CN109054014B - Polyphenol polymer and application thereof - Google Patents

Abstract

The invention relates to the field of medicine and pharmacology, and particularly relates to a polyphenol polymer and application thereof. The polyphenol polymer Unciaitiannin of the present invention has an average molecular weight of between 16 and 22 ten thousand and is prepared by the following process: taking catechu decoction pieces, crushing into coarse powder, adding 10 times of 80% ethanol, heating, refluxing or cold soaking for 3 times, each time for 1.5 hours, combining extracting solutions, filtering, concentrating the filtrate under reduced pressure until no alcohol smell exists, extracting with ethyl acetate, adsorbing the residual water layer obtained by extraction with an MCI column, eluting with water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol and 95% ethanol in sequence, collecting the 40% ethanol elution part, concentrating, loading on a Sephadex LH-20 chromatographic column, eluting with 80% ethanol, detecting by UPLC analysis, and combining pure Unciaritannin components shown by UPLC analysis to obtain the catechu. The polyphenol polymer can be used for preparing medicines for preventing and treating methicillin-resistant staphylococcus aureus infection.

Description

Polyphenol polymer and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a polyphenol polymer and application thereof.

Background

Staphylococcus aureus (Staphylococcus aureus for short) is the most common pathogenic bacterium causing human pyogenic infection, can directly cause local pyogenic infection, pneumonia, pseudomembranous enteritis, pericarditis and the like, and even septicemia, sepsis and other systemic infections, and seriously jeopardizes the life health of human beings. The types of infections by staphylococcus aureus can be divided into hospital-acquired infections and community-acquired infections, the latter further increasing the potential biohazard of this pathogen and the possibility of causing an infectious outbreak. Antibiotic therapy is the main treatment means of staphylococcus aureus infection, but in recent years, the infection caused by staphylococcus aureus has gradually become the focus of attention due to the characteristics of increasing the infection rate of staphylococcus aureus, accelerating the drug resistance, and leading staphylococcus aureus to become chronic, persistent and recurrent infection from acute infection, and the like, and particularly, the infection caused by methicillin-resistant staphylococcus aureus (MRSA) is urgent to discover a new drug action target and develop a new method for resisting the drug resistance staphylococcus aureus.

The therapeutic strategies of traditional antibiotics are mainly to kill or inhibit the growth of bacteria, their effects mainly include: inhibit bacterial DNA replication, protein synthesis and cell wall synthesis. Antibiotics discovered based on this strategy exert selective stress on bacteria after overuse, resulting in the emergence of drug-resistant strains. In recent years, research reports that a new mode of antibacterial infection treatment, namely antibacterial virulence drug (anti-viral drugs), is used for inhibiting the expression, transmission and adhesion of virulence proteins of target bacteria by targeting drugs, so that the virulence factor expression of the target bacteria is further targeted and regulated, and various infectious diseases are effectively prevented and treated. By reducing the pathogenicity of a pathogenic bacteria toxic protein by administering a drug to a patient so that the pathogenic bacteria toxic protein is harmless rather than killed, the occurrence of drug-resistant strains can be greatly reduced. Staphylococcus aureus regulatory protein (MgrA) is a global transcription factor, regulates more than 350 genes, shows various biological activities, and particularly plays an important role in regulating the expression of virulence factors of staphylococcus aureus. Early researches prove that MgrA plays an important regulating role in the pathogenicity of MRSA and can be used as a function target point for finding a novel anti-infective medicament for resisting the toxicity of MRSA.

The Catechu, radix Berchemiae Giraldianae, is dried soft extract of Uncaria gambir Roxb of Rubiaceae with leaf and twig. It is bitter and astringent in taste and cool in nature. Has the effects of astringing dampness, promoting wound healing, stopping bleeding, relieving pain, clearing heat and eliminating phlegm. The traditional Chinese medicine composition is mainly used for treating chronic ulcer and sore unhealing, eczema and running water, ulcerative gingivitis, aphtha, hemoptysis, hematemesis, hematuria, hematochezia, metrorrhagia, traumatic hemorrhage, hemorrhoid, carbuncle and swelling, phlegm-heat cough. The chemical component research shows that the component types in the medicinal materials or original plants are mainly flavanones, dimers of the flavanones, indole alkaloids, dimers of the flavanones and the indole alkaloids, and the like.

