CN110652505A

CN110652505A - Application of Trapa acornis nakai shell polyphenol extract in preparation of medicine for resisting HER-2 over-expression type breast cancer

Info

Publication number: CN110652505A
Application number: CN201810689155.9A
Authority: CN
Inventors: 高广春; 黄越燕; 蒋小红; 黄嬛; 朱琦峰; 汪淑贞; 张洁
Original assignee: Jiaxing University
Current assignee: Jiaxing University
Priority date: 2018-06-28
Filing date: 2018-06-28
Publication date: 2020-01-07
Anticipated expiration: 2038-06-28
Also published as: CN110652505B

Abstract

The invention belongs to the technical field of natural medicines, and particularly relates to application of a gallic acid and pentagalloyl glucose composition in a Trapa nanhuensis polyphenol extract in preparation of a medicine for resisting HER-2 over-expression breast cancer. The preparation method of the water chestnut hull polyphenol extract comprises the following steps: (1) pulverizing and sieving water chestnut shell of south lake; (2) ultrasonic extracting with ethanol, and concentrating under reduced pressure to obtain crude extract; (3) extracting the crude extract with ethyl acetate, loading onto macroporous adsorbent resin column, and concentrating under reduced pressure to obtain water chestnut shell polyphenol extract. The invention finds that the composition of gallic acid and pentagalloyl glucose in the Trapa nanhu polyphenol extract exerts a synergistic effect on SK-BR-3 cells and has a remarkable inhibition effect. The invention provides a certain basis for further explaining the anti-breast cancer effect of the water chestnut shell polyphenol extract, thereby providing technical support for developing and preparing anti-breast cancer medicaments aiming at different types of breast cancer.

Description

Application of Trapa acornis nakai shell polyphenol extract in preparation of medicine for resisting HER-2 over-expression type breast cancer

Technical Field

The invention belongs to the technical field of natural medicines, and particularly relates to a Trapa acornis nakai shell polyphenol extract, a preparation method and application thereof, and more particularly relates to application of a gallic acid and pentagalloylglucose composition in the Trapa acornis nakai shell polyphenol extract in preparation of a medicine for resisting HER-2 over-expression breast cancer.

Background

The Trapaceae (Trapaceae) Trapa (Trapa) plant has more than 70 species all over the world, and is distributed in the temperate zone from Europe, Asia and Africa to tropical zone. The pedicellus et pericarpium Trapae plant mainly contains flavone, polyphenol, sterol, alkaloid, polysaccharide, volatile oil, amino acid, etc., and has anticancer, antioxidant, alpha-glucosidase inhibiting, antimicrobial, analgesic, hepatoprotective, immunoregulatory, antiatherosclerotic, and blood glucose lowering effects.

Trapa acornis Nakano, also named Wujiao Trapa, Yuanbao Trapa, Yuanzu, Tuzu, Tujiao Trapa and Heshang Trapa, is a special aquatic economic crop in China, is named mainly in Zhejiang Jiaxing south lake, is famous for its unripe fruits, thin shell, delicious taste and rich nutrition, and has become a special economic crop in Hangzhou Jia lake plain water and countryside and has higher planting benefit. Water chestnut is eaten by only selecting water chestnut meat and water chestnut shells are usually discarded as waste, but a large number of researches show that the water chestnut shells are rich in active ingredients such as polyphenol, flavone, alkaloid and the like and have wide biological activity. The extracts of the Trapa acornis nakai are analyzed by thin layer chromatography and ultraviolet spectroscopy, and the Trapa acornis nakai is found to contain main secondary metabolites such as gallic acid and caffeic acid and is mainly present in water chestnut shells. The method adopts chromatography separation to separate different extraction parts of the water chestnut shells in the south lake, and discovers that the total extract of the water chestnut shells in the south lake, petroleum ether parts, ethyl acetate parts and the like have alpha-glucosidase inhibitory activity. The Ningying research finds that the south lake water chestnut shell extracting solution can be used for inhibiting the gastric cancer cell proliferation activity.

According to the existing research, the extract of the water chestnut shells in the south lake has obvious in-vitro inhibition effect on gastric cancer cells, and the active ingredients playing the anti-gastric cancer effect mainly comprise polyphenols, flavonoids and saponins, wherein the polyphenols and flavonoids have more obvious effect (Lincoln, analysis of the biological active ingredients of the water chestnut shells and research on anti-gastric cancer mechanism, doctor academic paper of Zhejiang university). However, the active ingredients which play a role in the traditional Chinese medicine composition are not deeply researched, and the action mechanism of the Trapa natans L polyphenol extract in the aspect of resisting the breast cancer is not reported in detail, and particularly the action mechanism of different types of breast cancer is not reported.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the composition of gallic acid and pentagalloyl glucose.

According to the invention, the water chestnut shells in the south lake are taken as raw materials, the inhibition effect of the polyphenol extract of the water chestnut shells in the south lake on HER-2 over-expression type breast cancer cells SK-BR-3 is researched, and the functional components in the polyphenol extract of the water chestnut shells in the south lake and the potential utilization value of the polyphenol extract are researched.

In some preferred modes, the composition of the gallic acid and the pentagalloyl glucose in the Trapa acornis shell polyphenol extract has the application of preparing a medicament for resisting HER-2 over-expression type breast cancer.

