CN115778998A - Application of radix Puerariae extract in preparing medicine for treating or preventing gastrointestinal diseases - Google Patents

Application of radix Puerariae extract in preparing medicine for treating or preventing gastrointestinal diseases Download PDF

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CN115778998A
CN115778998A CN202211496465.1A CN202211496465A CN115778998A CN 115778998 A CN115778998 A CN 115778998A CN 202211496465 A CN202211496465 A CN 202211496465A CN 115778998 A CN115778998 A CN 115778998A
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extraction
pce
cells
carbon dioxide
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赵辉
张美荣
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Medivix Technology Tianjin Co ltd
Tianjin University of Commerce
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Medivix Technology Tianjin Co ltd
Tianjin University of Commerce
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Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to an application of a kudzuvine root extract in preparation of a medicine for treating or preventing gastrointestinal diseases. The kudzu root extract of the invention is prepared by supercritical CO 2 Supercritical CO assisted by extraction or ethanol 2 The extraction rate is respectively 2.6 percent and 5.2 percent, and the content of the total flavone is respectively 1.52mg/g and 25.24mg/g. The kudzu root extract has strong free radical scavenging capacity and strong antioxidant activity. The kudzu root extract can effectively protect the damage of an intestinal epithelial barrier induced by aluminum, reduce the accumulation of ROS active oxygen caused by Al stimulation, reverse the down regulation of tight junction protein caused by aluminum stimulation, reduce the expression of P65 protein in an NF-kB metabolic pathway and inhibit the activation of P65 molecules.

Description

Application of radix Puerariae extract in preparing medicine for treating or preventing gastrointestinal diseases
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to an application of a kudzuvine root extract in preparation of a medicine for treating or preventing gastrointestinal diseases.
Background
Kudzu root (Pueraria) is a dried root of Pueraria lobata (Willd.) Ohwi of Leguminosae, is a traditional Chinese medicinal material in China, has high nutritional, edible, medicinal and health care values, is popular with researchers and consumers, and is better reputed by 'thousand-year ginseng'. Modern researches show that the kudzuvine root has rich biological activity, for example, isoflavone and flavonoid substances are main substance components with antioxidant and anti-inflammatory activities of the kudzuvine root, and saponins have a protective effect on liver injury.
Aluminum (Al) is the most abundant and prevalent metal element in the environment, and the main route of human exposure to aluminum is through food and water intake. Aluminum compounds are widely used in food additives, antacids, pharmaceutical and food packaging, cookware, etc., which also make possible excessive accumulation of aluminum in the human body, thereby threatening human health. Therefore, there is a high necessity to develop a drug for protecting gastrointestinal diseases caused by the metal element aluminum.
Disclosure of Invention
The invention aims to provide the application of a kudzuvine root extract in preparing a medicament for treating or preventing gastrointestinal diseases. The kudzu root extract has high biological activity and can inhibit the damage of the integrity of intestinal epithelium induced by aluminum.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of a kudzuvine root extract in preparing a medicament for treating or preventing gastrointestinal diseases, wherein the gastrointestinal diseases are caused by aluminum stimulation.
Preferably, the kudzu root extract is prepared by extracting kudzu root powder with supercritical carbon dioxide.
Preferably, the temperature of the supercritical carbon dioxide extraction is 20-40 ℃.
Preferably, the extraction pressure of the supercritical carbon dioxide extraction is 20 to 25MPa.
Preferably, the supercritical carbon dioxide extracted CO 2 The flow rate is 0.6-1L/min.
Preferably, the separation pressure of the supercritical carbon dioxide extraction is 6-10 Mpa.
Preferably, the separation temperature of the supercritical carbon dioxide extraction is 40-50 ℃.
Preferably, the extraction time of the supercritical carbon dioxide extraction is 2-4 h.
Preferably, the supercritical carbon dioxide extraction further comprises adding anhydrous ethanol.
Preferably, the dosage ratio of the kudzu root powder to the absolute ethyl alcohol is 400g.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an application of a radix puerariae extract in preparing a medicament for treating or preventing gastrointestinal diseases, wherein the invention utilizes RT-PCR to detect related molecules to explore a protection mechanism of the radix puerariae extract on an intestinal epithelial barrier, and the result shows that: radix Puerariae extract can reverse aluminum-induced intestinal epithelial barrier disruption caused by Occludin and Claudin-4 molecule down regulation, and its inflammatory factors TNF-alpha and IL-1 beta expression decrease; WB result is consistent with RT-PCR result, occludin and Claudin-4 protein expression is up-regulated after radix Puerariae extract treatment, and radix Puerariae extract can inhibit aluminum-induced intestinal epithelial integrity damage.
Furthermore, the extraction method is simple, the extraction efficiency is high, and the addition of absolute ethyl alcohol changes fluid CO 2 The polarity of the mixed solvent is improved, so that the polar flavonoid macromolecules can be dissolved in the mixed solvent (supercritical CO) 2 And absolute ethyl alcohol), the obtained pueraria extract contains more compounds, which not only contain weak polar small molecules, such as terpenes, lipids and the like, but also contain ethanol-soluble flavone macromolecular compounds. The kudzu root extract mainly comprises ethyl linoleate, 4-isopropylbenzaldehyde, 2, 3-dihydrothiophene, ligustrazine and other compounds, and has a protective effect on intestinal epithelial single-layer membrane barriers.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows supercritical CO 2 The extraction process single-factor experimental result is shown, wherein A is the influence of extraction pressure on the extraction rate, and B is the influence of extraction temperature on the extraction rate;
FIG. 2 is ethanol assisted supercritical CO 2 The single-factor result of the extraction process is shown, wherein A is the influence of extraction pressure on the extraction rate, and B is the influence of extraction temperature on the extraction rate;
FIG. 3 is a rutin standard curve;
FIG. 4 illustrates the radical scavenging ability of PCE and PAE, wherein A is to scavenge DPPH radicals, B is to scavenge ABTS radicals, and C is to scavenge hydroxyl radicals;
FIG. 5 shows the survival rate of cells after PCE acts on HT-29 cells for 24h under different concentrations;
FIG. 6 shows different AlCl concentrations 3 Survival of the treated cells;
FIG. 7 shows PCE antagonizing AlCl 3 Stimulation of HT-29 cell survival;
FIG. 8 is PCE antagonistic AlCl 3 Changes in TEER values due to stimulation of HT-29;
FIG. 9 shows PCE antagonism AlCl 3 Stimulation of monolayer barrier permeability change by HT-29;
FIG. 10 shows PCE antagonizing AlCl 3 Stimulation of oxidative stress by HT-29;
FIG. 11 shows PCE processing to suppress AlCl 3 Stimulating the expression levels of TNF-alpha and IL-1 beta by HT-29;
FIG. 12 shows PCE processing to suppress AlCl 3 Stimulation of the expression level of P65 by HT-29;
FIG. 13 is the mRNA expression level of HT-29Occludin, claudin-4;
FIG. 14 shows the expression levels of HT-29 tight junction protein Occludin, claudin-4.
Detailed Description
The invention provides an application of a kudzuvine root extract in preparing a medicament for treating or preventing gastrointestinal diseases.
In the present invention, the pueraria lobata extract is preferably prepared by extracting pueraria lobata powder with supercritical carbon dioxide.
In the present invention, unless otherwise specified, all the raw materials used are commercially available in the art.
In the present invention, the pueraria powder is preferably prepared by a method comprising the steps of: peeling radix Puerariae, oven drying, pulverizing, and sieving to obtain radix Puerariae powder.
The invention has no special requirements on the crushing mode, such as a crusher.
In the present invention, the screened mesh size is preferably 40 mesh.
