KR20210091949A - Composition containing extract of butterbur as an active ingredient for preventing, alleviating or treating sepsis - Google Patents

Abstract

The present invention relates to a composition containing a Petasites japonicus extract as an active ingredient for preventing, improving, or treating sepsis. Accordingly, by containing a material extracted from Petasites japonicus, that is a natural substance, as an active ingredient, burden with respect to rejection in human body can be reduced and sepsis can be efficiently prevented, alleviated, or treated.

Description

Composition containing extract of butterbur as an active ingredient for preventing, alleviating or treating sepsis

The present invention relates to a composition for preventing, improving, or treating sepsis containing a butterbur extract as an active ingredient, and more particularly, as an active ingredient of the butterbur extract, Bakkenolide D, 5-O-caffeoylquinic acid, 1,5-dicaffeoylquinic acid It relates to a composition capable of preventing, ameliorating or treating sepsis, including any one or more.

Among glycohydrolases, neuraminidase is an enzyme that hydrolyzes sialic acid at the end of a sugar complex on a cell surface, particularly a glycoprotein. Neuraminidase (NA) hydrolyzes the acetal bond between galactose and sialic acid, which are components of glycoproteins, and specifically selectively hydrolyzes the α2-3 position and the α2-6 position (Shinya et al. Nature). 2006, 440, 435). Neuraminidase is distributed in higher eukaryotes and many pathogenic viruses and bacteria (Vimr, E.; Lichtensteiger, C. Trends Microbiol. 2002, 10, 254.; Schauer, R. Glycoconjugate J. 2000, 17, 485.; Taylor, G. L. Curr. opin. Struct. Biol. 1996, 6, 830). The hydrolysis of sialic acid by neuraminidase plays a crucial role in cell-cell signaling and cell-cell factors.

Influenza virus is a highly contagious virus that causes acute respiratory disease, and is one of the viruses that cause severe respiratory symptoms in children, the elderly, and patients with cardiopulmonary disease (Hien, TT et al. N. Eng. J. Med., 350, 1179, 2004). Influenza virus belongs to Orthomyxovirus taxonomically, and there are three types, A, B, and C, and the most prevalent type is A and B. In the case of influenza virus, the viron proliferated in the infected host cell is conjugated with sialic acid at the end of the glycoprotein at the end of the cell. When sialic acid and byron are hydrolyzed by neuraminidase, the virus spreads to new cells, where inhibition of the function of neuramidase can inhibit the spread of the virus. Therefore, neuramidase inhibitors are being used as antiviral materials.

On the other hand, biofilm production is a key process when pathogenic bacteria infect host cells. Neuraminidase acts as a crucial enzyme in biofilm formation. Therefore, inhibiting the function of neuraminidase inhibits biofilm production and, as a result, can block bacterial infection (Soong, G. et al., J. Clin. Invest. 2006, 116, 2297). Streptococcus pneumoniae (Streptococcus pneumonia), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Clostridium perfringen (Clostridium perfringen) are representative pathogenic bacteria that infect through the biofilm stage generated by neuraminidase. In addition, sialic acid is closely related to the immune response. In general, after viral or bacterial infection, as sialic acid in the sugar complex on the cell surface is hydrolyzed by neuraminidase, cytokines in the cell are activated and an inflammatory reaction proceeds, leading to sepsis. Therefore, neuraminidase inhibitors are effective in preventing and treating pathogenic viruses and bacteria and inflammatory diseases accompanying infection.

In order to alleviate inflammation caused by these viral and bacterial infections, research on active substances derived from natural substances that inhibit neuraminidase, such as 'Patent Document 1', is being actively conducted.

10-0962334 B1 (2010. 06. 10.)

The present invention has been devised to solve the above problems, and an object to be solved in the present invention is to provide a composition capable of preventing, improving or treating sepsis by inhibiting neuraminidase of bacteria using butterbur.

The present invention for solving the above problems is a technical feature to provide a food composition for preventing or improving sepsis, characterized in that it contains butterbur extract as an active ingredient.