In order to solve the technical problems, the invention provides a preparation method of a water chestnut shell polyphenol extract, which comprises the following steps:

(1) peeling off the fruit of Trapa acornis nakai, leaving the shell, drying in the shade, pulverizing with a plant pulverizer, sieving with a 60-mesh sieve, collecting the powder, and standing in the shade and dry place;

(2) adding 60% ethanol solution according to a material-liquid ratio of 1:20(g/ml), extracting in a thermostatic water bath at 60 deg.C for 2h, taking out, ultrasonic treating in an ultrasonic instrument with ultrasonic power of 300KW for 20min, filtering while hot, collecting filtrate, extracting residue for 2 times, mixing filtrates, centrifuging the filtrate at 3000r/min for 10min, collecting supernatant, and concentrating under reduced pressure to obtain crude extract.

(3) The crude extract is dissolved back by water, extracted by ethyl acetate for three times, and concentrated under reduced pressure to obtain the water chestnut shell extract. Dissolving the extract with water, loading onto DM130 macroporous adsorbent resin column, eluting with 30% ethanol solution of 2 column volume, collecting fractions, and concentrating under reduced pressure to obtain water chestnut shell polyphenol extract.

The invention provides the water chestnut hull polyphenol extract prepared by the method, wherein the total polyphenol content of the extract is 81.5-84.9%.

As the application of the invention, the polyphenol in the extract comprises gallic acid, tri-galloyl glucose, tetra-galloyl glucose and pentagalloyl glucose.

As the application of the invention, based on the total weight of the extract, the content of gallic acid is 24.8-26.4%, the content of tri-galloylglucose is 12.5-14.0%, the content of tetra-galloylglucose is 7.6-8.3%, and the content of pentagalloylglucose is 25.2-28.7%.

The invention has the following advantages:

(1) provides the application of a gallic acid and pentagalloyl glucose composition in preparing a medicine for resisting HER-2 over-expression type breast cancer, the composition is derived from a Nanhu lake water chestnut shell polyphenol extract, and has the characteristics of wide source, clear composition, safety and reliability.

(2) The invention extracts and separates the effective components of the water chestnut shells of the south lake to obtain the extract which can effectively resist HER-2 over-expression type breast cancer, and the active components which can resist HER-2 over-expression type breast cancer in the polyphenol extract of the water chestnut shells of the south lake are further deeply analyzed to determine the active components which can resist HER-2 over-expression type breast cancer in the polyphenol extract of the water chestnut shells of the south lake and possible action modes among the active components. The invention finds that the composition of the gallic acid and the pentagalloyl glucose exerts a synergistic effect on SK-BR-3 cells and shows a remarkable inhibition effect, while the tri-galloyl glucose and the tetra-galloyl glucose possibly exert an antagonistic effect on the SK-BR-3 cells and can reduce the synergistic effect of the composition of the gallic acid and the pentagalloyl glucose. The invention provides a certain basis for further explaining the anti-breast cancer effect of the water chestnut shell extract, thereby providing technical support for developing and preparing anti-breast cancer medicaments for different types of breast cancer. When the south lake water chestnut shell polyphenol extract is used for preparing the anti-breast cancer medicament, active ingredients with specific inhibition effects on different breast cancer cells are separated and extracted aiming at different types of breast cancer, so that the anti-cancer activity and effect of the medicament are obviously improved.

Drawings

FIG. 1 is a UPLC-MS analysis spectrum of a polyphenol extract of trapa natans L.K.K. shell, wherein (A) is a chromatogram scanning spectrum, and (B) is a mass spectrum scanning spectrum.

FIG. 2 is an ultraviolet absorption spectrum and a mass spectrum of the polyphenols of the trapa natans l, wherein (A) is an ultraviolet absorption spectrum and a mass spectrum of gallic acid, (B) is an ultraviolet absorption spectrum and a mass spectrum of tri-galloyl glucose, (C) is an ultraviolet absorption spectrum and a mass spectrum of tetra-galloyl glucose, and (D) is an ultraviolet absorption spectrum and a mass spectrum of pentagalloyl glucose.

FIG. 3 shows the polyphenol extract 3 of Trapa acornis nakai shell at different concentrations^#Influence on the cell cycle of SK-BR-3 cells.

FIG. 4 shows the polyphenol extract 3 from Trapa natans L.K.K. shell at different concentrations^#Effect on apoptosis of SK-BR-3 cells.

FIG. 5 shows the polyphenol extract 1 from Trapa natans L.K.K. shell at different concentrations^#Effects on the cell cycle of MDA-MB-468 cells.

FIG. 6 shows the polyphenol extract 1 from Trapa natans L.K.K. shell at different concentrations^#Effects on apoptosis of MDA-MB-468 cells.

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the invention, all parts and percentages are weight units, and all equipment, raw materials and the like can be purchased from the market or are commonly used in the industry, if not specified. Unless otherwise indicated, the examples employ methods that are within the ordinary skill in the art.

The Trapa acornis nakai adopted in the embodiment is collected from Jiaxing in Zhejiang.

