CN111973580A - Application of caffeic acid in preparation of medicines for promoting glucose absorption - Google Patents

Abstract

The invention belongs to the technical field of medical application, and particularly relates to a new application of caffeic acid in preparing a medicament for promoting glucose absorption. The invention discovers a new application of caffeic acid through experiments, wherein the caffeic acid can promote the absorption of normal Caco-2 cells to glucose and can promote Caco-2 cells to take up a glucose tracer 2-NBDG, and the action mechanism of the caffeic acid can obviously promote the expression of SGLT1 and GLUT2 proteins and the transcription of mRNA. Therefore, caffeic acid can be used as a medicine for promoting glucose absorption of Caco-2 cells, namely, can be used for preparing a medicine for glucose absorption of small intestine epithelial cells.

Description

Application of caffeic acid in preparation of medicines for promoting glucose absorption

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a new application of caffeic acid in preparation of a medicine for promoting glucose absorption.

Background

Caffeic acid (English name: caffic acid), the structural formula is shown as follows:

the molecular formula is as follows: c₉H₈O4；

Molecular weight: 180.15, respectively;

the characteristics are as follows: yellow crystalline powder;

the source is as follows: caffeic acid can be purchased directly from common commercial products or extracted from flos Plumbaginis Zeylanicae according to the methods in the prior art.

Caffeic acid is one of active ingredients extracted from flos Plumbaginis Zeylanicae, and its pharmacological activity mainly includes antiinflammatory, blood lipid regulating, antioxidant and antidepressant effects; however, the effect of promoting Caco-2 glucose absorption is not reported. The application takes Caco-2 cells as a research object, discusses the influence of caffeic acid on glucose uptake and consumption of the Caco-2 cells, and explores the action mechanism of the caffeic acid.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a new application of caffeic acid in preparing a medicament for promoting glucose absorption.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of caffeic acid in preparing medicine for promoting glucose absorption is provided. Caffeic acid can be used as medicine for promoting glucose absorption of Caco-2 cells, namely, medicine for promoting glucose absorption of small intestine epithelial cells to promote nutrient absorption.

Specifically, the caffeic acid can remarkably promote the absorption of normal Caco-2 cells to glucose and can promote the Caco-2 cells to take up the glucose tracer 2-NBDG; the mechanism of the protein can play a role in promoting the glucose absorption of Caco-2 cells by promoting the expression of SGLTI and GLUT2 proteins and the transcription of mRNA of Caco-2 cell transporters.

The application and the caffeic acid can further remarkably promote the expression of SGLT1 and GLUT2 proteins and the transcription of mRNA.

The invention also provides a compound preparation which is prepared by compounding the caffeic acid and conventional auxiliary materials in the field and has the function of promoting glucose absorption.

Specifically, the compound preparation can be prepared into tablets, granules, pills, capsules, injections and the like.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, a new application of the compound caffeic acid and quercetin-3-O-beta-D-galactoside is discovered through a large number of researches and experiments, the compound caffeic acid and quercetin-3-O-beta-D-galactoside can obviously promote the absorption of normal Caco-2 cells to glucose and the uptake of a glucose tracer 2-NBDG by Caco-2 cells, and the action mechanism of the compound caffeic acid and quercetin-3-O-beta-D-galactoside can obviously promote the expression of SGLT1 and GLUT2 proteins and the transcription of mRNA. Therefore, caffeic acid and quercetin-3-O-beta-D-galactoside can be used for preparing a medicament for promoting glucose absorption of Caco-2 cells, namely can be used for preparing a medicament for promoting glucose absorption.