In the present invention, the temperature of the supercritical carbon dioxide extraction is preferably 20 to 40 ℃, more preferably 30 ℃. The temperature of the supercritical carbon dioxide extraction of the invention is easier to dissolve the effective substances in the kudzuvine root.
In the invention, the extraction pressure of the supercritical carbon dioxide extraction is preferably 20-25 MP, and the extraction pressure can fully extract the volatile oil component in the radix puerariae, and can save the using amount of carbon dioxide and the cost.
In the present invention, the supercritical carbon dioxide extracted CO 2 The flow rate is preferably 0.6 to 1L/min, more preferably 0.8L/min.
In the present invention, the separation pressure of the supercritical carbon dioxide extraction is preferably 6 to 10MPa, and more preferably 8MPa.
In the present invention, the separation temperature of the supercritical carbon dioxide extraction is preferably 40 to 50 ℃, more preferably 45 ℃.
In the present invention, the extraction time of the supercritical carbon dioxide extraction is preferably 2 to 4 hours, and more preferably 3 hours.
In the present invention, the supercritical carbon dioxide extraction preferably further comprises adding anhydrous ethanol. The addition of absolute ethanol can change the CO of the fluid 2 The polarity of the mixed solvent is improved, so that the polar flavonoid macromolecules can be dissolved in the mixed solvent, and the extraction rate is further improved.
In the invention, the dosage ratio of the pueraria powder to the absolute ethyl alcohol is preferably 400g.
In the present invention, it is preferable that the supercritical carbon dioxide extraction is followed by filtering the obtained liquid to obtain the pueraria lobata extract.
In order to further illustrate the present invention, the following detailed description of the application of the pueraria lobata extract provided by the present invention is made with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1 supercritical CO 2 Single factor experiment of extraction process
Pre-treating kudzu root: removing outer skin of radix Puerariae, cutting into strips, oven drying, pulverizing with a pulverizer, sieving with 40 mesh sieve to obtain radix Puerariae powder, and storing in refrigerator at 4 deg.C.
The extraction rate of the kudzu root extract is taken as a response value, and the factors influencing the extraction rate are mainly two: the extraction pressure and extraction temperature, the specific single factor test protocol is as follows:
1) Putting 400g radix Puerariae powder into extraction tank, setting extraction temperature at 25 deg.C, and CO 2 The flow is 0.8L/min, the separation pressure is 8MPa, the separation temperature is 45 ℃, the extraction time is 3h, and the extraction rates of the radix puerariae extract under the extraction pressures of 5MPa, 10MPa, 15MPa, 20MPa and 25MPa are respectively considered;
2) Putting 400g of radix Puerariae powder into an extraction tank, setting extraction pressure of 15MPa 2 The flow rate is 0.8L/min, the separation pressure is 8MPa, the separation temperature is 45 ℃, the extraction time is 3h, and the extraction rates of the radix puerariae extract at the extraction temperatures of 20 ℃,30 ℃,40, 50 and 60 ℃ are respectively considered.
Filtering the obtained radix Puerariae extract with a filter, weighing, and calculating the extraction rate, wherein the calculation formula is shown as formula I, and the result is shown in FIG. 1.
Figure BDA0003962476610000041
In the formula: a is the extraction rate of the kudzu root extract; m is the mass/g of the kudzu root extract; m is the mass/g of the kudzu root powder.
As can be seen from A in FIG. 1, the extraction rate of the radix Puerariae extract increases with the increase of the extraction pressure, which indicates that the extraction pressure has an obvious influence on the extraction rate; when the extraction pressure is increased from 5MPa to 20MPa, the extraction rate is gradually reduced along with the rising trend of the extraction pressure and is stabilized to about 2.6 percent, most of volatile oil components in the kudzuvine root are probably extracted by fluid carbon dioxide under the pressure of 20MPa, and in order to save the cost and the using amount of the carbon dioxide, the extraction pressure of 20MPa is selected to be the best.
As shown in B in FIG. 1, the temperature was raised from 25 ℃ to 30 ℃ to cause CO to flow 2 The fluid can dissolve effective substances in radix Puerariae more easily and with CO 2 Entering a separator; the temperature is further increased, the extraction rate is not increased, but is gradually reduced. Probably because when the temperature reached 31.2 deg.C, CO was reached 2 The critical temperature of the carbon dioxide is converted into a supercritical fluid state, the density of the carbon dioxide is approximate to that of liquid, the viscosity of the carbon dioxide is approximate to that of gas, and the diffusion coefficient of the carbon dioxide is 100 times that of the liquid. Therefore, the extraction temperature is increased from 20 ℃ to 30 ℃, the extraction rate is greatly improved, the molecular motion of the solvent in the system is accelerated, the dissolution, permeation and diffusion speeds of the solvent are high, the extraction of effective volatile components of the kudzuvine root is facilitated, and the extraction rate is high. After further temperature increase, CO is reduced 2 The density of the fluid reduces the polarity, and the capacity of dissolving effective substances in the kudzuvine root is reduced, so that the extraction rate is not increased or reduced. Therefore, the extraction temperature is preferably selected to be 30 ℃.
Example 2 Anhydrous ethanol assisted supercritical CO 2 Single factor experiment of extraction process
1) Putting 400g of radix Puerariae powder prepared in example 1 into an extraction tank, adding 100mL of anhydrous ethanol, and setting the extraction temperature at 25 deg.C and CO 2 The flow is 0.8L/min, the separation pressure is 8MPa, the separation temperature is 45 ℃, the extraction time is 3h, and the extraction rates of the radix puerariae extract under the extraction pressures of 5MPa, 10MPa, 15MPa, 20MPa and 25MPa are respectively considered;
2) 400g of the kudzu root powder obtained in example 1 was put into an extraction tank, 100mL of anhydrous ethanol was added thereto, the extraction pressure was set at 15MPa, and CO was added thereto 2 The flow rate is 0.8L/min, the separation pressure is 8MPa, the separation temperature is 45 ℃, the extraction time is 3h, and the extraction at 20 ℃,30 ℃,40, 50 and 60 ℃ is respectively consideredThe extraction rate of the kudzu root extract at the temperature.
Filtering the obtained radix Puerariae extract with a filter, weighing, and calculating the extraction rate, wherein the calculation formula is shown in formula II, and the result is shown in FIG. 2.
Figure BDA0003962476610000051
In the formula: a is the extraction rate of the kudzu root extract; m is the mass/g of the kudzu root extract; m is the mass/g of the kudzu root powder.
As can be seen from FIG. 2, the extraction yield was increased as a whole up to 5.3% by using anhydrous ethanol as an entrainer. The reason is that with the addition of anhydrous ethanol, the CO fluid is changed 2 The polarity of the mixed solvent is improved, so that the polar flavonoid macromolecules can be dissolved in the mixed solvent, the compound types in the kudzu root extract are increased, and the kudzu root extract not only contains low-polarity micromolecules such as terpenes, lipids and the like, but also contains the flavonoid macromolecular compounds dissolved in ethanol.
Supercritical fluid CO 2 Is a hydrophobic, non-polar solvent, and therefore, small molecules with low polarity are easily dissolved therein. The polarity of the mixture can be controlled by adding a certain amount of polar cosolvent such as ethanol, so as to improve the solubility of the solute difficult to volatilize and the polar solute and improve the extraction rate.
Test example 1 measurement of Total Flavonoids content
400g of kudzu root powder of example 1 was placed in an extraction tank, and the extraction pressure was set at 15MPa 2 The flow rate is 0.8L/min, the separation pressure is 8MPa, the separation temperature is 45 ℃, the extraction time is 3h, and the extraction temperature is 30 ℃, so that the radix puerariae extract is recorded as PCE.