Example 1 preparation of Trapa acornis Miq polyphenol extract and determination of Total Polyphenol content

1. Pretreatment of the water chestnut shells in the south lake: peeling off the fruit of Trapa acornis nakai, leaving the shell, drying in the shade, pulverizing with a plant pulverizer, sieving with 60 mesh sieve, collecting the powder, and standing in the shade and dry place.

2. Preparing an extract: adding 60% ethanol solution according to a material-liquid ratio of 1:20(g/ml), extracting in a thermostatic water bath at 60 deg.C for 2h, taking out, ultrasonic treating in an ultrasonic instrument with ultrasonic power of 300KW for 20min, filtering while hot, collecting filtrate, extracting residue for 2 times, mixing filtrates, centrifuging the filtrate at 3000r/min for 10min, collecting supernatant, and concentrating under reduced pressure to obtain crude extract. And (3) after the crude extract is redissolved by water, extracting for three times by using ethyl acetate, and concentrating under reduced pressure to obtain the water chestnut shell polyphenol extract.

3. Determination of total polyphenol content of extract

The Folin-Ciocalteu method is adopted to measure the content of total polyphenol in the extract.

(1) Preparation of the Standard Curve

Accurately weighing 8.5mg of gallic acid standard, dissolving with distilled water, diluting to 50mL, and shaking. Respectively sucking 0mL, 0.2 mL, 0.4mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, 1.4 mL, 1.6 mL, 1.8 mL, 2.0mL, 2.2 mL, 2.4 mL and 2.6mL into a 25mL volumetric flask, and preparing standard solutions with different concentrations by using distilled water to fix the volume. Precisely sucking 1mL of standard solutions with different concentrations, adding into a 25mL volumetric flask, adding 10mL of distilled water, shaking, adding 1.5mL of Folin reagent, mixing, and adding 10% Na within 5min₂CO₃6mL, shake up, add distilled water to constant volume to the mark, shake up. Standing for 3 hr, measuring absorbance (A) at 765nm, and drawing standard curve with gallic acid concentration as abscissa and absorbance as ordinate.

(2) Sample assay

Weighing about 10mg of extract to be detected, fixing the volume in a 25mL volumetric flask, respectively putting 0.5, 1.0 and 2.0mL in the 25mL volumetric flask, adding 10mL of distilled water, shaking up, adding 1.5mL of Folin reagent, mixing well, adding 10% Na within 5min₂CO₃6mL, shake up, add distilled water to constant volume to the mark, shake up. After standing for 3 hours, the absorbance (A) was measured at 765 nm. The samples to be tested were assayed in triplicate. Calculating total polyphenol content according to linear regression equation of gallic acid standard curve, wherein total polyphenol content (%) [ (A-0.0387)/90.328 × 25/0.5 × 50/10.4%]100%, a is absorbance.

Example 2 analysis of polyphenol Compound of Trapa acornis nakai Shell Polyphenol extract

The UPLC-MS is used for analyzing the main components of polyphenol substances in the water chestnut hull extract of the lake south.

Wherein, the chromatographic conditions are as follows:

the liquid phase is waters Acquity UPLC QSM; the mobile phase is acetonitrile (A) and 0.01 percent formic acid water solution (B); the gradient elution procedure was: 0min, 95% A + 5% B; 0.5min, 95% A + 5% B; 10min, 70% A + 30% B; 18min, 1% A + 99% B; 22min, 1% A + 99% B; 25min, 95% A + 5% B; the flow rate is 0.4 ml/min; the ultraviolet detector is a waters Acquity PDA UPLC eLambda at 800nm, and the scanning range is 190-600 nm.

The mass spectrometry conditions were as follows: ion source-ESI negative ions and ion detector was waters ADC.

As shown in FIGS. 1 and 2, it was concluded from the results of the analyses that the polyphenol compound of the water chestnut hull in south lake contained gallic acid ([ M-H ] -169.0134, ultraviolet absorption peaks. lamda. lamda.max 217.2nm and 270.2nm), trigalloylglucose ([ M-H ] -635.0908; ultraviolet absorption peaks. lamda.max: 224.2nm and 270.2nm), tetragalloylglucose ([ M-H ] -787.1103; ultraviolet absorption peaks. lamda.max: 221.2nm and 269.2nm), and pentagalloylglucose ([ M-H ] -939.1137; ultraviolet absorption peaks. lamda.max: 224.2nm and 278.2 nm).

Example 3 determination of polyphenol content in Trapa acornis nakai Shell Polyphenol extract

1. HPLC parameter setting

The instrument used for the experiment is an Agilent 1200 high performance liquid chromatograph; the column was Eclipse XDB-C18(5 μm, 4.6 x 150 mm); the eluent is methanol (A) and 0.5% acetic acid water solution (B); gradient elution conditions: 0min, 12% A; 5min, 20% A; 15min, 20% A; 25min, 70% A; the scanning wavelength is 280 nm; the sample amount is 5 mul; the flow rate is 1 ml/min; the column temperature was 30 ℃.

2. Water chestnut shell extract treatment

Weighing 0.8mg of the extract of the trapa natans l.K.was dissolved in 1ml of methanol, sonicated (50 ℃, P ═ 100w) for 1h, filtered (0.45 μm) through a microporous filter membrane for use, and the treatments were performed in triplicate.

