CN110237134A - A citrus citrus polyphenol capable of inhibiting the proliferation of intestinal cancer cells and its preparation method and application - Google Patents

Abstract

The invention discloses a citrus citrus polyphenol capable of inhibiting the proliferation of intestinal cancer cells, a preparation method and application thereof. The method comprises: pulverizing Fructus Fructus Fructus Fructus Fructus Fructus Fructus Fructus Aurantii to obtain powder, adding Fructus Fructus Fructus Fructus Fructus Fructus Fructus Fructus Fructus Fructus Powder into water, mixing evenly, and heating for extraction treatment to obtain extract liquid; centrifuging the Fructus Fructus Fructus Fructus Fructus Fructus Fructus Auratus, taking supernatant liquid, filtering to obtain filtrate, concentrating, Dry to obtain freeze-dried powder; dissolve the freeze-dried powder in water, prepare a mixed solution, add the macroporous resin to the mixed solution, perform adsorption treatment, filter with suction, add the adsorbed macroporous resin to the ethanol solution, and place it on a shaker desorption treatment to obtain a desorption solution; the desorption solution is suction-filtered, the filtrate is taken, concentrated, and freeze-dried to obtain the citrus citrus polyphenols that have the effect of inhibiting the proliferation of intestinal cancer cells. The citrus aurantium extract provided by the invention has the activity of inhibiting the proliferation of human colon cancer cells RKO, can be used for the development of drugs for the preparation of colon cancer, and provides an experimental basis and scientific basis for the application of traditional Chinese medicine compatibility in clinical antitumor drug research.

Description

A citrus citrus polyphenol capable of inhibiting the proliferation of intestinal cancer cells and its preparation method and application

technical field

The invention relates to the technical field of citrus aurantium extraction, in particular to a citrus citrus polyphenol capable of inhibiting the proliferation of intestinal cancer cells, a preparation method and application thereof.

Background technique

Citrus aurantium is the dry young fruit of Citrus aurantium L. and its cultivars or sweet orange C. sinensisOsbeck of the Rutaceae plant. "Famous Doctors Bielu" records about Citrus aurantium: "Remove chest and hypophlegm addiction, stop water, break stones, relieve fullness, urgency and pain in the heart...threat wind pain, calm stomach qi, stop diarrhea". "Shen Nong's Materia Medica" said it: "eliminate cold and heat, stop dysentery...". Citrus aurantium, bitter, pungent, sour, slightly cold in nature, is a classic qi-regulating medicine, which belongs to the spleen and stomach meridians, and has the effects of breaking qi and eliminating accumulation, resolving phlegm and dispelling swelling, that is, Citrus aurantium can regulate the whole body's qi , the role of eliminating stagnation and fullness, is widely used clinically to treat stagnant internal stagnation, fullness and pain, heavy diarrhea after diarrhea, obstructed stool, loose stool and other diseases.

Pharmacological studies have shown that the main active ingredients of Citrus aurantium are flavonoids, volatile oil and a small amount of alkaloids. Flavonoids such as naringin, hesperidin, and neohesperidin can best reflect the characteristics of Citrus aurantii, and are important active ingredients of Citrus Citrus Citrus to regulate qi, stagnation, relieve asthma, anti-inflammation, anti-oxidation and anti-allergy, and have gastric motility Improves gastric emptying and intestinal propulsion in rats. Alkaloids are the main components of Cardiac Boost in Aurantium citrifolia, mainly synephrine, N-methyltyramine, acetyl norsynephrine, norepinephrine, quinoline, narcotine, etc. Citrus aurantium was first recorded in "Shen Nong's Materia Medica", and was listed as a middle grade. It tastes bitter, pungent, slightly cold, and returns to the spleen, stomach, and large intestine meridian.

Citrus aurantium treats accumulated cold and prolapse of the anus: one piece of Citrus aurantium. Grind on the stone to make it smooth, drill it to install the handle, apply it with honey, and burn it to make it warm and iron it. ("Dry Gold Prescription"); Aurantium citrifolia treats postpartum abdominal pain, and you can't lie down when you are full: citrus aurantium (burning black, too much haste), and peony are divided into equal parts. The pestle is scattered. Take a square-inch dagger, three times a day. And the main carbuncle pus, with wheat porridge. ("Synopsis of the Golden Chamber" Citrus aurantium and Shaoyao powder); treat constipation: Divide aurantium citrus and acacia in equal parts. ("Shi Yi De Xiao Fang").