400g of kudzu root powder of example 1 was placed in an extraction tank, 100mL of absolute ethanol was added, extraction pressure was set at 15MPa, CO was added 2 The flow rate is 0.8L/min, the separation pressure is 8MPa, the separation temperature is 45 ℃, the extraction time is 3h, and the extraction temperature is 30 ℃, so that the obtained radix puerariae extract is marked as PAE.
1) Preparing rutin standard curve
Precisely weighing 2.5mg of Rutin standard substance dried to constant weight, dissolving in distilled water, diluting to a constant volume of 10mL, adding 60% ethanol by volume fraction, dissolving, diluting to a constant volume, and shaking to obtain a Rutin standard substance solution with a mass concentration of 0.25 mg/mL.
2) Color reaction
Precisely sucking 0, 0.2, 0.4, 0.8, 1.0 and 1.2mL of rutin standard substance solution into a 10mL volumetric flask, adding 30% ethanol solution to 2.4mL, and adding 5% NaNO 2 Shaking 0.4mL solution, standing for 5min, adding 10% Al (NO) 3 ) 3 0.7mL of solution is shaken up and kept stand for 5min, 4.0mL of 1mol/L NaOH solution is added, finally, the volume is determined to 10mL of scale mark by 30 percent of ethanol solution, shaken up and kept stand for 5min. Each group of concentrations was repeated 3 times.
3) Drawing a standard curve
The absorbance was measured at a wavelength of 510 nm. Taking the concentration (mug/mL) of the Rutin standard substance as the abscissa and the absorbance (Abs) as the ordinate, drawing a standard curve, as shown in FIG. 3, the Rutin standard curve equation is y =0.5956x +0.0278, R 2 =0.998, and the results of subsequent experiments were calculated according to this regression equation.
4) And (3) sample determination: taking supercritical CO 2 Extract (PCE) and ethanol assisted supercritical CO 2 The extract (PAE) was dissolved in 60% ethanol solution and tested, each sample was 3 parallel, the total flavone content in the sample was calculated according to the standard curve, the results are shown in Table 1.
TABLE 1PCE, PAE Total Flavonoids content
Sample (I) The content of total flavone is mg/g
PCE 1.52
PAE 25.24
As can be seen from Table 1, the total flavone content of PAE is as high as 25.24mg/g, which is much higher than that of PCE, probably because the addition of the anhydrous ethanol entrainer changes the polarity of the mixed solvent, increases the polarity, is beneficial to the dissolution of flavonoids, and makes the flavone content of PAE much higher than that of PCE.
Test example 2
0.5g of PCE sample is dissolved and diluted to 1mL by acetonitrile, 0.2g of PAE sample is dissolved and diluted to 1mL by acetonitrile, the mixed solution is respectively filtered by organic filter membranes with the pore diameter of 0.22 μm to be put into a sample bottle for GC-MS analysis, and the area normalization is used for calculating the content, and the results are shown in 2 and table 3.
GC-MS conditions
A chromatographic column: capillary chromatography (Thermo GR-5MS, 30m.times.0.25 mm.times.0.25 μm); the temperature of a sample inlet is 250 ℃; temperature rising procedure: initial temperature is 50 deg.C, keeping for 1min, heating from 50 deg.C to 200 deg.C at rate of 3 deg.C/min, and keeping for 5min; then increased from 200 ℃ to 280 ℃ at 20 ℃/min and held for 10min. The carrier gas is helium, the flow rate is 1.0mL/min, the split ratio is 60.
EI ionization energy: 70eV; ion source temperature: 250 ℃; the scanning mode is full scanning, and the scanning quality range is 50-450.
TABLE 2GC-MS analysis of PCE Compound composition
Figure BDA0003962476610000071
Figure BDA0003962476610000081
As can be seen from Table 2, the aroma components of the PCE detected by the gas chromatography-mass spectrometry combination instrument are 35 in total, wherein 10, 5, 1, 8, 5, 3 and 3 of hydrocarbons, acids, alcohols, lipids, aldehydes, ketones and other types are respectively detected, which occupy 71.36% of the PCE components, and the first ten components are ethyl linoleate, ethyl oleate, 4-isopropylbenzaldehyde, 4-isopropylcyclohexadene-1, 4-dienal, palmitic acid, pinene, gamma-terpinene, 2, 3-dihydrothiophene, dibutyl phthalate and ligustrazine.
TABLE 3GC-MS analysis of the composition of PAE compounds
Figure BDA0003962476610000082
Figure BDA0003962476610000091
Figure BDA0003962476610000101
From Table 3, it can be seen that supercritical CO with addition of absolute ethanol 2 The extracted substances are 43 kinds, and the quantity of the kinds is more than that of pure supercritical CO 2 Extracting, wherein the content of hydrocarbons, acids, alcohols, lipids, aldehydes, ketones and other species is respectively 9, 6, 1, 15, 4, 1 and 7, and the detected substances account for 72.35% of the PAE. The first ten main components are ethyl linoleate, 1, 3-butanediol, ethyl palmitate, 4-isopropylbenzaldehyde, 2, 3-dihydrothiophene, ethyl linolenate, 2, 5-isopropylbenzaldehyde, ligustrazine and beta-pinene.
Ethyl linoleate is a main compound of PCE and PAE, and the content of 4-isopropylbenzaldehyde, 2, 3-dihydrothiophene and ligustrazine in the PAE is obviously improved compared with that of PCE. Ethyl linoleate is a colorless to pale yellow oily liquid, has the effect of reducing cholesterol and blood fat in blood, is mainly used as a pharmaceutical raw material for preventing and treating atherosclerosis, is one of fatty acid ethyl esters, and more documents show that the fatty acid ethyl esters are toxic media of ethanol in vivo, such as pancreas, liver, heart and brain. Ethyl oleate is rapidly degraded in the digestive tract, so oral ingestion is safe. Oleic acid is a fatty acid naturally found in various animal and vegetable fats and oils, is a colorless, odorless oil, is used as an emulsifier in food and topical pharmaceutical preparations, can also be used as a transdermal enhancer, can be used to increase the bioavailability of poorly water-soluble drugs in tablets, and can be used as part of the matrix of soft capsules. 4-isopropylbenzaldehyde, 2, 3-dihydrothiophene are used widely as intermediates for medicine synthesis, the content of the two in PAE is increased, ligustrazine is an alkyl pyrazine extracted from Ligusticum wallichii, is often used as an essence in foods such as chips, bread, tea, dairy products, bean products and the like, is used for treating cardiovascular and cerebrovascular diseases for about 40 years, and is also used for treating various diseases such as coronary heart disease, diabetes, cancer and liver injury.
Application example 2 in vitro antioxidant experiment
1. Solution preparation
1) Preparation of DPPH solution (0.1 mmol/L): weighing 4mg to 100mL of absolute ethyl alcohol, and storing at room temperature in a dark place.
2) Ferrous sulfate solution (5 mmol/L): 66.8mg were weighed into 40mL of water.
3) Hydrogen peroxide solution (5 mmol/L): pipetting 18. Mu.L of 30% 2 O 2 Dissolved in 30mL of water.
4) Salicylic acid solution (5 mmol/L): 33.2mg was weighed out and dissolved in 40mL of absolute ethanol.
5) 0.2mM PBS (PH = 7.4): 716mg of dihydrate sodium dihydrogen phosphate is weighed and dissolved in 10mL of water, and the solution is marked as solution A; 312mg of disodium hydrogen phosphate dodecahydrate was weighed out and dissolved in 10mL of water, and the solution was designated as solution B. Prepared at a ratio of a: B =19, and diluted 1000-fold (0.1 mL of PBS +99.9mL of water).