3. Preparation of standard curve by external standard method

Weighing 8mg of gallic acid, tri-galloylglucose, tetra-galloylglucose and pentagalloylglucose standard respectively, placing in a 25ml volumetric flask, dissolving with methanol, fixing volume, and shaking to obtain standard stock solution. Transferring a certain amount of standard substance stock solution into another 10ml volumetric flask, fixing the volume with methanol, shaking up to obtain a gallic acid standard solution with mass concentration of 0.78125, 3.125, 12.5, 50, 100, 150, 200 mug/ml and a pentagalloyl glucose standard solution with mass concentration of 12.5, 25, 50, 100, 200, 400 mug/ml, removing impurity particles through a 0.45 μm microporous membrane, injecting into a 1.5ml sample injection bottle, numbering in sequence, performing HPLC analysis, and performing parallel determination for three times. And drawing a standard curve by taking the peak area as a vertical coordinate and the mass concentration of the sample injection as a horizontal coordinate to respectively obtain a regression equation.

4. Determination of active ingredients in polyphenol extracts

And (3) sampling and analyzing the extract according to the method in the step (3) to obtain peak areas of gallic acid, tri-galloyl glucose, tetra-galloyl glucose and pentagalloyl glucose in the extract, and respectively calculating the contents of the gallic acid, the tri-galloyl glucose, the tetra-galloyl glucose and the pentagalloyl glucose in the water chestnut shell extract according to the standard curve obtained in the step (3).

Example 4A Trapa natans L polyphenol extract 1^#Inhibition of breast cancer cells SK-BR-3 and MDA-MB-468 Function of

Adding 60% ethanol solution into pretreated water caltrop shells in a material-liquid ratio of 1:20(g/ml), extracting for 2h in a constant-temperature water bath at 60 ℃, taking out, putting into an ultrasonic instrument with ultrasonic power of 300KW, carrying out ultrasonic treatment for 20min, filtering while hot, collecting filtrate, extracting filter residues for 2 times again, combining the filtrate, centrifuging the filtrate at 3000r/min for 10min, collecting supernatant, and concentrating under reduced pressure to obtain a crude extract. And (3) after the crude extract is redissolved by water, extracting for three times by using ethyl acetate, and concentrating under reduced pressure to obtain the water chestnut shell polyphenol extract. The extract 1 obtained in this example was measured^#The content of polyphenol is 54.5-60.4%, the content of gallic acid is 2.6-2.8%, the content of tri-galloylglucose is 19.3-21.2%, the content of tetra-galloylglucose is 26.5-28.6%, and the content of pentagalloylglucose is 6.5-7.7%.

Respectively extracting with extract 1^#The breast cancer cells SK-BR-3 and MDA-MB-468 were treated at concentrations of 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 400. mu.g/ml and 800. mu.g/ml, and the inhibitory effects on the two breast cancer cells were examined for 24h, 48h and 72h, while cisplatin was used as a positive control.

Example 5A Trapa natans L polyphenol extract 2^#Inhibition of breast cancer cells SK-BR-3 and MDA-MB-468 Function of

Adding 60% ethanol solution into pretreated water caltrop shells in a material-liquid ratio of 1:20(g/ml), extracting for 2h in a constant-temperature water bath at 60 ℃, taking out, putting into an ultrasonic instrument with ultrasonic power of 300KW, carrying out ultrasonic treatment for 20min, filtering while hot, collecting filtrate, extracting filter residues for 2 times again, combining the filtrate, centrifuging the filtrate at 3000r/min for 10min, collecting supernatant, and concentrating under reduced pressure to obtain a crude extract. And (3) after the crude extract is redissolved by water, extracting for three times by using ethyl acetate, and concentrating under reduced pressure to obtain the water chestnut shell polyphenol extract. And (3) redissolving the extract by using water, loading the extract on an AB-8 macroporous adsorption resin column, eluting by using aqueous solution with the volume of 2 columns, collecting fractions, and concentrating under reduced pressure to obtain the water chestnut shell extract. The extract 2 obtained in this example was measured^#The polyphenol content is 30.5-32.3%, the gallic acid content is 10.5-11.6%, the tri-galloylglucose content is 4.5-5.8%, the tetra-galloylglucose content is 3.9-5.0%, and the pentagalloylThe glucose content is 6.3-8.5%.

Respectively extracting with extract 2^#The breast cancer cells SK-BR-3 and MDA-MB-468 were treated at concentrations of 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 400. mu.g/ml and 800. mu.g/ml, and the inhibitory effects on the two breast cancer cells were examined for 24h, 48h and 72h, while cisplatin was used as a positive control.