The present invention for solving the above problems is a technical feature to provide a pharmaceutical composition for preventing or improving sepsis, characterized in that it contains butterbur extract as an active ingredient.

In addition, the present invention for solving the above problems is technically characterized in that it contains any one or more of Bakkenolide D, 5-O-caffeoylquinic acid, and 1,5-dicaffeoylquinic acid as a butterbur extract.

The present invention can reduce the burden of rejection in the human body and effectively prevent, improve or treat sepsis by containing a material extracted from coltsfoot, which is a natural product, as an active ingredient.

1 is a graph showing the bacterial neuraminidase inhibitory activity of a substance extracted from a coltsfoot extract;
2 is a graph showing the hydrolytic activity of bacteria neuraminidase of a substance extracted from coltsfoot extract;
3 is a graph showing the evaluation of the inhibitory activity of bacterial neuraminidase of substance 2 by Lineweaver-Burk plot (left) and dixon plot (right);
4 is a graph showing the evaluation of the inhibitory activity of bacterial neuraminidase of substance 3 by Lineweaver-Burk plot (left) and dixon plot (right);
5 is a graph showing the evaluation of the inhibitory activity of bacterial neuraminidase of substance 4 by Lineweaver-Burk plot (left) and dixon plot (right);

The terms or words used in the present specification and claims conform to the technical idea of the present invention based on the "principle that the inventor can appropriately define the concept of a term in order to best describe his invention" It should be interpreted as the meaning and concept that

Therefore, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all the technical ideas of the present invention, so various equivalents that can replace them at the time of the present application It should be understood that there may be water and variations.

Example 1. Preparation of an extract of above ground coltsfoot

After mixing 1.6 kg of dried coltsfoot with methanol, it was extracted at room temperature for 7 days. A residue was obtained after removing the solvent under reduced pressure and a temperature of 45°C.

Experimental Example 1. Fractionation and analysis of active ingredients

The above-ground coltsfoot extract prepared in Example 1 was passed through a Diaion HP-20 column, n-hexane, chloroform, ethyl acetate, n-butanol, and Water was dispensed sequentially in order. Since the n-BuOH-soluble fraction showed enzyme inhibition against bacterial neuraminidase, this active fraction (11.4 g) was _{chromatographed on a silica gel column with CHCl 3} : MeOH = 25: 1, and a total of five fractions were obtained ( PB1-PB5).

PB3 (1.7 g) was chromatographed on Sephadex LH-20 with 95% methanol to obtain 5 sub-fractions. Subfraction 2 was rechromatographed on Sephadex LH-20 with 85% aqueous methanol. The remaining three subfractions were purified by Recycling Preparative HPLC with 70% aqueous methanol as a mobile phase to obtain material 1 (15 mg) and material 2 (17 mg).

PB5 (2.1g) was placed in a reversed-phase resin (ODS) column, separated into water with increasing methanol concentration (20-100%), and then rechromatographed with a Sephadex LH-20 column using 70% aqueous methanol. A total of three sub-fractions were obtained. The first subfraction was further separated by Recycling Preparative HPLC using 75% aqueous methanol, thus obtaining material 3 (32 mg) and material 4 (11 mg).

The separated materials 1 to 4 were ^{analyzed using 1} H and ¹³ C-nuclear magnetic resonance spectroscopy (NMR), and the results are as follows.

Substance 1.