2. Experimental methods
Measurement of DPPH radical scavenging ability
2mL of a sample solution at a concentration of 0.2, 0.4, 0.6, 0.8, 1.0mg/mL and 2mL of a DPPH solution (0.1 mmol/L) were added to a 10mL Ep tube, vortexed, shaken well, and stored at 37 ℃ for 30min in the dark. The absorbance value is determined at 517nm by taking absolute ethyl alcohol as a control and Vc as a positive control and taking the same concentration gradient with the sample liquid, and each test is carried out in parallel for 3 times. Clearance was calculated according to formula III:
Figure BDA0003962476610000111
in the formula: a. The 0 Absorbance of 2mL DPPH-ethanol solution +2mL absolute ethanol; a. The 1 The absorbance was 2mL of DPPH-ethanol solution +2mL of sample solution; a. The 2 The absorbance was 2mL of absolute ethanol +2mL of the sample solution.
2. Determination of the ability to scavenge hydroxyl radicals
2mL of FeSO was added to the tube 4 Solution (5 mmol/L), 2mLH 2 O 2 The solution (5 mmol/L), 2mL of Salicylic acid solution (5 mmol/L) and 2mL of samples with different concentrations (0-0.8 mg/mL) were reacted at 37 ℃ for 1h. Absorbance was measured at 517nm wavelength with distilled water as reference and Vc as positive control, and each test was performed in 3 replicates. The hydroxyl radical clearance calculation formula is the same as DPPH radical clearance (formula III), where: a. The 0 The absorbance is 2mL ferrous sulfate solution, 2mL hydrogen peroxide solution, 2mL salicylic acid and 2mL distilled water; a. The 1 The absorbance of the sample solution is 2mL of ferrous sulfate solution, 2mL of hydrogen peroxide solution, 2mL of salicylic acid and 2mL of sample solution; a. The 2 The absorbance of the sample solution is 2mL ferrous sulfate solution, 2mL distilled water, 2mL salicylic acid and 2 mL.
Determination of ABTS free radical scavenging Capacity
7.4mM ABTS solution and 2.6mM K were prepared 2 S 2 O 8 Mixing the solutions in equal volume, standing at room temperature in dark for 16h to form ABTS + And (4) stock solution. ABTS was diluted with 0.2M PBS (pH = 7.4) + The stock solution was such that its absorbance at 734nm was around 0.7 (error less than 0.2). 3mL of ABTS + The diluted solution was mixed with 1mL of sample solutions of different concentrations 0.2, 0.4, 0.6, 0.8, 1.0mg/mL, and the absorbance at 734nm was measured after reaction for 10min at room temperature in the dark. Vc was used as a positive control and repeated three times. The ABTS free radical scavenging clearance is the same as formula III, wherein: a. The 0 ABTS + diluent + water; a. The 1 ABTS + diluent + sample solution; a. The 2 PBS + sample solution.
3. Results of the experiment
The results are shown in FIG. 4, where A is scavenging DPPH free radicals, B is scavenging ABTS free radicals, and C is scavenging hydroxyl free radicals.
As can be known from A, the PCE and the PAE have stronger capacity of eliminating DPPH free radicals, the eliminating capacity of the PAE sample is gradually enhanced along with the increase of the concentration, and the eliminating capacity of the PAE sample is almost the same as that of ascorbic acid, probably because the flavone content is increased along with the addition of absolute ethyl alcohol, and the capacity of eliminating the free radicals is stronger; the clearance rate of PCE to DPPH free radical is increased gradually in a concentration-dependent mode and is equivalent to PAE, and when the concentration reaches 0.6mg/mL, the clearance rate is 83.42 +/-1.26%.
B, the capability of the kudzu root extract for eliminating ABTS free radicals is gradually increased in a concentration-dependent manner, wherein the PCE eliminating capability is obviously stronger than that of PAE, but is weaker than that of ascorbic acid, and the eliminating capability is 70-85% at the concentration of 1.0 mg/mL.
It can be known from C that the clearance rate of the kudzu root extract to hydroxyl free radicals reaches up to 60%, and compared with ascorbic acid, the clearance capacity of PCE and PAE gradually increases in a concentration-dependent manner.
The result of the in vitro antioxidant capacity shows that the kudzu root extract has strong capacity of removing free radicals, the antioxidant effect of PCE is better than that of PAE, and theoretical basis is provided for the following cell experiments.
Application example 1 intestinal epithelial barrier protection by aluminum
1. Solution preparation
Preparing a DMEM high-sugar culture medium: DMEM/High Glucose 44.9mL, fetal Bovine Serum (FBS) 5mL, streptomycin qing 100. Mu.L.
MTT solution
Accurately weighing 50.0mg MTT powder, dissolving with 10mL sterilized PBS, filtering with 0.22 μm water system membrane, removing impurities and bacteria in the solution, packaging, wrapping with tinfoil, and storing at 4 deg.C in dark place for two weeks; the temperature should be-20 ℃ for long-time storage, and repeated freeze thawing is avoided.
3.AlCl 3 And preparation of sample solution
AlCl 3 Stock solutions were prepared at 32mM in PBS and then diluted to 0, 1, 2, 4, 8 in PBS16mM working solution, and storing at 4 ℃ in the dark for later use.
The PCE is dissolved by an ethanol solution with the volume fraction of 60 percent of ethanol to prepare working solution with the concentration of 0, 25, 75, 125, 150, 175, 250 and 375 mu g/mL, and the working solution is stored at 4 ℃ in a dark place for later use.
Preparation of WB Experimental reagent
Preparing a TBST solution: diluting 20 × TBS solution 50mL with distilled water, diluting to constant volume of 1L, adding 1mL Tween-20, shaking thoroughly and shaking.
Preparation of 5 × electrophoretic solution: weighing 15.1g of Tris-base,94g of glycine and 5g of SDS,1L of distilled water, dissolving by a magnetic stirrer, and keeping at normal temperature for later use.
10 × preparation of membrane transfer liquid: 63.6g of Tris-base,146g of glycine and 1L of distilled water are weighed and dissolved by a magnetic stirrer, and the solution is reserved at normal temperature.
2. Experimental methods
HT-29 as a human colon cancer cell can form a compact monolayer membrane, simultaneously shows the similarity with a small intestine epithelial cell and can well simulate the intestinal epithelial barrier. Thus human colon cancer cells HT-29 were cultured as a cell model and AlCl was used 3 The extract can be used as inducer for stimulating oxidative stress of cells, so as to explore the protective effect of radix Puerariae extract.
HT-29 cell culture
1) Cell resuscitation
Taking out frozen HT-29 cells from a liquid nitrogen tank, placing the cells in a refrigerator at minus 80 ℃ for 30min, then taking out the cells from the refrigerator at minus 80 ℃, quickly placing the cells in a water bath at 37 ℃, continuously shaking the frozen tube by hands during the period to quickly thaw the frozen tube, then returning the frozen tube to a super clean bench, transferring the cell suspension into a 15mL centrifuge tube containing 5mL DMEM high sugar medium, centrifuging the cells at 1500r/min for 3min, discarding the supernatant, and then precipitating white cells at the bottom, and using 1mL new medium: containing a mixture of 10% Fetal Bovine Serum (FBS) and 1% streptomycin, the cells were resuspended again and pipetted into a petri dish containing complete medium.
2) Culture conditions
Following 1) the procedure, the petri dish was placed in an incubator at 37 ℃ and 5% CO 2 The culture medium contains 10% of fetal calfSerum (FBS) and 1% streptomycin in mixed solution in DMEM high-glucose medium, the medium was changed once a day.