Colorectal cancer (CRC) is one of the most common malignant tumors, ranking third in incidence and the leading cause of cancer-related death. According to the information released by the World Health Organization, the number of deaths caused by colorectal cancer in the world in 2015 was 774,000; in my country, the incidence of colorectal cancer shows a clear upward trend, and the mortality rate ranks second in the cause of death from malignant tumors.

Studies have shown that Fructus Aurantium has a certain protective effect on the gastrointestinal tract, and decoctions, tinctures and liquid extracts of Fructus Fructus Fructus Aurantium and Fructus Aurantium have inhibitory effects on the isolated intestines of mice and rabbits and the intestines of rabbits in vivo. The water decoction can make the gastrointestinal contraction rhythm of dogs with gastric and intestinal fistula strong, showing excitatory effect. Citrus aurantium extract has obvious antagonistic effect on intestinal contraction induced by acetylcholine and histamine.

The existing method for preparing polyphenols from Citrus Citrus is outdated, and is generally prepared from the crude extract obtained after simple extraction of Citrus Citrus medicinal materials, and the content of active ingredients is low; it shows that the present invention adopts a new method to extract and separate polyphenols from Citrus Citrus. The macroporous resin enriches the effective components in the extract, which improves the effective utilization rate of the raw materials of Citrus aurantium.

Contents of the invention

In order to overcome the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a citrus citrus polyphenol capable of inhibiting the proliferation of intestinal cancer cells, its preparation method and application.

The invention provides the application of Citrus aurantium extract in the preparation of traditional Chinese medicine extracts for inhibiting the proliferation of intestinal cancer cells, which can extract and enrich the active ingredients that can effectively inhibit the proliferation of intestinal cancer cells from the raw materials of Citrus aurantium, and realize the new application of Citrus citrus extract .

The object of the present invention is achieved at least by one of the following technical solutions.

A method for preparing Citrus citrus polyphenols with the effect of inhibiting the proliferation of intestinal cancer cells provided by the present invention comprises the following steps:

(1) crushing and sieving the dried medicinal material Fructus Fructus Fructus Fructus Fructus Aurantium, adding water to extract the Fructus Fructus Fructus Fructus Fructus Medicae powder, mixing evenly, heating for extraction treatment, and obtaining an extract;

(2) centrifuging the extract described in step (1), taking the supernatant, filtering to get the filtrate, concentrating, and freeze-drying to obtain freeze-dried powder;

(3) Add the freeze-dried powder described in step (2) into deionized water (redissolution), mix evenly to obtain a mixed solution, add the macroporous resin into the mixed solution (macroporous resin static adsorption), carry out adsorption treatment, pump Filter the filter residue, add the filter residue to the ethanol solution (use the ethanol solution to desorb the substance adsorbed in the macroporous resin), place it in a shaker for desorption treatment, and obtain a desorption solution;

(4) Suction filter the desorption liquid in step (3), take the filtrate, concentrate, and freeze-dry to obtain powder, that is, the aurantia citrus polyphenols that have the effect of inhibiting the proliferation of intestinal cancer cells. The polyphenol content in the powder obtained by freeze-drying is 55.92%-63.27%.

The citrus citrus polyphenols that have the effect of inhibiting the proliferation of intestinal cancer cells can be redissolved, and the inhibitory effect of citrus citrus extract on the proliferation of RKO cells can be explored by CCK8 experiment.

Further, the sieve size of the sieve in the step (1) is 20-100 mesh; the quality of the water in the step (1) is 10-20 times that of the Fructus Citrifolia powder mass; the heating in the step (1) is carried out to extract The temperature of the treatment is 60-80° C., and the heating time for the extraction treatment is 1-3 hours.