Drawings

FIG. 1 shows the values of transmembrane resistance of Caco-2 cell monolayers;

FIG. 2 is a graph of the effect of the compounds kaempferol (A), caffeic acid (B) and quercetin-3-O- β -D-galactoside (C) on normal Caco-2 glucose uptake and consumption; note: and blank group^***P<0.001；

FIG. 3 shows that kaempferol, caffeic acid, and quercetin-3-O-beta-D-galactoside, compounds promote uptake of glucose tracer 2-NBDG by Caco-2 cells; in the figure, (A) blank group, (B) 200. mu.M kaempferol, (C) 100. mu.M kaempferol, (D) 50. mu.M kaempferol; (E) 200. mu.M caffeic acid, (F) 100. mu.M caffeic acid, (G) 50. mu.M caffeic acid; (H)200 μ M quercetin-3-O- β -D-lactoside, (I)100 μ M quercetin-3-O- β -D-galactoside, (J)50 μ M quercetin-3-O- β -D-galactoside;

FIG. 4 shows a Western blotting method for determining the influence of caffeic acid (A) and quercetin-3-O-beta-D-galactoside (B) on SGLT1 and GLUT2 signal channel-associated protein expression (^***P<0.001，^**P<0.01，^*P<0.05)；

FIG. 5 is a graph of the significant promotion of transcription of key protein mRNA in the SGLT1(A) and GLUT2(B) pathways by the compounds caffeic acid and quercetin-3-O- β -D-galactoside using the qRT-PCR method; note: and blank group^***P<0.001，^**P<0.01，^*P<0.05；

The data in the above figures are expressed as mean. + -. SD, where Control is a blank group and 200, 100 and 50. mu.M are indicated for different concentration groups of the components of the Salvia miltiorrhiza Bunge.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Application test: the compound caffeic acid and quercetin-3-O-beta-D-galactoside can remarkably promote the absorption of normal Caco-2 cells to glucose and the uptake of glucose tracer by Caco-2 cells

First, experiment cell

Caco-2 cells, purchased from cell banks of the culture Collection of the national academy of sciences

Second, experimental samples

Kaempferol, caffeic acid, and quercetin-3-O-beta-D-galactoside were purified from the national food processing technology research and development professional center of southern university by the respective prior art references (Xiao Mei, Cao Ning, Fan just-hanging, et al.Studies on Flavonoids from the Leaves of Lindera aggregata [ J ] Journal of Chinese medical Materials,2011,34(1):62-64. Wu hanging-yi. et al.chemical Constants from Medicinal food Meal of Paeonia FFruicosa [ J ] Aciperonous Chinese Medicine and Li Shung-sea. et al.Flavond Consortions of sugar of wheat J.Pharma ] yellow chemical Materials and J.10. Journal of wheat J.10. Journal of purity).

Third, instrument and reagent

Fourth, experiment method

1. Cell culture

Caco-2 cells in complete medium containing 90% DMEM and 10% fetal bovine serum at 37 deg.C with 5% CO₂Cultured in an incubator.

2. Measurement of transmembrane resistance of colon cancer cell

Taking Caco-2 cells in logarithmic growth phase, and taking the cells at 10 degrees⁴One/well cell concentration was seeded in a Transwell chamber with a pore size of 0.4 μm in 24 wells, 3 multiple wells were set, and control wells (containing no cells, only complete medium) were set; the medium was changed 24h later (i.e. fresh complete medium), followed by a weekly change every other day, and a weekly change every day. The cell monolayer integrity and the compactness formation process are researched by measuring the transmembrane resistance value (TEER) by a cell resistance instrument at 8 th, 10 th, 12 th, 14 th, 16 th, 18 th and 21 th d of culture, and the culture is carried out to 21 th d.

And (3) measuring the resistance value: the cells are balanced for 30min at room temperature, the measuring electrode is irradiated for 30min under an ultraviolet lamp, then is soaked in 70% ethanol for 15min, and is taken out to be air-dried for 15 s. Discarding the supernatant of the upper chamber and the lower chamber in the Transwell chamber, washing twice with PBS, adding PBS for the third time, placing the cells in an incubator at 37 ℃ for balancing for 30min, and taking out; immersing the electrode and the culture plate into the culture plate at an angle of 90 degrees, and measuring and recording the resistance value of the electrode and the culture plate; measuring the resistance values of 3 control holes, measuring the resistance values of the experimental group, and calculating the average value; after the measurement, the PBS was discarded, and the complete medium was added to continue the culture. The results are shown in FIG. 1.