Example 6A Trapa natans L polyphenol extract 3^#Inhibition of breast cancer cells SK-BR-3 and MDA-MB-468 Function of

Adding 60% ethanol solution into pretreated water caltrop shells in a material-liquid ratio of 1:20(g/ml), extracting for 2h in a constant-temperature water bath at 60 ℃, taking out, putting into an ultrasonic instrument with ultrasonic power of 300KW, carrying out ultrasonic treatment for 20min, filtering while hot, collecting filtrate, extracting filter residues for 2 times again, combining the filtrate, centrifuging the filtrate at 3000r/min for 10min, collecting supernatant, and concentrating under reduced pressure to obtain a crude extract. The crude extract is dissolved back by water, extracted by ethyl acetate for three times, and concentrated under reduced pressure to obtain the water chestnut shell extract. Dissolving the extract with water, loading onto DM130 macroporous adsorbent resin column, eluting with 30% ethanol solution of 2 column volume, collecting fractions, and concentrating under reduced pressure to obtain water chestnut shell polyphenol extract. The content of polyphenol of the extract No. 3 obtained in this example was found to be 81.5 to 84.9%, the content of gallic acid was found to be 24.8 to 26.4%, the content of tri-galloylglucose was found to be 12.5 to 14.0%, the content of tetra-galloylglucose was found to be 7.6 to 8.3%, and the content of pentagalloylglucose was found to be 25.2 to 28.7%.

Respectively extracting with extract 3^#The breast cancer cells SK-BR-3 and MDA-MB-468 were treated at concentrations of 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 400. mu.g/ml and 800. mu.g/ml, and the inhibitory effects on the two breast cancer cells were examined for 24h, 48h and 72h, while cisplatin was used as a positive control.

Extract 1^#～3^#The measuring process and the result of the inhibition effect of the breast cancer cells SK-BR-3 and MDA-MB-468 are as follows:

1. reagents and materials

HER-2 overexpression type breast cancer cell SK-BR-3 and triple negative breast cancer MDA-MB-468 were given as a gift by Zhejiang university medical college and were inoculatedAdding into fresh DMEM incomplete high-sugar culture solution containing 10% fetal calf serum, and placing at 37 deg.C and 5% CO₂Cultured in an incubator.

Both the CCK-8 assay kit and the 96-well plate were purchased commercially.

2. Experimental treatment

(1) The breast cancer cells SK-BR-3 and MDA-MB-468 suspension were inoculated into 96-well culture plates (1 ten thousand cells/well), at 37 ℃ with 5% CO₂Culturing in incubator, and mixing the extracts 1^#～3^#Adding a cell culture plate, and processing for 24h, 48h and 72h respectively;

(2) after treating the cells, 10. mu.l of CCK-8 reagent was added to each well of the plate, and after the treated cells were mixed with the reagents, 5% CO was added₂Incubating for 1h in a carbon dioxide incubator at 37 ℃, and then measuring the OD value of the cell suspension at 450nm by using a Thermo, MuLTiSKAN MK3 enzyme labeling instrument;

(3) recording OD values of the samples to be detected and the blank control group read by the microplate reader at 450nm, recording the OD value of each sample to be detected as a measured value, recording the OD value of the blank control group as a blank value, setting a final value as a measured value and a blank value, and setting three parallels for each group of experiments;

(4) and (3) calculating an inhibition rate: inhibition ratio is 100% — sample group (OD value) ÷ negative control group (OD value) × 100%;

(5) the polyphenol extracts of trapa acornis shell 1 described in examples 4 to 6 were each assayed^#～3^#The results for inhibition of two breast cancer cells are shown in the following table:

TABLE 1 samples of different concentrations treated SK-BR-3 and MDA-MB-468 cells

As can be seen from the above results, for SK-BR-3, extract 3 was obtained^#Has strong inhibitory effect on it, probably due to extract 3^#Has a high total polyphenol content, and extract 2^#Has higher inhibitory effect than extract 1^#Although extract 2^#Has a total polyphenol content lower than that of extract 1^#This is probably due to the different content of each active ingredient in the total polyphenol that exerts different inhibitory effects. For MDA-MB-468, extract 1^#The inhibiting effect is much higher than that of the extract 2^#And extract 3^#. The total polyphenol content of the three extracts and the content of the four polyphenol active ingredients are different, and the inventor finds that the inhibition effect on two breast cancer cells is not increased along with the increase of the total polyphenol content, namely the inhibition effect is not positively correlated with the total polyphenol content, but is correlated with the content of the four polyphenol substances.

According to the above results, it is preliminarily concluded that although the content of total polyphenols has an effect on inhibiting breast cancer cells, different contents and ratios of the active ingredients in the extract may exert a more significant effect on inhibiting different cancer cells. The content of each active ingredient in the extract may affect the effect between each active ingredient in the extract, thereby affecting the inhibitory activity of the extract. Next, the inventors made further studies on the way in which the active ingredients specifically act.

Example 7 comparative test of four Polyphenol standards for inhibition of Breast cancer cells

According to the embodiments, the isolated nanhu trapa shell polyphenol extract mainly contains four polyphenol substances of gallic acid, tri-galloyl glucose, tetra-galloyl glucose and pentagalloyl glucose. In order to study the effect of each active ingredient on inhibiting different types of breast cancer cells, the inhibition effect of the four polyphenol standard substances and the inhibition effect of different standard substances on different breast cancer cells in combination are compared respectively, and the inhibition rate of 48h is detected.

TABLE 2 comparison test of four polyphenol standards for breast cancer cell inhibition

According to experimental data, the single gallic acid, the tri-galloyl glucose, the tetra-galloyl glucose and the pentagalloyl glucose standard respectively have certain inhibition effects on the breast cancer cells SK-BR-3 and MDA-MB-468.