Preferably, the extraction process can be extracted multiple times, the first extraction process is solid-liquid separation, the first extraction liquid and the first filter residue are obtained, the first filter residue is added to the water, and reheated to 60- 80°C, heating time is 1-3h, carry out the next round of extraction, solid-liquid separation, and then obtain the second extraction liquid and the second filter residue; and so on, can repeatedly extract the citrus aurantium powder, make full use of citrus aurantium The active ingredients in the powder, and then the extraction solution is combined before proceeding to the next step; the number of times of the extraction treatment is 1-3 times.

Further, the speed of the centrifugal treatment in step (2) is 2000-8000rpm, the time of the centrifugal treatment is 10-15min, and the temperature of the centrifugal treatment is 2-6°C; the filtration in the step (2) is the supernatant The liquid passes through a 60-mesh nylon filter cloth. Preferably, the supernatant is passed through a 60-mesh nylon filter cloth, and the number of layers of the nylon filter cloth is 4 layers.

Further, the method of concentration in step (2) includes rotary evaporation, and the temperature of the rotary evaporation is 50-65°C; the freeze-drying in step (2) is vacuum freeze-drying, and the freeze-drying time is 24-48h.

Further, the mass volume ratio of the lyophilized powder and deionized water in step (3) is 40-60 mg/mL; the models of the macroporous resin in step (3) include AB-8, ADS-17, D101 and NKA -9, etc.; the mass volume ratio of the dry base of the macroporous resin to the mixed solution is 1:10-1:20g/mL.

Further, the adsorption treatment in step (3) is carried out in a shaking table, the rotating speed of the shaking table is 50-200rpm; the time of the adsorption treatment is 3-15h, and the temperature of the adsorption treatment is 20-25°C .

Further, the volume fraction of the ethanol solution in step (3) is 80%-95%; the rotating speed of the shaking table placed in the shaking table for desorption treatment is 50-200rpm; the temperature of the desorption treatment is 20-25 ℃, the desorption treatment time is 3-12h.

Further, the method of concentrating in step (4) includes rotary evaporation.

The citrus citrus polyphenols provided by the invention with the effect of inhibiting the proliferation of intestinal cancer cells can be used in the preparation of drugs for inhibiting the proliferation of intestinal cancer cells (RKO).

The citrus citrus polyphenols provided by the present invention have the effect of inhibiting the proliferation of intestinal cancer cells. After the polyphenols are quantified by the Folin phenol method, they are added to the DMEM cell culture medium containing 10% FBS according to a certain concentration, which can be used to determine the effect of citrus citrus extract on Proliferation inhibitory effect of intestinal cancer cell RKO.

The present invention is verified by CCK8 inhibition of intestinal cancer RKO cell proliferation experiment, the polyphenol content in the crude extract freeze-dried powder A of Citrus aurantii must reach 250 μg/mL to inhibit the proliferation of more than 50% of intestinal cancer cells; the ethanol desorption AB prepared by the present invention Part of the freeze-dried powder C adsorbed by -8, the polyphenol content of which reaches 160 μg/mL can inhibit the proliferation of more than 50% of intestinal cancer cells. The experimental results showed that the efficacy of Aurantium citrus extract treated with AB-8 macroporous resin was better than that of untreated traditional Chinese medicine citrus citrus extract.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The preparation method provided by the present invention has simple steps. After being adsorbed by AB-8 macroporous resin and desorbed by ethanol, the obtained Citrus aurantium extract has the effect of inhibiting intestinal cancer cells, and can be used to prepare a Chinese medicine extract for the treatment of intestinal cancer , opened up a new approach to the treatment of intestinal cancer raw materials of traditional Chinese medicine, there are certain economic and social benefits.

Description of drawings

Fig. 1 is the histogram of the effect of freeze-dried powder group A, freeze-dried powder group B, and freeze-dried powder C group substances (polyphenol quantification) on the proliferation of human colon cancer RKO cells in Example 5;

Fig. 2 is the influence of Fructus Fructus Citrus Fructus extract at different concentrations on the morphology of human colon cancer RKO cells in Example 5, observed under an inverted microscope (Aoto Optical BDS200) (magnification is 20 times).

Detailed ways

The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be pointed out that, if there are any processes in the following that are not specifically described in detail, those skilled in the art can realize or understand with reference to the prior art. The reagents or instruments used were not indicated by the manufacturer, and they were regarded as conventional products that can be purchased from the market.