The resistance value (R) was calculated to be 0.33cm in the film area of the 24-well Transwell plate²Calculated as follows:

(R-R control wells) 0.33 ═ resistance value (Ω. cm)²)。

3. GOD-POD method for detecting influence of compound on glucose content in supernatant of Caco-2 cells

Blowing Caco-2 cells in logarithmic growth phase into cell suspension according to cell passage method, counting under an inverted microscope, and adjusting cell density to 10⁴Inoculating 500 μ L of the strain per well of 24-well plate, dividing into blank group and administration group, incubating and culturing in incubator for 16 days until resistance reaches 513 Ω. cm²The resistance is already greater than 500 omega.cm². At this point the cells have formed a tight cell monolayer. Adding compounds with different concentrations into the administration group, culturing for 12h and 24h, determining OD value at 505nm of an enzyme labeling instrument according to the operation of a glucose detection kit, judging the glucose intake amount according to the determined glucose content, and evaluating the influence of the flos Plumbaginis Zeylanicae extract on the glucose intake and consumption capacity. The results are shown in FIG. 2.

4. Fluorescent D-glucose homolog (2-NBDG) as tracer probe for detecting glucose uptake of Caco-2 cells

Caco-2 cells in logarithmic growth phase were seeded in 24-well plates (10)⁴One/well), divided into blank and administered groups, and cultured for 16d, at which time the cells have formed a compact cell monolayer structure. The drug administration groups are added with drugs with different concentrations, the culture and the culture are continued for 24h, 100 mu M2-NBDG is used for treating each group of Caco-2 cells (the blank group is added with the same amount of PBS), the cells are incubated for 30min in a dark place at 37 ℃ (the process is ensured to be finished in a sterile environment), the cells are washed for 3 times by using ice PBS, and the cells are photographed by an inverted fluorescence microscope. The results are shown in FIG. 3.

5. qRT-PCR detection of influence of Paeonia convolvulus components on mRNA transcription of SGLT1 signal channel of Caco-2 cells

(1) Cell culture:

blowing Caco-2 cells in logarithmic growth phase into cell suspension according to cell passage method, counting under an inverted microscope, and adjusting cell density to 10⁴And (4) inoculating 500 mu L of the cells per well in a 24-well plate, dividing the cells into a blank group and an administration group, incubating and culturing for 16d in an incubator, adding compounds with different concentrations in the administration group, culturing for 24h, and collecting the cells.

(2) Extraction of Total RNA

Lysis of cells: 1mL Trizol reagent was added to each well, blown with a pipette and collected into an enzyme-free EP tube, and lysed on ice for 5 min.

② RNA isolation: adding 200 μ L chloroform, turning upside down, mixing, and layering on ice for 3 min. After centrifugation at 12000rpm for 15min at 4 ℃, 250. mu.L of RNA supernatant was pipetted into a fresh enzyme-free EP tube.

③ precipitation of RNA: adding 250 μ L isopropanol, turning upside down, mixing, and standing on ice for 10 min. Centrifuging at 4 deg.C and 12000rpm for 15min, removing supernatant, and collecting the precipitate at the bottom of the tube as RNA.

Washing RNA: to the RNA precipitate, 1mL of 75% ethanol was added, the residual isopropanol was washed off by inverting, centrifuged at 12000rpm at 4 ℃ for 5min, the supernatant was discarded, and air-dried for 5 min.

Dissolving RNA: adding 30 μ L of non-enzyme water, mixing, and storing at-80 deg.C in refrigerator.

Sixthly, detecting the purity and concentration of RNA: RNA purity and concentration were determined using a microanalyzer and appropriate amounts of enzyme-free water were added to give an RNA concentration of about 500 ng/. mu.L.

(3) Removal of gDNA: the amount of RNA required to be added is 1000ng and the required volume of RNA, x, is calculated from the measured RNA concentration. The mixed reaction system is shown in table 1, and the reaction conditions are as follows: 42 ℃ for 3 min.

TABLE 1 reaction System for gDNA removal

Reagent	Content (wt.)
		gDNA Eraser	1μL
5×gDNA Eraser Buffer	2μL
		Total RNA	x
RNase Free H2O	Up to 10μL

(4) Reverse transcription into cDNA: the mixed reaction system is shown in tables 2-4, reverse transcription is carried out on a PCR instrument, and the reaction conditions are as follows: 37 ℃, 15min → 80 ℃, 5s → 4 ℃ and infinity.