Disclosure of Invention

The invention aims to provide a polyphenol polymer and application thereof.

Specifically, the first aspect of the present invention provides a polyphenol polymer Uncariitannin, which has an average molecular weight of 16 to 22 ten thousand, and which is produced by the following method: taking catechu decoction pieces, crushing into coarse powder, adding 10 times of 80% ethanol, heating, refluxing or cold soaking for 3 times, each time for 1.5 hours, combining extracting solutions, filtering, concentrating the filtrate under reduced pressure until no alcohol smell exists, extracting with ethyl acetate, adsorbing the residual water layer obtained by extraction with an MCI column, eluting with water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol and 95% ethanol in sequence, collecting the 40% ethanol elution part, concentrating, loading on a Sephadex LH-20 chromatographic column, eluting with 80% ethanol, detecting by UPLC analysis, and combining pure Unciaritannin components shown by UPLC analysis to obtain the catechu.

In a preferred embodiment, the polymer is acid degraded in excess of phloroglucinol and is represented by formula I:

the invention also provides application of the polyphenol polymer Uncariitiannin in preparation of medicines for preventing and treating methicillin-resistant staphylococcus aureus infection.

The details of various aspects of the invention are set forth in subsequent sections. The features, objects, and advantages of the invention will be apparent from the description and from the claims.

Drawings

FIG. 1 is a drawing of Uncariianin¹³C NMR chart

FIG. 2 is an effluent spectrum of gel permeation chromatography

FIG. 3 is a UPLC chromatogram and BPI plot of a mixture from acid degradation of Uncariianin in excess of phloroglucinol

FIG. 4 is a graph showing the effect of Unciaitiannin on the normal growth of the Newman strain

FIG. 5 is a graph of the interaction of Uncaria with dose-dependent inhibition of MgrA-hla demonstrated by gel migration

FIG. 6 shows that Unciaitannin inhibits protein expression of Newman and USA300 LAC strain Hla

FIG. 7 shows Unciaitannin inhibits mRNA expression of Newman and USA300 LAC strain hla

Fig. 8 is the effect of Uncariitannin on Newman bacteria (MSSA) infected mice (n ═ 7). P < 0.01; p < 0.001; Mann-Whitney test, two tails. Each symbol represents data from 1 mouse, and the horizontal line represents the mean

FIG. 9 shows the effect of Unciaitannin on USA300 LAC bacteria (MRSA) infected mice (n 11). P < 0.01; p < 0.001; Mann-Whitney test, two tails. Each symbol represents data from 1 mouse, and the horizontal line represents the mean

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. All percentages, ratios, proportions, or parts are by weight unless otherwise specified.

In the following embodiments, the catechu is a dry paste obtained by concentrating a leachate obtained by boiling a small branch with leaves of uncaria gambir (gambir) roxb. of uncaria gambir (gambir) belonging to the genus uncaria of the family rubiaceae, and then concentrating the leachate.

The MCI column was CHP20P MCI gel (75-150 μm, Mitsubishi Chemical; preparation, Japan);

the gel column selects Sephadex LH-20(GE Healthcare Bio-Sciences AB, Sweden) as a filler;

the mass spectrum is Waters ACQUITY UPLC/Synapt G2-Si MS ultra-high performance liquid chromatography/electrospray quadrupole-time-of-flight mass spectrum combination, and the chromatographic column is Waters ACQUITY HSS T3(2.1mm x 100mm, 1.8 μm);

HPLC column for analysis was HST 3 column (4.6 × 250mm,5 μm);

HPLC column for preparation is HST 3 column (19 × 250mm,5 μm)