Example 1

A method for preparing citrus citrus polyphenols with the effect of inhibiting the proliferation of intestinal cancer cells, comprising the steps of:

(1) After crushing the medicinal material Fructus Fructus Fructus Aurantii, pass through a 20-mesh sieve to obtain Fructus Fructus Fructus Fructus Fructus Fructus Powder, add described 10g Fructus Fructus Fructus Fructus Fructus Fructus Fructus Powder into 200mL deionized water, mix evenly, heat and carry out extraction processing twice, the temperature of extraction processing is 80 ℃, The time for the extraction treatment is 1 hour, and the two extraction products are combined to obtain the extract;

(2) Centrifuge the extract described in step (1) (2°C, 2000rpm for 10min), take the supernatant, pass through 4 layers of 60-mesh nylon filter cloth to collect the filtrate, concentrate by rotary evaporation at 50°C, and vacuum freeze-dry to obtain freeze-dried powder;

(3) Add the freeze-dried powder described in step (2) into 20 mL of deionized water, mix well, and prepare a mixed solution (the concentration of the freeze-dried powder in the mixed solution is 50 mg/mL), macroporous resin AB-8 dry basis (Activated) according to the mass volume ratio of 1:10g/mL added to the mixed solution for static adsorption treatment, the adsorption treatment is carried out in a shaking table, the rotating speed of the shaking table is 200rpm, and the temperature of the adsorption treatment is 25°C , the time of adsorption treatment is 15h, suction filtration separates filtrate and filter residue (filter residue is the macroporous resin after adsorption), and filter residue is added in 20mL ethanol solution that volume fraction is 95%, is placed in shaker and carries out desorption process, shaker The rotating speed is 200rpm, the temperature of the desorption treatment is 25°C, the time of the desorption treatment is 12h, and the desorption liquid is obtained;

(4) suction filter the desorption liquid described in step (3), take the filtrate, evaporate and concentrate, and freeze-dry to obtain a freeze-dried powder. The total amount of polyphenols is 83.33mg).

Example 2

A method for preparing citrus citrus polyphenols with the effect of inhibiting the proliferation of intestinal cancer cells, comprising the steps of:

(1) After crushing the medicinal material Fructus Fructus Fructus Aurantii, pass through a 50-mesh sieve to obtain Fructus Fructus Fructus Fructus Fructus Fructus Powder, add described 10g Fructus Fructus Fructus Fructus Fructus Fructus Fructus Powder into 120mL deionized water, mix evenly, heat and carry out extraction treatment 3 times, the temperature of extraction treatment is 70 ℃, The extraction treatment time is 2 hours, and the extraction products are combined three times to obtain the extract;

(2) Centrifuge the extract described in step (1) (4 °C, 5000 rpm for 13 min), take the supernatant, pass through 4 layers of 60-mesh nylon filter cloth to collect the filtrate, concentrate by rotary evaporation at 60 °C, and vacuum freeze-dry to obtain freeze-dried powder;

(3) Add the freeze-dried powder described in step (2) into 20mL deionized water to prepare a mixed solution (in the mixed solution, the concentration of the freeze-dried powder is 50 mg/mL), non-polar macroporous resin D101 dry basis ( Activated) was added to the mixed solution according to the mass volume ratio of 1:12g/mL for static adsorption treatment. The adsorption treatment was carried out in a shaking table, the rotating speed of the shaking table was 100rpm, and the temperature of the adsorption treatment was 23°C. The time of adsorption treatment is 10h, and suction filtration separates filtrate and filter residue (filter residue is the macroporous resin after adsorption), and filter residue is added in 20mL volume fraction and is 80% ethanol solution, is placed in shaker and carries out desorption process, shaker The rotating speed is 100rpm, the temperature of desorption treatment is 23°C, and the time of desorption treatment is 6h, and the desorption liquid is obtained;

(4) suction filter the desorption liquid described in step (3), take the filtrate, evaporate and concentrate, and freeze-dry to obtain a freeze-dried powder. The total amount of polyphenols is 51.15mg).