TABLE 2 reaction System for reverse transcription

Reagent	Content (wt.)
		Removal of gDNA product	10μL
RT Prime Mix	1μL
		Prime Script RT Enzyme Mix I	1μL
5×Prime Script Buffer 2	4μL
		RNase Free H2O	4μL

The cDNA fragment obtained by the above reverse transcription was subjected to PCR amplification in the following manner. The results are shown in FIG. 5.

The concentration of each primer was diluted to 100. mu. mol/L, and the primer sequences of each amplified gene are shown in Table 3 below. The PCR reaction system is shown in Table 4 below.

TABLE 3 primer sequences

TABLE 4 PCR amplification reaction System

Reagent	Content (wt.)
		TB Green premix Ex Taq Ⅱ	12.5μL
PCR Reverse prime	1μL
		PCR Forward prime	1μL
cDNA solution	2μL
		RNase Free H2O	8.5μL

6. Western blotting detection of influence of compound on Caco-2 cell SGLT1 signal pathway protein expression

Preparing a solution required by an experiment:

preparation of 10 × TBS: weighing 80g of NaCl and 24.2g of Tris-base, adding 900mL of double distilled water, and placing on a magnetic stirrer to stir and dissolve. The pH was adjusted to 7.6 with concentrated hydrochloric acid, and the solution was taken up in a 1000mL volumetric flask and stored at room temperature for further use.

Preparing TBST solution: the 10 xTBS is diluted to 1 xTBS, and 2.5mL of Tween 20 is added to every 500mL of 1 xTBS, and the mixture is stored at room temperature for later use.

③ 1.5M Tris HCl preparation: 18.17g Tris-base was weighed out accurately, 80mL double distilled water was added, and the mixture was dissolved by stirring on a magnetic stirrer. Adjusting the pH value to 8.8 by concentrated hydrochloric acid, fixing the volume to 100mL, and storing at room temperature for later use.

Preparing 1.0M Tris-HCl: weighing 12.11g of Tris-base, adding into 80mL of double distilled water, stirring uniformly by using a single-direction and double-direction magnetic heating stirrer, metering the volume to 100mL by using a 100mL volumetric flask, finally adjusting the pH value to 6.8 by using concentrated hydrochloric acid, and storing at room temperature for later use.

Preparing 30% acrylamide: 29g of acrylamide and 1g of N, N-methylenebisacrylamide were added to 100mL of distilled water, and then completely dissolved using a magnetic stirrer, and finally stored at 4 ℃ for later use.

Preparing 1 times protein electrophoresis buffer solution: taking 3.03g of Tris-base, 18.77g of glycine and 1g of SDS, adding distilled water to the constant volume to 1000mL, and storing at room temperature for later use.

Seventhly, preparing a membrane transferring buffer solution: 30.3g of Tris-base and 142.6g of glycine are taken, 900mL of distilled water is added, a magnetic stirrer is used for stirring and dissolving, the volume is constant to 1L, and the mixture is stored at room temperature for standby.

Preparing a buffer solution of eighty 1 times rotating film: adding 350mL of distilled water and 100mL of methanol into 50mL of 10 Xmembrane buffer solution, mixing uniformly, and storing at 4 ℃ for later use.

Ninthly, preparing a mixed solution for cell lysis: the RIPA strong lysate, phosphatase inhibitor and PMSF are prepared and mixed evenly according to the proportion of 100:1:1, and the mixture is placed on ice to be prepared as it is.

The experimental method comprises the following steps:

(1) cell culture:

blowing Caco-2 cells in logarithmic growth phase into cell suspension according to cell passage method, counting under an inverted microscope, and adjusting cell density to 10⁴And (4) inoculating 500 mu L of the cells per well in a 24-well plate, dividing the cells into a blank group and an administration group, incubating and culturing for 16d in an incubator, adding compounds with different concentrations in the administration group, culturing for 24h, and collecting the cells.

(2) Extraction of total cellular protein:

the following components are taken as RIPA lysate: PMSF: phosphatase inhibitor ═ 100:1:1, 200. mu.L of total protein lysate was added to each cell sample, resuspended, and then lysed on ice for 30 min.

② centrifuging at 12000rpm for 10min at 4 ℃, sucking supernatant and transferring to a new EP tube, namely obtaining total protein.

