help of a ruler, and the distance between each line is about 0.5cm, so that each hole of the 6-hole plate has 5 straight lines passing through.

(2) The cells digested were centrifuged and then counted in 8X 10 resuspension⁵One well was seeded in 6-well plates, each set of 2 multiple wells.

(3) When the cells are fully paved in the whole hole after the cells are attached to the wall on the next day, the cells are treated by mitomycin (0.5 mu g/m1), the treatment concentration of DU145 is 10 mu g/ml, the treatment concentration of PC is 320 mu g/ml, after 3 hours, a straight ruler is used for drawing marks on the back surface of the six-hole plate uniformly by using a 10 mu l gun head, and the width of each mark is ensured to be consistent and the straight line vertical to the back surface is ensured.

(4) The medium containing mitomycin was aspirated off with a vacuum pump, the six well plates were washed 2 times with DPBS and discarded, and complete medium was added. The concentration of the administration group was 12.5. mu.g/ml, and the blank group was complete medium. Pictures were taken under the microscope at 22h, respectively, to ensure consistent location of each well.

(5) And analyzing the wound area by using image J, and calculating the wound healing rate. The distance the cells crawled, i.e. the width of 0 hours minus the width of the time point. Mobility ═ 1-wound area at the time of photographing/0 hour wound area × 100%. Statistical analysis is carried out by SPSS 21 test, compared with a blank group, P is less than 0.01, the difference is significant, and GraphPad Prism 5 is used as a histogram;

2.4 apoptosis

(1) Cells in logarithmic growth phase were digested and inoculated into six-well plates, and the next day of dosing was performed, setting two concentration groups, one control group. After 24h, 48h, 72h of drug treatment, cells were treated with BD cell cycle kit.

(2) An appropriate amount of 10 Xbuffer salt solution in the BD apoptosis kit is prepared into 1 Xbuffer solution. Three groups of cells were taken out at 24, 48, 72h, respectively, the supernatant was discarded, and a pre-chilled DPBS wash was added. Adding 0.25% pancreatin to digest the cells, sucking the culture solution by using a pipette to blow the cells off the wall of the culture bottle, transferring the cells to a 15ml centrifuge tube, placing the centrifuge tube in a centrifuge for 1000 rpm, centrifuging the cells for 3 minutes, and removing the supernatant.

(3) 6 EP tubes, designated A-F, were taken and the cells of each group were resuspended in 1X buffer saline and adjusted to 1X 10 after counting⁶One/ml, 100. mu.l of cell suspension from the blank group was taken from each of the A-D four EP tubes, and 100. mu.l of cell suspension from the two administration groups was taken from each of the E and F tubes.

(4) Add 5. mu.l each of AV and PI reagents from the BD apoptosis kit to tubes A, E, and F, respectively. Add 5. mu.l AV reagent to tube B, add 5. mu.l PI reagent to tube C, mix well and store in dark room temperature for 15min in the dark.

(5) 400 μ l of 1 × buffer salt solution was added to each EP tube, mixed and left for 15min, and then detected by a flow cytometer.

(6) The analysis was histogram by the software GraphPad Prism 5.

2.5 statistical analysis

SPSS statistical software was used to process the analytical data and perform a comparison between groups, where P < 0.05 and the difference is statistically significant, and where P < 0.01 and the difference is statistically significant.

3. Results of the experiment

3.1 results of cell proliferation experiments

The n-butanol fraction extract of Tung flower leaf has proliferation inhibiting effect on human colon cancer cells HT-29, SW480, and DLD-1, IC50 is shown in Table 1, and cell activity curve is shown in FIG. 1.

TABLE 1 IC50(μ g/ml) of n-butanol fraction extract of Tung-flower leaves on Colon cancer cells

3.2 Experimental results of Single cell clonogenic assays

The n-butyl alcohol part extract of the tung tree leaves can obviously inhibit the cloning formation of HT-29, SW480 and DLD-1 of human colon cancer cells, and has concentration dependence with the administration concentration. The clone formation rate of a control group of HT-29 cells is 91.50%, and the clone formation rates of a high-concentration administration group and a low-concentration administration group are 0.25% and 50.63% respectively; the clone formation rate of the DLD-1 cell in a control group is 91.38 percent, and the clone formation rates of the high-concentration administration group and the low-concentration administration group are 41.00 percent and 80.88 percent respectively; the clone formation rate of SW480 cells in the control group was 88.25%, and the clone formation rates of the high and low concentration administration groups were 16.50% and 54.88%, respectively. The number of cell clones formed is shown in FIGS. 2 and 3.