Example 3

A method for preparing citrus citrus polyphenols with the effect of inhibiting the proliferation of intestinal cancer cells, comprising the steps of:

(1) After crushing the medicinal material Fructus Fructus Fructus Fructus Aurantii, pass through a 100-mesh sieve to obtain Fructus Fructus Fructus Fructus Fructus Fructus Citrifolia powder, add described 10g Fructus Fructus Fructus Fructus Fructus Fructus Fructus Powder into 100mL deionized water, mix evenly, heat and carry out extraction treatment twice, the temperature of extraction processing is 60 ℃, The time of extraction treatment is 1.5h, and the two extraction products are combined to obtain the extract;

(2) Centrifuge the extract described in step (1) (4 °C, 5000 rpm for 15 min), take the supernatant, pass through 4 layers of 60-mesh nylon filter cloth to collect the filtrate, concentrate by rotary evaporation at 55 °C, and vacuum freeze-dry to obtain freeze-dried powder;

(3) Add the freeze-dried powder described in step (2) into 20 mL of deionized water, mix well, and prepare a mixed solution (the concentration of the freeze-dried powder in the mixed solution is 40 mg/mL), and follow the weak polarity macroporous resin ADS The mass volume ratio of -17 dry base (activated) to the mixed solution is 1:20g/mL, add the dry base of macroporous resin ADS-17 into the mixed solution for static adsorption treatment, and the adsorption treatment is carried out in a shaking table , the rotating speed of shaking table is 50rpm, the temperature of adsorption treatment is 20 ℃, the time of adsorption treatment is 10h, suction filtration separates filtrate and filter residue (filter residue is the macroporous resin after adsorption), and filter residue is added into 20mL volume fraction is 80% In the ethanol solution, place it in a shaking table for desorption treatment, the rotation speed of the shaking table is 50 rpm, the temperature of the desorption treatment is 25 ° C, and the time of the desorption treatment is 3 hours, to obtain the desorption solution;

(4) suction filter the desorption liquid described in step (3), take the filtrate, evaporate and concentrate, and freeze-dry to obtain a freeze-dried powder. The total amount of polyphenols is 67.04mg).

Example 4

A method for preparing citrus citrus polyphenols with the effect of inhibiting the proliferation of intestinal cancer cells, comprising the steps of:

(1) After crushing the medicinal material Fructus Fructus Fructus Fructus Aurantii, pass through 80 mesh sieves to obtain Fructus Fructus Fructus Fructus Fructus Fructus Fructus Powder, add described 10g Fructus Fructus Fructus Fructus Fructus Fructus Fructus Powder into 150mL deionized water, mix evenly, heat and carry out 2 times of extraction treatment, the temperature of extraction treatment is 75 ℃, The time for the extraction treatment is 2 hours, and the two extraction products are combined to obtain the extract;

(2) Centrifuge the extract described in step (1) (4 °C, 5000 rpm for 15 min), take the supernatant, pass through 4 layers of 60-mesh nylon filter cloth to collect the filtrate, concentrate by rotary evaporation at 65 °C, and vacuum freeze-dry to obtain freeze-dried powder;

(3) Add the freeze-dried powder described in step (2) into 20 mL of deionized water, mix well, and prepare a mixed solution (the concentration of the freeze-dried powder in the mixed solution is 60 mg/mL), according to the polar macroporous resin NKA- 9 The mass volume ratio of the dry base (activated) to the mixed solution is 1:16g/mL, and the polar macroporous resin NKA-9 dry base is added to the mixed solution for static adsorption treatment. carried out in , the rotating speed of the shaker was 120rpm (the temperature of the adsorption treatment was 25°C, the time of the adsorption treatment was 8h, the filtrate and the filter residue were separated by suction filtration (the filter residue was the macroporous resin after adsorption), and the filter residue was added to 20mL with a volume fraction of 92% ethanol solution, placed in a shaker for desorption treatment, the rotation speed of the shaker is 120rpm, the temperature of the desorption treatment is 20°C, and the time of the desorption treatment is 12h, to obtain the desorption solution;

(4) suction filter the desorption liquid described in step (3), take the filtrate, evaporate and concentrate, and freeze-dry to obtain a freeze-dried powder. The total amount of polyphenols is 54.35mg).

Implementation effect verification

The above provides 4 examples of implementation schemes. These 4 examples of implementation schemes use different macroporous resins respectively. The following combines relevant formulas and data to explore the adsorption and desorption rates of the active ingredients of Fructus Aurantium Fructus in the 4 kinds of macroporous resins.