3) Cell counting
Because HT-29 is adherent cells, firstly digesting the cells by pancreatin, then adding 1mL of fresh culture medium to stop digestion, centrifuging at 1500r/min for 3min, discarding supernatant, adding 1mL of fresh culture medium, gently blowing and mixing by using a 1mL pipette to resuspend the cells, mixing proper cell suspension with trypan blue solution according to the proportion of 1, taking 10 mu L of mixed solution, adding the mixed solution onto a cell counting plate for counting, as shown in formula IV,
Figure BDA0003962476610000131
4) Cell passage
The original old culture medium is removed by suction, the original culture medium is washed twice by PBS, the original culture medium is thoroughly washed, 1mL of pancreatin is added into each dish for digestion, the culture dish is placed back into the incubator for 2-3 min, then 1mL of fresh culture medium is added to stop digestion, the cells are blown down by a pipette, the cell suspension is transferred into a 15mL centrifuge tube, the centrifuge is carried out at 1500r/min for 3min, the supernatant is discarded, the cells are resuspended by 1mL of fresh culture medium, and the cell suspension is transferred into the fresh culture medium according to the proportion of 1.HT-29 cells require medium replacement once a day.
5) Cell cryopreservation
Dimethyl sulfoxide (DMSO) was mixed with the prepared culture medium at a ratio of 1.HT-29 cells are digested by pancreatin, then the digestion is stopped by a culture medium, the cells are transferred into a 15mL centrifuge tube, then centrifugation is carried out for 3min at 1500r/min, supernatant is discarded, 1mL complete culture medium is used for re-suspending the cells, cell sap and a prepared DMSO solution are mixed according to the ratio of 1.
2, MTT method for detecting toxicity of kudzu root extract on cells
1) Seeding cells
Taking a 96-well plate, adding 200 μ L of 5 × 10 4 cell/mL cell suspension, the number of cells in each well of the plate is 10 4 In addition, a well to which only the medium was added was reserved in advance as a blank, and appropriate duplicate wells were set. After the cells were added, the plate was gently shaken by the cross shaking method to make the cells uniformly distributed, 200. Mu.L of PBS was added to the wells around one week to eliminate the edge effect, the cells were observed under a microscope, and the cells were placed in an incubator at 37 ℃ and 5% CO 2 Overnight.
2) Adding a sample to be tested
Dissolving PCE with 60% ethanol, preparing corresponding concentration, adding samples into 96-well plate to make final concentration of 0.05, 0.1, 0.2, 0.4, 0.8, 1.0, 1.6 μ g/mL and 0.25, 0.75, 1.25, 1.5, 1.75, 2.5, 3.125, 3.75 μ g/mL, adding equal amount of complete culture medium into blank control group, and culturing for 4h.
3) Addition of MTT
MTT was dissolved in a sterilized PBS solution to prepare a 5mg/mL PBS solution containing MTT, 20. Mu.L of the solution was added to each well, and the mixture was incubated in an incubator for 4 hours.
4) Adding DMSO
Absorbing the original culture medium, adding 200 μ L DMSO to dissolve the precipitate, and standing in incubator for 15min or at room temperature for 30min to dissolve the blue precipitate completely.
5) Detection of absorbance by enzyme-linked immunosorbent assay
Putting the 96-well plate into an enzyme-labeling instrument, detecting the absorbance at 570nm, taking the hole only added with the culture medium as a blank control, and obtaining the formula shown in the formula V,
Figure BDA0003962476610000151
3. establishing a cell inflammation model
First, alCl is measured 3 And (5) toxicity, and establishing a cell oxidative stress model.AlCl was calculated from the median inhibitory concentration 3 Half inhibitory concentration of (a).
Median Inhibitory Concentration (IC) 50 ) The calculation method is an improved Kouyan method calculation, the formula is shown as a formula VI,
lgIC 50 ＝X m -I(P-(3-P m -P n ) 4) formula VI, wherein the formula is as follows,
wherein I-lg (maximum dose/adjacent dose), xm-lg maximum dose, the sum of P-positive reaction rates, pm-maximum positive reaction rate, pn-minimum positive reaction rate.
PCE antagonistic AlCl 3 Experiment of
And dissolving the PCE in 60% ethanol to prepare a solution for later use. Cells were treated at 5X 10 8 The density of (2) was inoculated in a 6-well plate, PCE was added to make the final concentrations 0.8. Mu.g/mL and 0.25. Mu.g/mL, respectively, when the cells were plated in a six-well plate, and a blank (60% ethanol) and a control (AlCl) were set separately 3 Intervention).
5. Establishment and evaluation of cell tight junction model
The monolayer of intestinal epithelium forms an intestinal epithelial barrier that controls the transport of molecules through transcellular and paracellular pathways. The tight junction barrier, advocated for its impaired or leaky function, increases the penetration of luminal antigens, toxins and bacteria into the blood, and mimics the integrity of the intestinal barrier in vitro cell experiments, usually in a manner that measures transepithelial resistance and cell monolayer passage rate, the HT-29 cell line being a human colon cancer cell, structurally and functionally similar to a differentiated small intestine epithelial cell. Morphologically similar to small intestinal epithelial cells and with the same polarity and intercellular tight junctions, HT-29 cells were used as an in vitro intestinal barrier model.
Establishing a tight connection model: plasma cells at 8X 10 4 The density per cell was determined by inoculating the cells at a pore size of 0.4 μm and a membrane area of 0.33cm 2 The Transwell plates of (1) were plated with 600. Mu.L of DMEM medium in the BL layer outside the chamber and 10. Mu.L of cell suspension in the AP layer inside the chamber, and the plating solution was changed every other day for the first week after inoculation and for the next day for up to 21 days after the start of the second week. After the cells are differentiated for 21 days to establish a cell tight junction model,serum-free culture for 24h, pre-treatment with 2. Mu.L of PCE in AP layer of Transwell chamber for 4h, and addition of 6mM AlCl 3 And (3) processing, wherein the resistance value change of the cell is measured after processing for 2h, 4h, 8h, 12h, 24h and 48h respectively. Before measurement, the electrodes of the instrument are soaked in 75% alcohol for 15min and then placed in sterilized PBS for later use. During measurement, the culture plate is firstly placed in a super clean platform for balance for half an hour to ensure the stability of the numerical value, and then the resistance values of the small chambers in different three directions are measured, and the average value is taken.
6. Cell monolayer passage rate
Measurement of the passage rate of FITC-dextran (FITC-dextran) which is a fluorescent macromolecular substance through monolayer cells was used to evaluate the permeability of the monolayer cells. After 21 days of HT-29 cell differentiation, the Transwell chamber was gently rinsed 2 times with 37 ℃ PBS solution, then 0.1mg/mL FITC 100. Mu.L was added to the AP layer of the chamber, 600. Mu.L PBS buffer was added to the BL layer, and the cell culture plate was placed in an incubator at 37 ℃,5% CO 2 And incubating for 1h, respectively taking 100 mu L of liquid from the BL layer and the AP layer of the Transwell, calculating absorbance (wherein the excitation wavelength is set to 480nm, and the emission wavelength is set to 520 nm) by using a multifunctional microplate reader, and calculating the permeability rate of FITC and the apparent permeability coefficient of transmembrane transport (Papp), wherein the calculation formula is shown as formula VII.
Papp＝ΔQ/(Δt*A*C 0 )(cm*s -1 ) A compound of the formula VII,
wherein Δ Q is a transport amount of Δ t; a is the membrane area; c 0 The initial concentration of AP layer in HT-29 cells.
7. Cellular reactive oxygen species production
Oxidative stress refers to a state in which oxidation and reduction are unbalanced in the body, resulting in insufficient antioxidant capacity of cells, an increase in cytotoxic Reactive Oxygen Species (ROS) in cells, generation of oxygen radicals, and attack of biological macromolecules, thereby causing damage to cells and tissues, and is considered as an important index for aging and diseases. Thus, the present invention uses 2',7' -dichlorodihydrofluorescein diacetate (DCFH-DA, sigma-Aldrich) to detect aluminum-induced levels of cellular Reactive Oxygen Species (ROS) production.