For SK-BR-3 cells, the inhibitory effect of gallic acid and pentagalloyl glucose is stronger than that of tri-galloyl glucose and tetra-galloyl glucose; when gallic acid, tri-galloylglucose, tetra-galloylglucose and pentagalloylglucose are used in a pairwise matching way, the matching use of the gallic acid and the pentagalloylglucose is found to show a strong inhibition effect, the inhibition rate is far higher than that of other combinations, and the conclusion can be drawn that the composition of the gallic acid and the pentagalloylglucose has a synergistic effect on the SK-BR-3 cell inhibition, but other compositions do not show a synergistic effect; tests are respectively carried out by adopting a composition of tri-galloylglucose + gallic acid + pentagalloylglucose and a composition of tetra-galloylglucose + gallic acid + pentagalloylglucose, the inhibition rate of the combination of the tri-galloylglucose, the gallic acid and the pentagalloylglucose is found to be obviously lower than the inhibition rate of the combination of the gallic acid and the pentagalloylglucose, and the fact that antagonism is possibly generated between the tri-galloylglucose, the tetra-galloylglucose and the composition of the gallic acid and the pentagalloylglucose results in obviously reduced inhibition effect of the combination of the tri-galloylglucose, the tetra-galloylglucose.

For MDA-MB-468 cells, the inhibitory effect of tri-galloylglucose and tetra-galloylglucose is stronger than that of gallic acid and pentagalloylglucose; when gallic acid, trigalloyl glucose, tetragalloyl glucose and pentagalloyl glucose are used in a pairwise matching way, the matching use of the trigalloyl glucose and the tetragalloyl glucose is found to show a strong inhibition effect, the inhibition rate is far higher than that of other combinations, and the conclusion can be drawn that the composition of the trigalloyl glucose and the tetragalloyl glucose has a synergistic effect on MDA-MB-468 cell inhibition, but other compositions do not show a synergistic effect; tests are respectively carried out by adopting a composition of gallic acid + tri-galloylglucose + tetra-galloylglucose and a composition of tri-galloylglucose + tetra-galloylglucose + pentagalloylglucose, the inhibition rate of the combined use of the three is found to be obviously lower than that of the combined use of the tri-galloylglucose and the pentagalloylglucose, and the inventor guesses that the gallic acid, the pentagalloylglucose, the tri-galloylglucose and the pentagalloylglucose have antagonism possibly, so that the inhibition effect of the combined use of the three is obviously reduced.

When the four polyphenol substances are mixed and used in the same proportion, the inhibition rates on SK-BR-3 cells and MDA-MB-468 cells are 75.7 percent and 74.3 percent respectively. It can be seen that the inhibition rate of gallic acid and pentagalloylglucose on SK-BR-3 cells can reach 97.2%, the inhibition rate of trigallaryl glucose and tetragalloylglucose on MDA-MB-468 cells can reach 95.6%, and the effect of the four polyphenol substances mixed in the same proportion is obviously different from that of the two combined groups. The specific inhibition effect of different active ingredients in the polyphenol extract of the water chestnut shells of the south lake on different types of breast cancer cell lines is proved. The four active ingredients may produce different inhibitory effects by means of synergism or antagonism aiming at different breast cancer cells.

Example 8A Trapa acornis nakai shell polyphenol extract 3^#Effect on the cell cycle and apoptosis of SK-BR-3

The polyphenol extract 3 of the water chestnut shells of the lake south described in example 6 is adopted^#Breast cancer cells SK-BR-3 were treated and the effect of the extract on cell cycle and apoptosis was studied.

1. Cell treatment:

(1) after the cultured SK-BR-3 cells are digested by pancreatin, cell suspension is prepared. 10ul of cell suspension was taken out for cell counting and then diluted with complete medium to a concentration of 100000 cells/ml.

(2) Adding the cells into a 6-well culture plate according to the volume of 2ml per well, discarding the cell culture solution when the cells grow to reach 40% confluence, replacing the cells except the normal control group with a complete culture solution containing different concentrations of the extract of the water chestnut shell (the extract concentrations are respectively 100 mu g/ml, 400 mu g/ml and 800 mu g/ml), culturing for 48h, and collecting the cells to obtain the treated cell culture solution.

TABLE 3 sample treatment of SK-BR-3 cells at different concentrations

Group of	Concentration of extract	Time of treatment
			Treatment group
1	100μg/ml	48h
			Treatment group
2	400μg/ml	48h
			Treatment group
3	800μg/ml	48h
			Control group
	0	48h

2. Experimental treatment

(1) Annexin V-FITC apoptosis assay:

a. sucking the cell culture solution out of the centrifugal tube, washing adherent cells once by PBS, and adding a proper amount of pancreatin cell digestive juice (which can contain EDTA) to digest the cells.

b. Mix slightly, transfer to centrifuge tube, 1000g centrifugal 5 minutes, discard the supernatant, collect cells, use PBS to gently resuspend cells and count.

c. 5-10 ten thousand of the resuspended cells were centrifuged at 1000g for 5 minutes, the supernatant was discarded, and 195. mu.l of Annexin V-FITC conjugate was added to gently resuspend the cells.

d. Add 5. mu.l Annexin V-FITC staining solution and mix gently.

e. Add 10. mu.l Propidium Iodide (PI) staining solution and mix gently.

f. Incubate at room temperature (20-25 ℃) for 10-20 minutes in the dark, followed by placing in an ice bath. Light protection can be performed using aluminum foil. The cells can be resuspended 2-3 times during incubation to improve labeling.

g. Flow cytometry detection was then performed.