The ultraviolet spectrophotometer is a UV-2401 PC spectrophotometer;

infrared spectroscopy was measured using a Perkin-Elmer 577 spectrometer;

nuclear magnetic resonance was measured using a Bruker AV-600 spectrometer;

the liquid phase uses acetonitrile as chromatographic grade, water is distilled water, and formic acid as analytical grade;

the mass spectrum uses acetonitrile as mass spectrum grade, water is distilled water treated by Mill-Q, and formic acid is mass spectrum grade;

other reagents were synthetic grade.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

Example 1 isolation and purification of Uncaria caninnin from Acacia catechu

a) Pulverizing 2.5kg of catechu, adding 10 times of 80% (V/V) ethanol, performing hot reflux extraction for 3-5 times, and evaporating the extracting solution under reduced pressure to obtain an extract;

b) suspending the extract obtained in the step a) in water, and extracting with ethyl acetate to obtain an ethyl acetate extraction part and a water part of the catechu extract respectively;

c) separating the water part of the catechu extract obtained in step b) by MCI column, eluting with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 95% ethanol in sequence, concentrating, testing activity, selecting the 40% ethanol elution part with strongest activity, and further separating

d) Separating the 40% ethanol elution fraction obtained in step c) with Sephadex LH-20 column, eluting with 80% EtOH, evaporating each eluate under reduced pressure, performing UPLC analysis, and mixing the pure Unciaitannin fractions revealed by UPLC analysis to obtain Unciaitannin (3.5 g). To proceed it with¹³C NMR, suggesting it is a polyphenol polymer, as shown in figure 1.

EXAMPLE 2 SEC-RI-LS determination of the molecular weight of the Polyphenol Polymer Uncariianin

The purity and molecular weight of the polyphenol polymer Uncaria nin were analyzed using a Viscotek GPCmax + RI (differential refractive index detector) + LS (light scattering detector) multi-detector GPC/SEC system from Malvern, under the following analysis conditions: mobile phase: HCOONH4/DMF (15 mM); flow rate: 0.7 mL/min; injection volume: 50. mu.L; chromatography column: I-MBMMW-3078 (10. mu.M, 300mM L × 8.0.0 mM ID); column temperature: 45 ℃ C.; sample solvent: HCOONH4/DMF (15 mM); analysis results were calculated using the method of absolute molecular weight; measurement results are as follows (FIG. 2):

number average molecular weight (M)_n)＝160192

Weight average molecular weight (M)_w)＝195463

Z-average molecular weight (M)_z)＝219322

Polydispersity index (M)_w/M_n)＝1.22。

Example 3 acid degradation of Unciaiiiannin with excess of phloroglucinol and identification of major degradation products

The reaction conditions were as follows: acetyl chloride (120mL) was added slowly to methanol (180mL), followed by Uncariaianin (100mg), ascorbic acid (250mg) and phloroglucinol (1.8g), and the reaction was refluxed at 80 ℃ for 4 hours. After the reaction, the mixture was concentrated under reduced pressure, water (400ml) was added to the mixture to suspend the mixture, ethyl acetate (600ml) was added thereto and extracted 5 times, and the ethyl acetate extract layer was washed with water to neutrality and concentrated under reduced pressure to obtain a degradation product (2 g).

Taking 1mg of sample to redissolve with methanol, filtering with a 0.22 mu m microporous filter membrane, and analyzing by UPLC-PDA-QTOF-MS. The chromatographic conditions were as follows: a: acetonitrile; b: 0.1% formic acid water; the gradient program was: 10-18% A (0-6min), 18-25% A (6-11min), 25-30% A (11-13min), 30-40% A (13-20min), 40-50% A (20-30min), 50-100% A (30-32 min); detection wavelength: 280 nm; column temperature: at 40 ℃.

The mass spectrometry conditions were as follows:

desolventizing air flow rate: 800L h^-1

Desolventizing temperature: 500 deg.C

Taper hole air flow rate: 50L h^-1

Ion source temperature: 150 ℃ C

Capillary voltage: 2500V

An acquisition mode: negative ion

The chromatogram and mass spectrum of the degradation product are shown in FIG. 3.