1) Stock solutions of 1-10 mM DMSO were prepared, and unused stock solutions of DMSO were aliquoted into single use vials and stored at-20 ℃ protected from light.
2) The working concentration of the dye was 1-10. Mu.M in physiological buffer PBS.
3) HT-29 cells were cultured in complete medium at 4X 10 4 The cells were seeded at a density in a six-well plate, placed in an incubator, and the state of the cells was observed at that time, and when HT-29 was 80% at a density in the six-well plate, the next operation was performed.
4) And (4) replacing with a fresh DMEM medium, and adding PCE for pretreatment for 4h.
5) Discarding the original medium, washing twice with PBS, adding fresh DMEM medium, adding AlCl to the final concentration of 6mM 3 The solution was subjected to an intervention for 8h.
6) The original medium was discarded, the medium was washed clean with incubated PBS, 1mM DCFH-DA was added and placed in the incubator for 30min.
7) Washing twice with PBS at 4 deg.c to stop staining, adding PBS in corresponding volume, and incubating in culture box for 40 min.
8) The original PBS was discarded, and the fluorescence intensity of the cells was measured using a Radiance 2100 Fluoromicroplate reader (Bio-Rad, hercules, calif., USA) at an emission wavelength of Ex/Em =485nm/535 nm.
8.RT-PCR
1) Extraction of RNA
HT-29 cell pretreatment: taking out the six-hole plate from the incubator, using a pipette to suck and remove the original culture medium, then using a 1mL liquid transfer gun to respectively suck 1mL PBS in the six holes, washing the original culture medium completely, then adding 1mL pancreatin into each dish, placing the culture medium in the incubator to incubate for 3min, taking out the culture medium to a clean bench, adding 1mL complete culture medium to stop digestion, using the liquid transfer gun to blow down adherent cells, transferring the cell suspension into a 1.5mL centrifuge tube, and centrifuging at 1500r/min for 3 min.
a. Cell lysis: in a 1.5mL Ep tube, 1mL of the trizol solution was added, and after standing on ice for 15min, the next step was performed.
b. Chloroform: adding 200 mu L of trichloromethane, rapidly and violently shaking for 15-30 s by a vortex shaking instrument, and standing for 10min on ice.
c. Centrifuging and layering: centrifuging in a high-speed centrifuge: 12000r/min,4 ℃,15min, and after completion, the liquid in the Ep tube clearly separated into three layers, a lower pink organic phase, a thin white protein layer, and an upper clear aqueous phase with RNA dissolved.
d. The upper clear aqueous phase was carefully and slowly transferred to a new Ep tube with a Non-clean tip, without aspirating intermediate proteins, and 500. Mu.L of isoproyl alcohol (guaranteed purity) was added, gently mixed and allowed to stand at room temperature for 10min.
e. Upon centrifugation again under the same conditions, a thin white precipitate, which is the extracted RNA, was clearly observed at the bottom of the Ep tube.
f. Discarding the supernatant, washing RNA precipitate with 75% ethanol prepared from 1mL of DEPC water, washing the previous organic solution as clean as possible, centrifuging at 10000r/min,4 ℃,5min, and repeating the operation for two to three times.
g. Absorbing the supernatant as much as possible, drying the inner chamber of the super clean workbench with warm air or putting the super clean workbench into an incubator at 37 ℃ for drying, and enabling the white precipitate to become transparent after about 20 min.
h. Visually observing the amount of the white RNA precipitate, dissolving the precipitate with an appropriate amount (10-30 mu L) of DEPC water, then placing the RNA in a constant-temperature metal bath at 55 ℃, and heating for 10min to promote the dissolution of the RNA.
i. Taking DEPC water for dissolving RNA as a blank control, adding 1 mu L of sample on a Nano Drop instrument to directly measure the concentration of the RNA and the value of A260/280, wherein the value of A260/280 is between 1.8 and 2.0, otherwise, protein and reagent pollution or RNA degradation possibly exists to influence the extraction quality, taking 1 mu g of RNA for reverse transcription, and storing the rest sample at-80 ℃ for later use.
2) Reverse transcription reaction
The experiment was carried out according to the method recommended by the product manual of reverse transcription kit of Roche, and the reaction system was as follows: mgCl 2 -4μL；10×buffer-2μL；RNAsin-0.5μL；10mM dNTP-2μL；Random primer-1μL；Rtranscriptase-0.5μL；total RNA-1μg；RNAFree H 2 O-Make up to 20 μ L. Reaction conditions are as follows: 25-10 min; 50-60 min; 85-5 min;4 ℃ for an infinite time.
And carrying out reverse transcription on the extracted RNA according to a Roche reverse transcription kit to obtain a product cDNA, measuring the cDNA concentration by ultra-trace, diluting to 100 ng/mu L according to the requirement of a PCR reaction system, and storing in a refrigerator at the temperature of-20 ℃.
3) PCR reaction system
Performing RT-PCR reaction by using a Roche PCR kit, wherein the reaction system comprises the following steps: 2 μ L of cDNA; 1 mu L of upstream primer; 1 μ L of downstream primer; h 2 O-6 mu L;2 × SYBR Green-10 μ L; the total volume of the reaction is-20 mu L;
the PT-PCR reaction was performed on Roche LC96 with the following reaction program:
pre-denaturation at 95 ℃ for 10min; (denaturation at 95 ℃ for 15s, extension at 60 ℃ for 1 min) 40 cycles.
GAPDH is used as an internal reference, each sample is provided with 3 multiple holes, a con group, an Al group and a PCE group, and the average value is calculated to be used as an RQ value. And determining corresponding primers according to experimental design, adding the primers into the 8 connecting pipes, and paying attention to the identification direction. After each PCR reaction, melt cut analysis was performed to confirm the specificity of the amplified product.
According to the PCR instruction and the related requirements of the instrument, the PCR system is completed, wherein the PCR system is pre-denatured at 95 ℃ for 30s, denatured at 95 ℃ for 5s, and extended at 60 ℃ for 20s, and the PCR system is cycled for 40 times. After completion, the mixture was put in an LC96 PCR instrument for real-time quantitative PCR.
8.Western-Blot
1) And (3) cleaning the glass plate: firstly, the glass clamping plate is washed clean by tap water, residual water stains are removed by lens wiping paper, the glass clamping plate is particularly noted that residual dirt does not exist in the gap between the glass clamping plate and the glass clamping plate, and then the glass clamping plate is washed clean by distilled water and placed on a support to be naturally dried or placed in a constant-temperature drying box to be dried.
2) Leak detection is then performed: the two glass plates are fixed by a clamping plate and are arranged on a glue distribution bracket. Firstly, injecting distilled water into the two plates, observing whether the liquid level of the upper layer drops too much, pouring the water out if the leakage detection is successful, and sucking the water by using filter paper.
3) Preparing glue: separating glue (15%)
Sample adding sequence: tris-buffer 1.5M PH 8.8dd H2O 30% acrylamide 10% SDS 10% ammonium persulfate TEMED (both produce cement, should pour cement immediately).
4) Glue pouring: and after the glue leakage is determined, sucking and separating the glue solution by using a 1mL liquid transfer gun, adding the glue along one side of the glass plate, and stopping filling the separation glue until the glue surface rises to the height of 1cm below the comb without worrying about bubbles.
5) Pressing rubber: and a layer of isopropanol is injected on the upper layer of the separation gel, and the function is to press the plane of the separation gel straight and drive out the generated bubbles. And after the separation gel is solidified, the time is 30-50 min, the isopropanol on the upper layer of the gel is discarded, the gel is turned over and kept stand for 5min, and the isopropanol is volatilized.
6) Preparing concentrated glue, adding the concentrated glue until the concentrated glue overflows from the glass plate, inserting a comb, avoiding bubbles, and standing for 30-50 min.