(2) Cell cycle and apoptosis assays

a. The cell culture fluid was collected into a centrifuge tube and the cells were digested with trypsin. Centrifuging at about 1000g for 3-5 min to precipitate cells. The supernatant was carefully aspirated, and about 50. mu.l of the culture medium was left to avoid aspiration of the cells. Approximately 1ml of ice-cooled PBS was added, the cells were resuspended, and transferred to a 1.5ml centrifuge tube. The cells were pelleted by centrifugation again and the supernatant carefully aspirated, approximately 50 microliters or so of PBS could remain to avoid aspiration of the cells. Lightly flicking the bottom of the centrifugal tube to properly disperse the cells, and avoiding cell agglomeration.

b. Cell fixation: adding into 1ml ice bath precooled 70% ethanol, lightly blowing and mixing evenly, and fixing for 2 hours at 4 ℃. Centrifuge at 1000g for 3-5 minutes to pellet the cells. The supernatant was carefully aspirated, and about 50. mu.l or so of 70% ethanol remained to avoid aspiration of the cells. Approximately 1ml of ice-cold PBS was added to resuspend the cells. The cells were pelleted by centrifugation again and the supernatant carefully aspirated, approximately 50 microliters or so of PBS could remain to avoid aspiration of the cells. Gently flick the bottom of the centrifuge tube to properly disperse the cells and avoid cell clumping.

c. Dyeing: 0.5 ml of propidium iodide staining solution was added to each tube of cell sample, and the cell pellet was slowly and sufficiently resuspended and incubated at 37 ℃ in the dark for 30 minutes. Subsequently, the cells can be stored at 4 ℃ or in an ice bath protected from light. Flow detection was completed within 24 hours after staining was complete.

d. Flow detection and analysis: the red fluorescence was detected with a flow cytometer at the 488nm excitation wavelength, with the light scattering detected. Analysis software was used for cellular DNA content analysis and light scattering analysis.

3. Results of the experiment

(1) Extract 3^#Effect on SK-BR-3 cell cycle distribution

The propidium iodide and DNA are combined to emit red fluorescence, the PI fluorescence intensity detected by a flow cytometer can directly reflect the DNA content in cells, and the cell cycle is further reflected by the DNA content. The G0/G1 phase is the prophase of synthesis, and the DNA content is 2N; the G2/M phase is the anaphase and the mitosis phase, and the DNA content is 4N; the DNA content in S phase is between the two. Thus, cell cycle differentiation can be achieved by flow-based detection of DNA content.

As can be seen from FIG. 3, the polyphenol extract 3 of Trapa acornis husk was obtained at various concentrations^#Can disturb the cell cycle process of breast cancer cells SK-BR-3, suppress most cells in S phase, prevent cells from further division, and induce apoptosis.

(2) Effect of extracts on apoptosis of SK-BR-3

Normal cells, apoptotic cells and dead cells can be distinguished on a histogram of a flow cytometer by using FITC-labeled Annexin V and PI as dual fluorescent probes. As can be seen from FIG. 4, the breast cancer SK-BR-3 cells had a large amount of apoptosis in all treatment groups, and the increase of apoptotic cells was observed with the increase of the concentration of the extract from the Trapa natans L.K.K.400 ug/ml, and the most significant apoptosis was observed (note: region B1 represents dead cells, region B2+ B4 represents apoptotic cells, and region B3 represents normal cells), which probably caused the active components in the extract to induce the cleavage of PARP, thereby promoting the occurrence of apoptosis.

The extract 3# contains 81.5-84.9% of polyphenol, 24.8-26.4% of gallic acid, 12.5-14.0% of tri-galloylglucose, 7.6-8.3% of tetra-galloylglucose and 25.2-28.7% of pentagalloylglucose, wherein the content of gallic acid and pentagalloylglucose in the extract isThe content of sugar is obviously higher than the total amount of tri-galloylglucose and tetra-galloylglucose, so that the high-activity chitosan oligosaccharide has strong inhibition effect on SK-BR-3 cells and IC thereof₅₀It was 81.0. mu.g/ml (based on 48h of treatment). The extract has no significant inhibitory effect on MDA-MB-468 cells, and its IC is not significant enough₅₀242.5. mu.g/ml.

Example 9A Trapa acornis nakai shell polyphenol extract 1^#Effect on cell cycle and apoptosis of MDA-MB-468

The polyphenol extract 1 of the water chestnut shells of the lake of example 4 is adopted^#Breast cancer cells MDA-MB-468 were treated and the effect of the extract on cell cycle and apoptosis was studied. Cell processing and experimental processing were as described in example 8. The experimental results are as follows:

(1) extract 1^#Effect on MDA-MB-468 cell cycle distribution

As can be seen from FIG. 5, the concentrations of the polyphenol extract 1 from Trapa acornis husk were varied^#Can disturb the cell cycle process of breast cancer MDA-MB-468, suppress most cells in S phase, prevent cells from further division, and induce apoptosis.