③ adding 5 Xloading buffer of 1/4 protein volume, mixing, then water bathing at 100 ℃ for 5min to denature the protein, and storing at-20 ℃.

(3) Determination of protein concentration by BCA method:

the method comprises the following steps of: copper reagent 50: preparing a BCA working solution according to the proportion of 1.

Adding 2 mu L of protein samples into a 96-well plate respectively, and diluting by 10 times by using PBS diluent, namely, adding 18 mu L of PBA diluent into each well in advance, and setting 3 multiple wells for each sample.

And thirdly, adding 200 mu L of BCA working solution into each hole, fully and uniformly mixing to avoid generating bubbles, placing the mixture in a 37 ℃ constant temperature box for 30min, and measuring the absorbance value at 562nm by using an enzyme-linked immunosorbent assay instrument.

And fourthly, substituting the measured absorbance value into the standard curve y of 0.766x +0.998 to calculate the protein concentration.

(4)SDS-PAGE

Firstly, glue preparation: to a 10% SDS polyacrylamide gel (using 8% separation gel prepared in Table 1, incubated at 37 ℃ for 30min, then 5% concentrated gel was added, incubated at 37 ℃ for 30min, to obtain a 10% SDS polyacrylamide gel), an indicator Marker was added, and the same amount of protein sample (30/60. mu.g) was separated. The protein was concentrated in a vertical electrophoresis tank containing 1 Xelectrophoresis buffer at 80V for 30 min. And when the protein runs to the separation gel, regulating the voltage to 120V for 60min, and separating the protein.

TABLE 5, 10% SDS Polyacrylamide gel formulation

Secondly, film turning: after electrophoresis is finished, cutting off glue within a required protein molecular weight range according to a Marker, cutting off a PVDF membrane with the same size as the glue, soaking the PVDF membrane in methanol to activate the PVDF membrane, placing and clamping a plate in the sequence of an anode, a spongy cushion, filter paper, the PVDF membrane, the glue, the filter paper, the spongy cushion and a cathode, placing the plate in a membrane rotating groove, adding a membrane rotating buffer solution, placing the plate in a refrigerator at the temperature of-4 ℃, and transferring at 72V for 60 min.

(5) Immunoblot color development

Sealing: after the membrane conversion is finished, the PVDF membrane is taken out and soaked in TBST buffer solution containing 5% skimmed milk powder, and is placed on a shaking table for sealing for 2 hours.

Incubation of primary antibody: the reaction solution of iNOS/COX-2/beta-actin: TBST 1: 1000, cutting PVDF membrane according to the molecular weight of the corresponding protein, soaking in the corresponding primary antibody diluent, and incubating overnight at 4 ℃. The PVDF membrane was washed 5 times with PBST, 5min each time.

③ incubating secondary antibody: rabbit/mouse antibodies: TBST 1: 2000, the PVDF membrane was soaked in the corresponding secondary antibody dilution and incubated for 2h at room temperature. The PVDF membrane was washed 5 times with PBST, 5min each time.

Fourthly, color development: and (3) dropwise adding a developing solution on the PVDF film, and placing the PVDF film in a chemiluminescence imager for development. Finally, Image J was used to measure the gray scale values for quantitative analysis. The results are shown in FIG. 4.

The above experiments were repeated 3 times, and the results were statistically analyzed using SPSS 19.0 software. Experimental data on

And (4) showing. The two groups were compared using t-test, and the groups were compared using analysis of variance (One-Way ANOVA) with P<0.05 is statistically significant.

Fifth, experimental results

As shown in FIG. 1, TEER values are an important indicator of response to cell monolayer integrity by measuring the crossing of Caco-2 cell monolayers in cultures 8, 10, 12, 14, 16, 18 and 21dThe membrane resistance, which was found to increase with the culture time, was already > 500. omega. cm at 16d TEER values². After 16d, the resistance value continuously increases and gradually becomes flat, which indicates that the colon cells form a compact cell monolayer, and the modeling is successful.