In order to characterize the structure of the degradation products, they were isolated and purified. The sample was purified by preparative HPLC to give compound Uncariaideradation A (3mg), Uncariaideradation B (2.5mg) (formula I). The chromatographic conditions were as follows: a: acetonitrile; b: 0.1% formic acid water; the gradient program was: 15-25% A (0-5min), 25% A (5-15min), 25-40% A (15-15.1min), 40% A (15.1-45min), 40-100% A (45-45.1min), 100% A (45.1-60 min).

Structural identification data for compound Uncariaidegraration A:

UV(MeOH)λ_max(logε)211(4.37),261(3.72),289(4.01)nm；

IR(KBr)ν_max3374,2956,2925,1665,1638,1499,1451,1394,1209,1138,1080,1023,993,825,787cm^-1；

HRESIMS m/z 387.0724[M-H]^-(calcd for C₁₉H₁₆O₉,387.0716)；

¹H NMR(DMSO-d₆600MHz) and¹³C NMR(DMSO-d₆100MHz) are shown in table 1.

Numbering	H	C
				2		163.5
3		107.9
			4		105.3
5		150.4
			6	6.17，d(2.0)	98.8
7		157.5
			8	6.43，d(2.0)	89.6
9		155.3
			10		167.7
11	3.36，t(7.4)	23.3
			12	3.43，t(7.4)	40.6
13		202.3
			14		103.7
15		164.5
			16	5.81，s	94.6
17		165.2
			18	5.81，s	94.6
19		164.5
			10-OCH3	3.89，s	53.1

Table 1 shows the degradation products Uncariaideradation A¹H NMR and¹³data of C NMR

Structural identification data for compound Uncariaidegraration B:

UV(MeOH)λ_max(logε)207(4.36)，262(3.61)，292(3.97)nm；

IR(KBr)v_max3421，2954，1673，1631，1385，1295，1255，1198，1167，1138，1112，1079，1024cm^-1；

HRESIMS m/z 401.0877[M-H]^-(calcd for C₂₀H₁₇O₉，401.0873)；

¹H NMR(DMSO-d₆600MHz) and¹³C NMR(DMSO-d₆100MHz) is shown in table 2.

No	H	C
				2		163.3
3		108.0
			4		105.4
5		150.5
			6	6.17，d(2.0)	98.8
7		157.5
			8	6.43，d(2.0)	89.6
9		155.4
			10		167.7
11	3.34，m	23.4
			12	3.34，m	40.8
13		202.0
			14		104.0
15		163.1
			16	5.98，d(2.1)	91.7
17		166.1
			18	5.88，d(2.1)	95.9
19		163.3
			10-OCH3	3.88，s	53.1
15-OCH3	3.79，s	55.9

Table 2 shows the degradation products Uncariaideradation B¹H NMR and¹³data of C NMR

Example 4 Effect of Uncariitannin on Normal growth of Newman Strain

Staphylococcus aureus monoclonals (Newman) were picked and added to Trypticase Soy Broth (TSB) and incubated overnight at 37 ℃ on a constant temperature shaker at 250 rpm. Diluting the bacterial liquid by 100 times with fresh TSB culture medium, shaking at 37 deg.C, and culturing at 250rpm for about 2.5h to OD₆₀₀And (3) when the concentration reaches 0.2-0.3, subpackaging, adding medicines with different concentrations, adding DMSO with the same volume into a control group, uniformly mixing, taking 10 mu l of bacterial liquid to measure and coat the plates, continuously culturing the rest bacterial liquid in a constant temperature shaking table at 37 ℃ for 12h, taking 10 mu l of bacterial liquid to coat the plates every 2h, counting, and measuring the influence of Unciaitannin on the normal growth of the staphylococcus aureus.

The results show that Unciaitiannin had no significant effect on the normal growth of Newman bacteria at 800. mu.g/ml (FIG. 4).