Calculate the adsorption and desorption rates of different resins according to the following formula (refer to the literature "Isolation, Purification, Structure Identification and Related Activity Research of Arborvitae Leaf Polyphenols").

Polyphenol adsorption rate R1(%)=(C0-Ce)/C0*100;

Resin desorption rate R2(%)=Cd*V2/(Qe*m)*100;

Resin adsorption capacity Qe(mg/g)=(C0-Ce)*V0/m;

Resin desorption amount D(mg/g)=Cd*V2/m;

Wherein, C0: polyphenol concentration in the extract described in step (1), mg/mL;

Ce: after the adsorption treatment described in step (3), the concentration of polyphenols in the filtrate obtained by suction filtration after adsorption equilibrium, mg/mL;

R1: adsorption rate, 100%;

Qe: adsorption capacity, mg/g;

V0: sample volume, mL;

M: Resin mass, g;

R2: Desorption rate, 100%;

D: desorption amount, mg/g;

Cd: the concentration of polyphenols in the filtrate obtained after desorption equilibrium obtained after the desorption solution described in step (4), mg/mL;

V2: volume of desorption solution, mL;

The test results of the adsorption rate and desorption rate of the active ingredients of Aurantium Fructus Fructus Fructus Fructus in 4 kinds of macroporous resins are shown in the following table 1, and table 1 is the test data table of the adsorption rate and desorption rate of 4 kinds of macroporous resins.

Table 1

Comprehensive above-mentioned 4 example embodiments, in these 4 kinds of macroporous resins, the adsorption rate and the desorption rate to the Fructus Fructus Aurantii active ingredient are maximum all is AB-8 resin (embodiment 1), illustrate the AB-8 macroporous resin of weak polarity The ability to adsorb and desorb the active ingredients of Citrus aurantium is the strongest. Considering the adsorption rate and desorption rate comprehensively, the AB-8 macroporous resin with an adsorption rate of 63.27% and a desorption rate of 96.33% was selected as the resin for preparing the test substance for inhibiting intestinal cancer cell proliferation in subsequent experiments.

The freeze-dried powder described in step (2) in the preparation method provided by Example 1 is named as freeze-dried powder A; the described suction filtration of step (3) in the preparation method provided by Example 1 separates the filtrate and the filter residue, and the obtained filtrate , carry out freeze-drying treatment, and the powder obtained is named as freeze-dried powder B; the polyphenols of Fructus Fructus Fructus Fructus Fructus Fructus Polyphenols described in step (4) in the preparation method provided by Example 1, which has the effect of inhibiting the proliferation of intestinal cancer cells (RKO), are named as freeze-dried powder B. Dry powder C. The CCK8 experiment was carried out to explore the inhibitory effect of these three extracts of Citrus aurantium (lyophilized powder A, freeze-dried powder B and freeze-dried powder C) on the proliferation of RKO cells.

Example 5 Experimental Study on the Inhibition of the Proliferation of Human Colon Cancer Cell RKO Cells by the Freeze-dried Powder of Citrus Citrus Extract

Experimental Materials

Experimental cell line

The human colon adenocarcinoma RKO cells were routinely cultured in DMEM medium with a volume fraction of 10% FBS (culture conditions: 37° C., CO ₂ volume fraction 5%).

Test substance stock solution

Weigh the freeze-dried powder of Citrus aurantium extract (lyophilized powder A, freeze-dried powder B and freeze-dried powder C), respectively dissolve in PBS buffer (GIBCO, product number: 10010023), pass through a 0.22 μm sterile filter membrane, 4 ° C It is stored and diluted to the working concentration with DMEM containing 10% FBS by volume fraction before administration. The concentrations of the test substances involved in the experiment are all quantified by the Folin-phenol method.