FIG. 2 uses glucose oxidase kit to measure the glucose content in the supernatant after 12h and 24h of administration of three compounds with different concentrations. The results show that: the glucose content of caffeic acid and quercetin-3-O-beta-D-galactoside in supernatants of 200 mu M, 100 mu M and 50 mu M concentrations is obviously reduced in 12h and 24h after administration, and both the caffeic acid and the quercetin-3-O-beta-D-galactoside can obviously improve the glucose consumption of Caco-2 cells (P <0.001), so that the caffeic acid and the quercetin-3-O-beta-D-galactoside have the function of promoting the Caco-2 cells to absorb the glucose. Kaempferol at 200. mu.M, 100. mu.M and 50. mu.M had no significant effect on the glucose content in the supernatant.

FIG. 3 shows that Caco-2 cells were treated with the compound for 24 hours, and groups of Caco-2 cells were treated with 100. mu.M 2-NBDG, and photographed by an inverted fluorescence microscope, and the amount of 2-NBDG uptake was proportional to the fluorescence intensity, thereby measuring the sugar uptake ability of Caco-2. There was no significant change in green fluorescence signal in Caco-2 cells after exposure to kaempferol at 200. mu.M, 100. mu.M and 50. mu.M (FIGS. 3-B, C, D) compared to the blank (FIG. 3-A). After the action of 200 mu M, 100 mu M and 50 mu M (figure 3-H, I, J) of quercetin-3-O-beta-D-galactoside and 200 mu M, 100 mu M and 50 mu M (figure 3-E, F, G) of caffeic acid, green fluorescence signals in Caco-2 cells are remarkably enhanced, and the results prove that the caffeic acid and the quercetin-3-O-beta-D-galactoside can promote the rapid uptake of 2-NBDG on the Caco-2 cells, the intracellular accumulation and the fluorescence excitation of the 2-NBDG and are in direct proportion to the fluorescence intensity in a certain dosage range.

In order to explore the action mechanism of caffeic acid and quercetin-3-O-beta-D-galactoside for promoting Caco-2 glucose absorption, the application researches the influence of the caffeic acid and the quercetin-3-O-beta-D-galactoside on the expression of SGLT1 signal channel related protein. Figure 4 results show that: compared with the blank group, 100 μ M caffeic acid (fig. 4-a) significantly promoted the expression of GLUT2 protein at a concentration of 200 μ M (× P <0.001), and SGLT1 protein at a concentration of 200 μ M. quercetin-3-O- β -D-galactoside significantly increased SGLT1 and GLUT2 protein expression at concentrations of 200 μ M, 100 μ M and 50 μ M (fig. 4-B) (. P <0.001,. P <0.01,. P < 0.05).

FIG. 5 the effect of caffeic acid and quercetin-3-O- β -D-galactoside on SGLT1 signaling pathway gene expression was examined using real-time fluorescent quantitative PCR. The results showed that caffeic acid significantly promoted the transcription of SGLT1 and GLUT2 mrnas at concentrations of 200 and 100 μ M (. about.p <0.001,. about.p <0.01,. about.p <0.5), whereas caffeic acid significantly promoted the transcription of SGLT1mRNA at a concentration of 50 μ M and was significant (. about.p <0.5) (fig. 5-a). At the same time quercetin-3-O- β -D-galactosides also significantly increased the expression of SGLT1 and GLUT2 genes at concentrations of 200, 100 and 50 μ M (. P <0.001,. P <0.01,. P <0.5) (fig. 5-B). Therefore, caffeic acid and quercetin-3-O-beta-D-galactoside can increase glucose uptake of Caca-2 cells by up-regulating gene levels of SGLT1 and GLUT2 and protein expression thereof.

Claims (5)

1. Application of caffeic acid in preparing medicine for promoting glucose absorption is provided.

2. The use of claim 1, wherein caffeic acid significantly promotes glucose uptake by Caco-2 cells and uptake of the glucose tracer 2-NBDG by Caco-2 cells.

3. The use as claimed in claim 1, wherein caffeic acid is capable of significantly promoting expression of SGLT1 and GLUT2 proteins and transcription of mRNA.

4. Caffeic acid and conventional adjuvants in the field are compounded to make into compound preparation with effect of promoting glucose absorption.

5. The compound preparation of claim 4, wherein the compound preparation is in the form of tablets, granules, pills, capsules or injections.

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