Example 5 gel migration experiments demonstrated that Uncariaritannin can dose-dependently inhibit the interaction of MgrA-hla

Gel migration assay (EMSA) is a technique for studying the interaction of DNA binding proteins with their associated DNA binding sequences and can be used for qualitative and quantitative analysis. The method comprises the following specific steps: mu.l MgrA (final concentration 2.5. mu.M), 2. mu.l of the test substances at different concentrations and 2. mu.l hla DNA (final concentration 0.1. mu.M) were incubated in 14. mu.l binding buffer (10mM Tris-HCl [ pH7.4 ]]，50mM KCl，5mM MgCl ₂10% glycerol). After standing at room temperature for 30min, the sample was analyzed by electrophoresis on a pre-electrophoresed 6.5% native polyacrylamide gel (120V, 40min), and after electrophoresis, the gel was soaked in a gel red dye for 30min and then detected by fluorescence development. The results show that Unciaitiannin can inhibit Mg dose-dependentlyBinding of rA protein to the DNA promoter of hla (FIG. 5).

Example 6 Unciaitiannin inhibits protein expression by Newman and USA300 LAC Strain Hla

α -hemolysin (Hla) is a key downstream virulence factor regulated by MgrA, and can analyze the protein expression of Hla by Western blot, and analyze the influence of Uncariitannin on the virulence of sensitive bacteria Newman and drug-resistant bacteria (USA300 LAC). The specific steps are picking the monoclonal antibody of Staphylococcus aureus, adding it into TSB culture medium, shaking at 37 deg.C, culturing overnight at 250rpm, diluting bacterial liquid with fresh TSB culture medium 100 times, shaking at 37 deg.C, culturing at 250rpm for 3h, subpackaging, adding different concentrations of drugs, adding same volume of DMSO to control group, continuing culturing at 37 deg.C for 3h, centrifuging bacterial culture (10000 × g, 4 deg.C, 5min), collecting supernatant, adding 5 × SDS-PAGE sample buffer, mixing, heating at 99 deg.C for 5min, separating the sample on 12% SDS-PAGE, incubating with 25V voltage, 1A current, 30min, transferring to membrane, incubating again with TBN, and detecting the protein in TBN, and washing with washing membrane at room temperature, and using TBN, and washing membrane for 5 times of washing, and using TBS at room temperature to detect the sample by using TBS-1.C.

The experimental results show that Unciaitannin is effective against Newman as a sensitive bacterium and can also inhibit the expression of the Hla protein of MRSA strain USA300 LAC in a dose-dependent manner (FIG. 6).

Example 7 Unciaitiannin inhibits mRNA expression by Newman and USA300 LAC Strain hla

Selecting staphylococcus aureus monoclonal, adding the staphylococcus aureus monoclonal into a TSB culture medium, culturing overnight at a constant temperature of 37 ℃ by a shaking table at a rotating speed of 250rpm, diluting a bacterial liquid by 100 times by using a fresh TSB culture medium, subpackaging, simultaneously adding medicines with different concentrations, adding DMSO with the same volume into a control group, culturing for 3 hours at a constant temperature of 37 ℃ by a shaking table at a rotating speed of 250rpm, centrifuging a bacterial culture (10000 × g, 4 ℃ and 10 minutes), discarding supernatant of the bacterial liquid, collecting cells and extracting RNA, suspending the cells in a lysate, adding 0.5g of glass beads, violently shaking and breaking cell walls, centrifuging, collecting supernatant, adding chloroform, violently shaking for 30S, standing for 2min, centrifuging (10000 × g and 15min), then adding absolute ethyl alcohol with a 1/2 volume, extracting RNA by using a UNIQ-10 column type total RNA extraction Kit, reversely converting the RNA into cDNA by using a PrimeScipt RT Kit, adding primers, and performing fluorescence quantitative detection by using a SYBR Master Mix Kit, wherein each reaction system is 10 mul and 16. mu.l rRNA.

The experimental results showed that Unciaitiannin was effective against Newman as a sensitive bacterium and was also able to dose-dependently inhibit hla mRNA expression from MRSA strain USA300 LAC (FIG. 7).