7) Lifting the comb: after the concentrated gel is solidified, the comb is vertically and slightly pulled out upwards by pinching with two hands without shaking left and right.
8) Installing a rubber running plate: the glue was mounted on glue running clamps with the small glass plate facing inwards and the large glass plate facing outwards, and 1 running buffer was injected (this process can check for leakage from the splint).
9) Loading: the sample was taken out of the-20 ℃ refrigerator, the metal plate was heated, and the protein sample was shaken up with a vortex shaker. The sample is not required to be loaded too fast, so that the sample is easy to be punched out of the sample loading hole.
10 Electrophoresis: the sample is aligned by using 90V voltage, when the sample is run to the separation gel, the voltage is changed to 120V, electrophoresis is stopped until the sample just runs out of the glass clamping plate, and the next operation is carried out.
11 Film transfer: after electrophoresis is finished, separating gel near a protein sample with a proper molecular weight is taken down and soaked in a membrane transferring solution, a PVDF membrane with a proper size with a colloid is cut off, the PVDF membrane is placed in fresh methanol for soaking for 1.5min, the membrane is placed in the membrane transferring solution, a black plate of a splint faces downwards, a white plate faces upwards, sponge-filter paper-glue-membrane-filter paper-sponge (black glue white membrane) are sequentially arranged from bottom to top, bubbles are pressed out, the membrane transferring solution is placed in a membrane transferring groove, current is switched on for 200mA in a low-temperature environment, 90min is carried out, and whether bubbles are produced or not is observed.
12 Sealing: blocking with 5% skimmed milk or BSA blocking solution prepared from TBS + Tween-20 for 1h, and preparing the blocking solution as it is.
13 Hatch primary antibody: according to the size of the membrane and the antibody specification, 5% of skimmed milk or antibody diluent is used for diluting the primary antibody according to a certain proportion, the primary antibody is prepared at present, and the shaking table is used overnight at 4 ℃.
14 Hatch secondary antibody: the membrane was removed, allowed to equilibrate in the chamber for 30min, and then eluted with 1 × tbst for 5min, which was repeated three times.
15 Secondary antibody for washing: the secondary antibody was diluted with 5% skim milk at a certain ratio, incubated for 1h, and then eluted with 1 × tbst for 5min, and this operation was repeated three times.
16 Color development): ECL color development, exposure.
3. Results of the experiment
Cytotoxicity of PCE
According to the in vitro antioxidant result, selecting the radix puerariae extract PCE to carry out a cell experiment, and carrying out a cytotoxicity experiment on different concentrations of PCE in order to determine that the protection effect of the radix puerariae extract on cells is carried out under the condition that the radix puerariae extract does not inhibit the cells and ensure that the subsequent experiments do not have the condition that the cell number is reduced caused by the PCE. The number of cells after PCE acts on HT-29 cells for 24h under different concentrations is analyzed by MTT method, and the experimental result is shown in FIG. 5.
From the results of MTT experiments, it can be seen that when the PCE concentration is 0.25-3.75 mug/mL, the cell survival rate is not reduced, and the growth of the cells is promoted to a certain extent. Thus within a suitable concentration range, PCE is not toxic to cells.
2.AlCl 3 Cell toxicity of (2)
Aluminum is the most common and widespread metal element in the environment, and aluminum compounds are widely used in food additives, antacids, medicines, food packaging, cooking utensils, and the like. The mode of taking in the aluminum is mainly through food and water. Excessive intake of aluminum can accumulate in the intestinal tract, causing damage to intestinal tissue. Thus to AlCl 3 Performing cytotoxicity test, and determining AlCl by using median inhibitory concentration formula 3 Concentration, FIG. 6 shows AlCl concentrations 3 Survival of treated cells (compare to group 0, "-" indicates P<0.05, ". Indicates P<0.01, ". Indicates P<0.001, ". Indicates P<0.0001，Mean±SD，n＝5)。
As can be seen from FIG. 6, alCl was observed in the MTT test 3 At a concentration of 4mM, cell viability was not affected compared to control (PBS). High AlCl concentration compared to control (PBS) 3 Cell viability was significantly reduced with treatment (8-16 mM). Calculating AlCl after calculating by a median inhibitory concentration formula 3 Is 6mM, so that AlCl is selected at a concentration of 6mM 3 And (5) establishing a cell inflammation model.
PCE antagonistic experiment results
Taking the cell survival rate as an index, adding PCE with different concentrations to pretreat the cells for 4h, and then adding 6mM AlCl 3 Acting for 8h, adding MTT to determine cell survival rate, determining the optimal PCE antagonistic concentration for subsequent experiments, and FIG. 7 shows PCE antagonistic AlCl 3 Stimulation of HT-29 cell survival (compared to group 0, "+" indicates P)<0.05, ". Indicates P<0.01, ". Indicates P<0.001, ". Indicates P<0.0001，Mean±SD，n＝5)。
As can be seen from FIG. 7, when the AlCl is intervened by PCE with a concentration of 0.25. Mu.g/mL 3 The cell survival rate is higher when the strain is oxidized than when only AlCl is added 3 Stimulated survival rates showed significant differences (P)<0.0001 Comparable to the control group). Therefore, the PCE of 0.25 mug/mL is selected for subsequent experiments by the invention.
Protection of a monolayer of cell membranes by PCE
TER is an indicator used to assess the permeability of the intestinal epithelial barrier to ions. Generally speaking, a method for simulating an in vitro intestinal epithelial barrier by forming a compact monolayer cell barrier by cells is adopted, and the cell layer is sparser as the resistance value is smaller; conversely, the more intact the cell layer. FIG. 8 shows PCE antagonizing AlCl 3 The change in TEER values by stimulation of HT-29.
As can be seen from fig. 8, the TER value of the Al group started to significantly decrease after 8h of aluminum stimulation compared to the control group, while the group pretreated with the PCE group for 4h could significantly inhibit the decrease of the TER value; the PCE has a quite good effect on the protection of a monolayer cell membrane within 24h of aluminum stimulation. Therefore, when aluminum stimulation can damage the monolayer dense barrier of the cells, the resistance value is reduced, and the PCE pretreatment group can obviously inhibit the reduction of the TER value caused by the aluminum stimulation, which indicates that the PCE has a protection effect on the intestinal epithelial monolayer barrier.
PCE reduction of cell monolayer transmission
Defective intestinal epithelial cell barriers are an important part of the intestinal barrier dysfunction, and macromolecular toxins, bacteria and the like easily penetrate through the intestinal wall to cause inflammatory reactions, multiple organ dysfunction syndromes and the like. The barrier function of the intestinal epithelium is closely related to the complete tight connection of the intestinal epithelium, and once the protein of the epithelium is apoptotic and aged, the tight connection is lost or mutated, the intercellular space is enlarged, the permeability is increased, and the barrier function of the intestinal tract is damaged.
In the experiment, HT-29 cells are cultured for 21 days to form a compact single-layer membrane, then are cultured in the serum-free mode for 24 hours, PCE is added for pretreatment for 4 hours, and AlCl with the final concentration of 6mM is added 3 Treating for 24h, and directly adding AlCl into the model group 3 And treating for 24h. After 24 hours, 600. Mu.L of PBS buffer preheated to 37 ℃ in advance was added to the BP layer, 1mg/mL of FITC-dextran diluted with PBS buffer was added to the AP layer, and the mixture was put in an incubator for 1 hour, and then 100. Mu.L of the solution was aspirated from the AP layer and BP layer, respectively, and the solution was added to a completely black 96-well plate, and the absorbance was measured.