3.3 flow-type apoptosis assay results

The n-butanol fraction extract of the tung tree leaves can induce apoptosis of HT-29, SW480 and DLD-1 of human colon cancer cells, and the specific results are shown in fig. 4 to 7, wherein the high-concentration group has a significant difference (P is less than 0.01) compared with the control group.

Research on anti-cancer mechanism of n-butyl alcohol part extract of tung tree leaf

1. Experimental Material

Cell line-human colon cancer cell HT-29

Medicine-tung tree leaf n-butanol fraction extract

Main reagents and consumables

Main agent-

RIPA protein lysate: sigma, USA;

protease inhibitors: sigma, USA;

30% polyacrylamide: sigma, USA;

ammonium Persulfate (APS): sigma, USA;

tetramethylethylenediamine (TEMED): sigma, USA;

protein loading buffer (4 ×): sigma, USA;

protein pre-staining Marker: sigma, USA;

tween-20 (20%): sigma, USA;

mitomycin: sigma, USA;

10% neutral formalin: sigma, USA;

phosphatase inhibitors (cocktails) 2 and 3: sigma, USA;

RIPA1640 medium: gibco, USA;

7.5% sodium bicarbonate: gibco, USA;

4-hydroxyethyl piperazine ethanesulfonic acid (HEPES): gibco Inc. of USA

Fetal bovine serum: gibco, USA;

tris (hydroxymethyl) aminomethane (Tris base): 6enb Bioscience, USA;

sodium Dodecyl Sulfate (SDS): genbase Bioscience, USA;

polyvinylidene fluoride membrane (0.22 μmPVDF membrane): millipore, USA;

ECL chemiluminescence detection kit: thermo corporation, usa;

and (3) skim milk powder: FOODCLUB, USA;

BCA protein quantification kit: bio bi yun tian, china;

methanol: china Shaoxing chemical plant;

main consumables (food) -X

6cm cell culture dish, 10cm cell culture dish: BD corporation, usa;

15ml centrifuge tube, 50 ml centrifuge tube: BD corporation, usa;

a pipette: american corn corporation;

gun head box, EP pipe, PCR pipe: AXYGEN corporation, usa;

main Instrument-

-80 ℃ bio ultra-low temperature refrigerator: thermo scientific, usa;

superclean bench: suzhou jinjing corporation;

37 ℃ bacteria incubator, oven: china Jinghong corporation;

cell scraping: manufactured by BD corporation of usa;

inverted fluorescence microscopy: produced by OLYMPUS corporation of japan;

palm centrifuge: thermo Fisher Scientific, USA;

an ice crushing and making machine: sanyo corporation, Japan;

a computer: china Association company;

antibody- (Ab) -Ab

Rabbit anti-human Bcl-2 cloned antibody (Cell Signaling Technology, cat # 2870S, USA);

rabbit anti-human Bax monoclonal antibody (Cell Signaling Technology, cat # 5023S, USA);

rabbit anti-human Puma monoclonal antibody (Cell Signaling Technology, cat # 4976S, U.S.);

rabbit anti-human Bim monoclonal antibody (Cell Signaling Technology, cat # 2933S, USA);

rabbit anti-human p-AKT monoclonal antibody (Cell Signaling Technology, cat # 9271S, USA);

rabbit anti-human AKT monoclonal antibody (Cell Signaling Technology, cat # 9272S, USA);

rabbit anti-human GSK3 beta cloned antibody (Cell Signaling Technology, cat # 9315S, USA);

rabbit anti-human P-GSK3 beta cloned antibody (Cell Signaling Technology, cat # 9323S, USA);

rabbit anti-human PARP monoclonal antibody (Cell Signaling Technology, cat # 9542S, USA);

rabbit anti-human Cleaved PARP monoclonal antibody (Cell Signaling Technology, cat # 5625S, USA);

rabbit anti-human cleared Caspase-9 cloned antibody (Cell Signaling Technology, cat # 7237S, USA);

rabbit anti-human Caspase-7 cloned antibody (Cell Signaling Technology, cat # 9492S, USA);

rabbit anti-human cleared-Caspase-7 cloned antibody (Cell Signaling Technology, cat # 8438S, USA);

rabbit anti-human Caspase-3 cloned antibody (Cell Signaling Technology, cat # 9662S, USA);