Example 8 Effect of Unciaitiannin on Newman bacteria (MSSA) infected mice

Selecting staphylococcus aureus Newman in TSB culture medium, culturing overnight in a shaker at 37 ℃, diluting 20 times with fresh TSB culture medium, culturing for 3 hours in a shaker at 37 ℃, centrifugally collecting thalli, washing twice with PBS, suspending in PBS, adjusting the concentration of the thalli to proper concentration, performing intraperitoneal injection of Unciarnianin (50mg/kg) and corresponding blank solvent (20% PEG400) one day in advance for experimental group and control group BALB/c mice (6 weeks old), administering 4 hours after next day, performing intraperitoneal injection of 200 mu l of suspended bacteria (1.1 × 10)⁸). Thereafter, the administration was performed once every 24 h. On day 3 post-infection, the animals were sacrificed by carbon dioxide asphyxiation, and heart, liver, kidney, spleen tissues were ground in 800. mu.l of PBS (0.1% Triton X-100 added), diluted 10-fold into 6-step, 10. mu.l of ground diluted solution was plated on TSA agar medium, incubated overnight at 37 ℃ and counted.

As shown in FIG. 8, the number of colony counts in liver, kidney, spleen and heart of the group administered with mice infected with Newman bacteria was significantly decreased after administration of Uncariianin treatment, indicating that Uncariianin can also decrease the infectivity of Staphylococcus aureus in vivo.

Example 9 Effect of Uncariitannin on USA300 LAC bacteria (MRSA) infected mice

Selecting Staphylococcus aureus USA300 LAC, placing in TSB culture medium, shaking table culturing at 37 deg.C overnight, diluting with fresh TSB culture medium 20 times, and shaking table culturing at 37 deg.C for 3 hrBALB/c mice (6 weeks old) of the experimental and control groups were intraperitoneally injected with Unciaitannin (50mg/kg) and the corresponding blank solvent (20% PEG400) one day in advance, 4 hours after the next day of administration, 200. mu.l of resuspended cells (6.2 × 10) were intraperitoneally injected⁸). Thereafter, the administration was performed once every 24 h. On day 3 post-infection, the animals were sacrificed by carbon dioxide asphyxiation, and liver, kidney, spleen tissues were ground in 800. mu.l of PBS (0.1% Triton X-100 was added), diluted 10-fold into 6-step, 10. mu.l of ground diluted solution was plated on TSA agar medium, cultured overnight at 37 ℃ and counted.

As shown in FIG. 9, the number of colony counts in liver, kidney and spleen of the administered group was significantly reduced after mice were infected with USA300 LAC bacteria and given Uncaritanin treatment, indicating that Uncaritanin can also reduce the infectivity of Staphylococcus aureus in vivo.

The various aspects of the invention are addressed above. It should be understood, however, that equivalent changes and modifications may be made thereto by those skilled in the art without departing from the spirit of the present invention, and that such changes and modifications are intended to be covered by the appended claims.

Claims (3)

1. A polyphenol polymer catechu tannin (Unciarnitanin), characterized by: the average molecular weight of the polyphenol polymer is between 16 and 22 ten thousand, and the polymer is prepared by the following method: taking catechu decoction pieces, crushing into coarse powder, adding 10 times of 80% ethanol, heating, refluxing or cold soaking for 3 times, each time for 1.5 hours, combining extracting solutions, filtering, concentrating the filtrate under reduced pressure until no alcohol smell exists, extracting with ethyl acetate, adsorbing the residual water layer obtained by extraction with an MCI column, eluting with water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol and 95% ethanol in sequence, collecting the 40% ethanol elution part, concentrating, loading on a Sephadex LH-20 chromatographic column, eluting with 80% ethanol, detecting by UPLC analysis, and combining pure catechuinin (Uncarantiannin) components shown by UPLC analysis to obtain the catechu tannin.

2. A polyphenol polymer catechuinnin (uncariianin) as claimed in claim 1 wherein the product of acid degradation of the polyphenol polymer under conditions of excess phloroglucinol is according to formula i:

3. use of a polyphenol polymer catechu tannin (Uncariitannin) as defined in claim 1 in the manufacture of a medicament for the prevention and treatment of methicillin-resistant staphylococcus aureus infection.

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