experimental reagent

DMEM medium (GIBCO 11965-092), fetal bovine serum (GIBCO 10099-141-FBS), Trpsin (GIBCO 25200072), Penicillin-Streptomycin (GIBCO 15140163), PBS (GIBCO 10010023), CCK-8 solution (Dojindo)

experiment equipment

Chongqing Auto Optical Inverted Microscope (BDS200), UV Microplate Detector (BioteK Synergy H1), Carbon Dioxide Cell Incubator (Thermo), Ultra-clean Bench (Sujing Antai), Automatic Autoclave (SanyoMLS3020, Japan) , desktop electric blast drying oven (Shanghai Precision Experimental Equipment DHG9123A), refrigerator (Siemens KG18V21TI), liquid nitrogen tank (MVE), low-speed centrifuge (Shanghai Anting Scientific Instrument KA100).

experimental method

RKO cells were cultured in DMEM with a volume fraction of 10% FBS based on 37°C and a volume fraction of 5% CO ₂ for routine culture (100mm culture dish). When the cells were fused to 80%, the cells were collected, the culture medium was discarded, and Wash with PBS buffer three times, add 1mL Trpsin-EDTA to digest for 1min, add 2mL complete medium (DMEM) to stop the digestion reaction, pipette the cells and transfer them to a centrifuge tube, centrifuge at 1000rpm for 5min, adjust the concentration of the cell suspension to 2.5×10 ⁴ /mL.

The cells were seeded into a 96-well culture plate, 100 μL of cell suspension was added to each well, and the 96-well culture plate was placed in a 37° C., 5% CO ₂ cell incubator for culture.

Observe the growth of the cells. After culturing for 12 hours, they can grow to 50-60%. Add 100 μL of citrus citrus lyophilized powder solution (lyophilized powder A, lyophilized powder B or lyophilized powder C), and continue to cultivate for 24 hours.

After 24 hours, suck out all the medium in the well, replace it with 100 μL serum-free medium, add 10 μL CCK-8 solution, place in a carbon dioxide incubator (37°C, _CO2 volume fraction is 5%), and continue to cultivate for 2 hours. The absorbance of each well was measured at 450 nm in a microplate reader, and 4 replicate wells were set for each group.

The results are expressed as the survival rate of the drug on the cells:

according to The formula was used to calculate the effect of Citrus aurantium extract on the proliferation of human colon cancer RKO cells.

The experimental groups are as follows:

Quantification by polyphenols (determination by Folin's phenol method):

Experimental group a: the crude extract of Citrus aurantium before macroporous resin adsorption (lyophilized powder A); ②experimental group b: the crude extract of Citrus aurantium in aqueous solution after macroporous resin adsorption (lyophilized powder B); ③experimental group c (desorption group): Aurantium citrus extract (freeze-dried powder C) desorbed by macroporous resin with ethanol; ④ control group: RKO cells and complete medium (DMEM); ⑤ blank group: complete medium (DMEM).

Statistical processing

All data were independently measured in parallel three times, and the data results were expressed in the form of mean ± standard deviation (mean ± SD). SPSS 24.0 software was used to conduct statistical analysis on the test data, and p<0.05 was regarded as a statistically significant difference. Graphpad Prism 6.0 software was used to analyze the variance of the data and make graphs.

Experimental results

The results are shown in Figure 1. After quantification of polyphenols, the freeze-dried powder A of the crude extract of Citrus aurantium can significantly inhibit the proliferation of RKO cells at 125 μg/mL; the remaining solution freeze-dried powder B after AB-8 adsorption 640 μg/mL can significantly inhibit the proliferation of RKO cells; ethanol desorption AB-8 adsorbed part of the lyophilized powder C can significantly inhibit the proliferation of RKO cells at 40 μg/mL, as shown in Figure 2, can significantly change at 160 μg/mL The morphology of RKO cells promotes the growth of cells in clusters.

In addition, the polyphenol content in the freeze-dried powder A of the crude extract of Citrus aurantii must reach 250 μg/mL to inhibit the proliferation of more than 50% of intestinal cancer cells; 640μg/mL can inhibit more than 50% of intestinal cancer cell proliferation; ethanol desorbed AB-8 freeze-dried powder C, its polyphenol content reaches 160μg/mL can inhibit more than 50% of intestinal cancer cell proliferation.