2. experimental methods

(1) And (3) inoculating the human colon cancer cells HT-29 in the logarithmic growth phase into a 6cm culture dish respectively, administering the drug the next day, directly adding an equal volume of fresh complete culture medium into a blank group, treating for 24 hours, 48 hours and 72 hours, and collecting the protein.

(2) Protein concentration was determined by BCA method and denatured.

(3) Separating gel is prepared and collected, and then sample loading is carried out electrophoresis, wherein the sample loading amount is 50 mu g/hole.

(4) The film was rotated in an ice box with 0.2um PVDF film at a voltage of 90V for 3 hours.

(5) The membrane was blocked with 5% skim milk for one hour on a shaker at room temperature.

(6) Washing with TBST for 5min for three times, incubating the corresponding primary antibody, and incubating overnight on a refrigerator shaker.

(7) The next day, the cells were washed three times with TBST for 5min each time, incubated with the corresponding secondary antibody, and incubated for 1h on a shaker at room temperature. The washing with TBST was then repeated three times for 5min each.

(8) Chemiluminescence is performed, and the chemiluminescence is analyzed by software such as Image J.

3. Results of the experiment

The n-butyl alcohol part extract of the tung tree leaves can induce cells to generate mitochondrial apoptosis and caspase-dependent apoptosis. As shown in FIG. 8, the n-butanol fraction extract of Tung-flower leaves can up-regulate the expression of Cleaved caspase3, Cleaved caspase7, Cleaved caspase8, Cleaved caspase9, Cleaved PARP, P-Bcl-2, Puma, Bad, Bim, Bak, Bik, and down-regulate Bcl-2.

Claims

1. The application of the n-butyl alcohol extract of the tung tree leaves in the preparation of the medicine for treating colon cancer is characterized in that the extract is prepared by the following method: sun drying and pulverizing the tung tree leaves into coarse powder, adding 25 times of 95% ethanol, and soaking for 7 days; concentrating under reduced pressure to obtain total ethanol extract of the leaf of the tung tree, dissolving the total ethanol extract with pure water to obtain suspension, volatilizing until no ethanol smell exists, adding petroleum ether for extraction to obtain a lower-layer water phase, adding ethyl acetate into the lower-layer water phase for continuous extraction, adding n-butanol into the obtained lower-layer water phase for continuous extraction to obtain an upper-layer n-butanol part extract, and drying the upper-layer n-butanol part extract in a water bath to obtain the n-butanol extract of the leaf of the tung tree; the colon cancer is derived from human colon cancer cells HT-29, SW480 and DLD-1.

2. Use according to claim 1, characterized in that: the medicine is capsule, tablet, pill, granule, paste, mixture or suspension.

3. A preparation method of a tung tree leaf n-butyl alcohol extract for treating colon cancer is characterized by comprising the following steps: sun drying and pulverizing the tung tree leaves into coarse powder, adding 25 times of 95% ethanol, and soaking for 7 days; concentrating under reduced pressure to obtain total ethanol extract of the leaf of the tung tree, dissolving the total ethanol extract with pure water to obtain suspension, volatilizing until no ethanol smell exists, adding petroleum ether for extraction to obtain a lower-layer water phase, adding ethyl acetate into the lower-layer water phase for continuous extraction, adding n-butanol into the obtained lower-layer water phase for continuous extraction to obtain an upper-layer n-butanol part extract, and drying the upper-layer n-butanol part extract in a water bath to obtain the n-butanol extract of the leaf of the tung tree.

4. The n-butanol extract of the tung tree leaves prepared by the preparation method according to claim 3.