(2) Extract 1^#Effect on apoptosis of MDA-MB-468 cells

Normal cells, apoptotic cells and dead cells can be distinguished on a histogram of a flow cytometer by using FITC-labeled Annexin V and PI as dual fluorescent probes. As can be seen from FIG. 6, the breast cancer MDA-MB-468 shows a great deal of apoptosis in all treatment groups, and the increase of apoptotic cells is observed with the increase of the concentration of the extract from the water chestnut shell of the south lake, and the apoptosis is most significant when the concentration of the extract reaches 400ug/ml (note: region B1 represents dead cells, region B2+ B4 represents apoptotic cells, and region B3 represents normal cells), which may be that the active components in the extract can induce the breakage of PARP, thereby promoting the occurrence of apoptosis.

Extract 1^#The polyphenol content of the extract is 54.5-60.4%, the gallic acid content is 2.6-2.8%, the tri-galloylglucose content is 19.3-21.2%, the tetra-galloylglucose content is 26.5-28.6%, the pentagalloylglucose content is 6.5-7.7%, and the extract contains tri-galloylglucose, galloylThe contents of galloylglucose and tetragalloylglucose are obviously higher, so that the galloylglucose and tetragalloylglucose have stronger inhibition effect on MDA-MB-468 cells, and the IC of the galloylglucose and tetragalloylglucose is IC₅₀105.3. mu.g/ml (based on 48h of treatment). The extract has poor inhibitory effect on SK-BR-3 cells, and its IC₅₀It was 358.7. mu.g/ml.

In view of the above embodiments, the present invention provides an application of a Trapa acornis nakai shell polyphenol extract in the preparation of an anti-HER-2 over-expression type breast cancer drug and a triple negative breast cancer drug, wherein the extract mainly contains four polyphenol substances of gallic acid, tri-galloylglucose, tetra-galloylglucose and pentagalloylglucose. Through further analysis on active ingredients which play a role in resisting HER-2 over-expression type breast cancer and a role in resisting triple-negative breast cancer, the gallic acid and the pentagalloyl glucose composition play a synergistic role in SK-BR-3 cells and show a remarkable inhibition effect, while the tri-galloyl glucose and the tetra-galloyl glucose possibly play an antagonistic role in the SK-BR-3 cells and can reduce the synergistic effect of the gallic acid and the pentagalloyl glucose composition; meanwhile, the composition of the tri-galloylglucose and the tetra-galloylglucose is found to exert a synergistic effect on the MDA-MB-468 cells and show a remarkable inhibition effect, while the gallic acid and the pentagalloylglucose possibly exert an antagonistic effect on the MDA-MB-468 cells and reduce the synergistic effect of the composition of the tri-galloylglucose and the tetra-galloylglucose. The invention provides a certain basis for further explaining the anti-breast cancer efficacy of the water chestnut shells, thereby providing technical support for developing and preparing anti-breast cancer drugs for different types of breast cancer. When the south lake water chestnut shell polyphenol extract is used for preparing the anti-breast cancer medicament, active ingredients with specific inhibition effects on different breast cancer cells are separated and extracted aiming at different types of breast cancer, so that the anti-cancer activity and effect of the medicament are obviously improved. How to separate and extract the active monomers in the water chestnut hull polyphenol extract in the lake south is the direction in which further research needs to be carried out next.

Claims

1. The application of a composition of gallic acid and pentagalloylglucose in preparing a medicament for resisting HER-2 over-expression type breast cancer is disclosed, wherein the composition of the gallic acid and the pentagalloylglucose is from a Trapa acornis shell polyphenol extract, and the preparation method of the Trapa acornis shell polyphenol extract comprises the following steps:

(2) adding 60% ethanol solution according to a material-liquid ratio of 1:20(g/ml), extracting in a thermostatic water bath at 60 ℃ for 2h, taking out, placing in an ultrasonic instrument with an ultrasonic power of 300KW for ultrasonic treatment for 20min, filtering while hot, collecting filtrate, repeatedly extracting filter residue for 2 times, mixing filtrates, centrifuging the filtrate at 3000r/min for 10min, collecting supernatant, and concentrating under reduced pressure to obtain crude extract;

(3) dissolving the crude extract with water, extracting with ethyl acetate for three times, and concentrating under reduced pressure to obtain pedicellus et pericarpium Trapae extract; dissolving the extract with water, loading onto DM130 macroporous adsorbent resin column, eluting with 30% ethanol solution of 2 column volume, collecting fractions, and concentrating under reduced pressure to obtain water chestnut shell polyphenol extract.

2. The use as claimed in claim 1, wherein the total polyphenol content of the Trapa acornis shell polyphenol extract is 81.5-84.9%.

3. The use of claim 1, wherein the polyphenols in the polyphenol extract of trapa natans l husk comprise gallic acid, trigalloylglucose, tetragalloylglucose and pentagalloylglucose.

4. The use as claimed in claim 3, wherein the content of gallic acid is 24.8-26.4%, the content of tri-galloylglucose is 12.5-14.0%, the content of tetra-galloylglucose is 7.6-8.3%, and the content of pentagalloylglucose is 25.2-28.7%, based on the total weight of the extract.