FIG. 9 shows PCE antagonizing AlCl 3 The monolayer barrier permeability change upon stimulation of HT-29 (compared to the Al group, "+" indicates P)<0.05, ". Indicates P<0.01, ". Indicates P<0.001, ". Indicates P<0.0001, mean ± SD, n = 3). As can be seen from FIG. 9, only AlCl was added 3 The stimulated group can increase the permeability of the cells, while the group with PCE pretreatment 4,h can significantly inhibit the increase of the permeability of the cells. Therefore, the PCE can protect the single-layer membrane barrier of the cell from being damaged and has a certain protection effect on the membrane barrier.
6. Cellular ROS production results
The metal ions are important inducers of in vivo oxidative stress, which is an imbalance state of in vivo oxidation and oxidation resistance, tends to oxidize, generates ROS reactive oxygen species including hydroxyl radicals, oxygen anions, hydrogen peroxide and other free radicals, directly or indirectly oxidizes and damages DNA, proteins, lipids and the like, and is an important inducer of cell aging and death.
Oxidative stress activates the production of ROS reactive oxygen species, often associated with dysbiosis due to incomplete reduction of oxygen. Furthermore, ROS-induced oxidative stress increases the inflammatory response, possibly through a positive feedback mechanism, leading to a continued increase in ROS production and subsequent tissue damage and synthesis of inflammatory proteins. It was therefore examined whether PCE pretreatment could reduce aluminum-induced ROS accumulation in HT-29 cells. FIG. 10 shows PCE antagonizing AlCl 3 Oxidative stress by stimulation of HT-29 (in comparison with the Al group, "+" indicates P)<0.05, ". Indicates P<0.01, ". Indicates P<0.001, ". Indicates P<0.0001, mean ± SD, n = 3). Intracellular ROS levels of Al group were significantly higher than those of Control group, while PCE group was significantly lower than those of Al group (P)<0.01). It can be seen that PCE pretreatment can significantly inhibit the accumulation of ROS in HT-29 cells.
PCE reduces the expression of inflammatory factors in HT-29 cells
FIG. 11 shows PCE processing to suppress AlCl 3 Stimulation of TNF-alpha and IL-1 beta expression levels by HT-29, FIG. 12 shows PCE treatment inhibits AlCl 3 Expression level of P65 by stimulation of HT-29 (": compare with Al group," - "indicates P<0.05, ". Indicates P<0.01, ". Indicates P<0.001, ". Indicates P<0.0001，Mean±SD，n＝3)。
As can be seen in fig. 11, aluminum stimulation can cause intestinal barrier dysfunction by causing the production of inflammatory cytokines, especially TNF- α, in human colonic epithelial cells. After PCE treatment, the expression levels of TNF-alpha and IL-1 beta in the cells are detected by RT-PCR, and the expression levels of TNF-alpha and IL-1 beta are found to be remarkably improved after aluminum stimulation of the cells, while the expression levels of TNF-alpha are remarkably reduced after PCE treatment.
As can be seen from FIG. 12, under the stimulation of Al, the P65 component in the NF- κ B signal pathway is activated, while PCE (P < 0.01) can obviously reduce the activation of P65, which indicates that the kudzu root extract can indeed regulate the NF- κ B pathway to inhibit the intestinal epithelial injury caused by Al.
PCE regulates expression of tight junctions
The tight junction is mainly located at the top of epithelial cells and comprises Occupudin, claudin-4 protein and the like, wherein Occupudin is a membrane expansion protein, the abnormal expression of claudin-4 is closely related to the occurrence and development of malignant tumors and various diseases, and the final result of the abnormal expression of the two proteins is to increase the permeability of intestinal mucosa and influence the barrier function of intestinal tracts. Previous researches show that inflammatory substances, intestinal microorganisms, cytokines and the like can cause the abnormal expression of the tight junction proteins such as Occludin, claudin-4 and the like. Therefore, occludin and Claudin-4 are selected as observation indexes in the research.
Figure 13 is HT-29Occludin, claudin-4 mRNA expression level, figure 14 is HT-29 tight junction protein Occludin, claudin-4 expression level (": compare with the group con,", represents P <0.05, ", represents P <0.01,", represents P <0.001, ", represents P <0.0001, mean + -SD, n = 3). To further confirm this phenomenon, it was further confirmed at the protein level, based on the demonstration that Occludin and Claudin-4 could be upregulated by PCE at the gene level. The expression of the claudin is detected by using a western blotting method. The results show that the tight junction protein of the aluminum group is obviously down-regulated; the addition of PCE may inhibit the down-regulation of the expression of claudin. This result is consistent with gene level assays, both of which demonstrate the regulatory effect of PCE on tightly linked molecules.
The removal capacity of the PCE, PAE of the present invention for DPPH, OH, and ABTS radicals increases in a concentration-dependent manner.
The method performs a cell toxicity test on the PCE to ensure that the PCE does not generate inhibition effect on cells under corresponding concentration; establishing Al oxidative stress in vitro intestinal tract cell model and determining AlCl 3 Has a median inhibitory concentration of 6mM; finally, an antagonistic experiment is carried out, and the optimal protective dose of the PCE is determined to be 0.25 mu g/mL.
The TEER test and the cell permeability test of the invention are important indexes for detecting whether the in vitro intestinal epithelial single-layer barrier integrity of cell simulation is damaged, and the experimental result preliminarily shows that the PCE can inhibit the damage to the intestinal epithelial barrier integrity caused by aluminum stimulation and has the potential of protecting the integrity. The PCE can reduce the accumulation of ROS reactive oxygen species and slow down cell aging and death.
According to the invention, the expression conditions of the tight junction protein and the inflammatory factor are explored through RT-PCR, and then Western-Blot is used for verifying the PCR results of the tight junction proteins Occludin and claudin-4, so that PCE can be further proved to reverse the down regulation of the tight junction protein caused by aluminum stimulation, the expression of P65 protein in an NF-kB metabolic pathway is reduced, and the result shows that PCE can inhibit the activation of P65 molecules.
The invention is to supercritical CO 2 The extraction is optimized, the extraction rate of the radix puerariae extract is improved, the content of total flavonoids in PAE is far higher than that of PCE, and the variety and content of compounds in PAE are increased through GC-MS analysis. The in vitro antioxidant result shows that the PCE and the PAE have strong free radical scavenging capacity. By establishing AlCl 3 The oxidative stress cell model experiment carries out PCE antioxidant stress and intestinal epithelial barrier research, and shows that the kudzu root extract can protect the integrity of the intestinal epithelial barrier caused by aluminum stimulation.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive work according to the embodiments of the present invention, and the embodiments are within the scope of the present invention.

Claims (10)

1. Use of a pueraria lobata extract for the preparation of a medicament for treating or preventing gastrointestinal disorders caused by aluminum stimulation.
2. The use as claimed in claim 1, wherein the pueraria lobata extract is prepared by extracting pueraria lobata powder with supercritical carbon dioxide.
3. Use according to claim 2, wherein the temperature of the supercritical carbon dioxide extraction is 20-40 ℃.
4. Use according to claim 2, wherein the pressure of the supercritical carbon dioxide extraction is 20 to 25MPa.
5. Use according to claim 2, wherein the supercritical carbon dioxide extracted CO 2 The flow rate is 0.6-1L/min.
6. Use according to claim 2, wherein the separation pressure of the supercritical carbon dioxide extraction is 6 to 10Mpa.
7. Use according to claim 2, wherein the separation temperature of the supercritical carbon dioxide extraction is 40-50 ℃.
8. Use according to claim 2, wherein the supercritical carbon dioxide extraction is carried out for an extraction time of 2 to 4 hours.
9. The use of claim 2, wherein the supercritical carbon dioxide extraction further comprises adding anhydrous ethanol.
10. The use as claimed in claim 9, wherein the dosage ratio of the kudzu root powder to the absolute ethyl alcohol is 400g.
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