The experimental results show that the method of adsorption by AB-8 macroporous resin and desorption by absolute ethanol can effectively enrich the active ingredients in the crude extract of Citrus aurantium which inhibit the proliferation of intestinal cancer cell RKO. It may be due to the complex composition of the crude extract of Citrus aurantium, which contains active ingredients that can inhibit the proliferation of intestinal cancer cells, but the abundance of effective substances per unit polyphenol concentration is low, and AB-8 is a macroporous with weak polarity. Resin, which can be used to enrich less polar phenolic substances. The active components of Aurantium citrus fruit adsorbed by AB-8 and desorbed by absolute ethanol should belong to phenols with weak polarity, and this part of substances has a good effect on inhibiting the proliferation of intestinal cancer cell RKO.

The above examples are only preferred implementations of the present invention, and are only used to explain the present invention, rather than limit the present invention. Changes, replacements, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention shall belong to the present invention. protection scope of the invention.

Claims (10)

1. a kind of preparation method with the dried immature fruit of citron orange polyphenol for inhibiting colon-cancer cell proliferation function, which is characterized in that including walking as follows It is rapid:

(1) by dried immature fruit of citron orange grinding and sieving, Immature Orange Fruit is obtained, the Immature Orange Fruit is added to the water, is uniformly mixed, heating carries out Extraction process obtains extracting solution；

(2) step (1) extracting solution is subjected to centrifugal treating, takes supernatant, filters to take filtrate, be concentrated, freeze-drying obtains Freeze-dried powder；

(3) step (2) described freeze-dried powder is added to the water, is uniformly mixed, obtains mixed liquor, mixed liquor is added in macroreticular resin In, adsorption treatment is carried out, suction filtration takes filter residue, and filter residue is added in ethanol solution, is placed in shaking table and carries out desorption processing, solved Imbibition；

(4) by step (3) the stripping liquid suction filtration, filtrate is taken, is concentrated, being freeze-dried described in obtaining, there is inhibition colon-cancer cell to increase Grow the dried immature fruit of citron orange polyphenol of effect.

2. preparation method according to claim 1, which is characterized in that the slot size of step (1) described sieving is 20- 100 mesh；The quality of step (1) described water is 10-20 times of Immature Orange Fruit quality；Described heat extracts the temperature of processing and is 60-80 DEG C, the time that the heating extracts processing is 1-3h.

3. preparation method according to claim 1, which is characterized in that the rate of step (2) described centrifugal treating is 2000- 8000rpm, the time of centrifugal treating are 10-15min, and the temperature of the centrifugal treating is 2-6 DEG C；It is described to be filtered into supernatant Cross the nylon filtering cloth of 60 mesh.

4. preparation method according to claim 1, which is characterized in that the mode of step (2) described concentration includes that rotation is steamed Hair, the temperature of the rotary evaporation are 50-65 DEG C；The freeze-drying is vacuum freeze drying, and the time of freeze-drying is 24- 48h。

5. preparation method according to claim 1, which is characterized in that the quality volume of step (3) freeze-dried powder and water Than for 40-60:1mg/mL；The model of the macroreticular resin includes AB-8, ADS-17, D101 and NKA-9；The macroreticular resin with The mass volume ratio of mixed liquor is 1:10-1:20 g/mL.

6. preparation method according to claim 1, which is characterized in that step (3) described adsorption treatment be in shaking table into Capable, the revolving speed of the shaking table is 50-200 rpm；The time of the adsorption treatment is 3-15h, and the temperature of adsorption treatment is 20- 25℃。

7. preparation method according to claim 1, which is characterized in that the volume fraction of step (3) described ethanol solution is 80%-95%, the mass volume ratio of macroreticular resin and ethanol solution is 0.1g/mL after the adsorption saturation；It is placed in shaking table and carries out The shaking speed of desorption processing is 50-200 rpm；The temperature of the desorption processing is 20-25 DEG C, and the time for desorbing processing is 3- 12h。

8. preparation method according to claim 1, which is characterized in that the mode of step (4) described concentration includes that rotation is steamed Hair.

9. a kind of have the trifoliate orange for inhibiting colon-cancer cell proliferation function as made from the described in any item preparation methods of claim 1-8 Real polyphenol.

10. the dried immature fruit of citron orange polyphenol with inhibition colon-cancer cell proliferation function as claimed in claim 9 inhibits colon-cancer cell to increase in preparation The application in drug grown.