¹ H-NMR (700 MHz, Chloroform- d )δ 5.10 (1H, m, H-1), 1.78 (2H, m, H-2), 1.66 (1H, m, H-3), 1.34 (1H, m, H-3), 1.55 (1H, m, H-4), 1.91 (1H, d, H-6), 2.21 (1H, m, H-6), 5.72 (1H, d, H-9) , 2.78 (1H, dd, H-10), 4.63 (2H, m, H-12), 5.15 (2H, d, H-13), 0.87 (3H, d, H-14), 1.09 (3H, s) , H-15), 1.91 (3H, s, H-2′), 5.91 (1H, dd, H-3″), 1.85 (3H, dd, H-4″), 1.75 (3H, s, H-) 5″). ¹³ C-NMR (175 MHz, Chloroform-d) δ 70.5 (C-1), 26.8 (C-2), 29.5 (C-3), 35.2 (C-4), 43.4 (C-5), 45.8 ( C-6), 54.9 (C-7), 177.5 (C-8), 80.8 (C-9), 51.4 (C-10), 147.7 (C-11), 70.6 (C-12), 108.3 (C) -13), 15.5 (C-14), 19.5 (C-15), 169.9 (C-1′), 20.9 (C-2′), 167.3 (C-1″), 128.2 (C-2″), 136.7 (C-3″), 15.5 (C-4″), 20.3 (C-5″).

Substance 2.

¹ H-NMR (900 MHz, Chloroform- d )δ 5.15 (1H, m, H-1), 1.84 (1H, m, H-2), 1.76 (1H, m, H-2), 1.67 (1H, dd, H-3), 1.37 (1H, dd, H-3), 1.57 (1H, m, H-4), 2.24 (1H, d, H-6), 1.96 (1H, d, H-6) , 5.76 (1H, d, H-9), 2.75 (1H, dd, H-10), 4.67 (2H, m, H-12), 5.21 (1H, s, H-13), 5.17 (1H, s) , H-13), 0.90 (3H, d, H-14), 1.11 (3H, s, H-15), 2.02 (3H, s, H-2′), 5.62 (1H, d, J = 10.14 Hz) , H-2″), 7.04 (1H, d, J = 10.14 Hz, H-3″), 2.39 (3H, s, H-4″). ¹³ C-NMR (225 MHz, Chloroform- d ) δ 70.3 (C-1), 26.8 (C-2), 29.5 (C-3), 35.3 (C-4), 43.3 (C-5), 45.8 ( C-6), 54.9 (C-7), 177.5 (C-8), 80.8 (C-9), 51.7 (C-10), 147.8 (C-11), 70.5 (C-12), 108.2 (C) -13), 15.5 (C-14), 19.5 (C-15), 169.9 (C-1′), 21.2 (C-2′), 165.6 (C-1″), 112.4 (C-2″), 152.8 (C-3″), 19.2 (C-4″).

Substance 3.

¹ H-NMR (700 MHz, Methanol- d ₄ )δ 2.25 (2H, d, H-2), 5.46 (1H, m, H-3), 4.00 (1H, dd, H-4), 5.42 (1H) , m, H-5), 2.19 (1H, m, H-6), 2.35 (1H, dd, H-6), 7.08 (2H, s, H-2′,2″), 6.79 (1H, s) , H-5″), 6.81 (1H, s, H-5′), 6.97 (2H, d, H-6′,6″), 7.59 (1H, d, H-7″), 7.64 (1H, d, H-7′), 6.28 (1H, d, H-8″), 6.37 (1H, d, H-8′). ¹³ C-NMR (175 MHz, Methanol- d ₄ ) δ 73.5 (C-1), 36.4 (C-2), 71.3 (C-3), 69.4 (C-4), 70.6 (C-5), 34.7 (C-6), 176.3 (C-7), 126.4 (C-1″), 126.5 (C-1′), 113.9 (C-2′,2″), 145.3 (C-3′,3″) , 148.0 (C-4″), 148.1 (C-4′), 115.1 (C-5′,5″), 121.7 (C-6′,6″), 145.7 (C-7″), 145.9 (C -7′), 113.7 (C-8″), 114.2 (C-8′), 167.0 (C-9″), 167.5 (C-9′).

Substance 4.

¹ H-NMR (900 MHz, Methanol- d ₄ ) δ 2.18 (1H, m, H-2), 2.08 (1H, m, H-2), 5.36 (1H, m, H-5), 3.75 (1H) , dd, H-3), 4.20 (1H, m, H-4), 2.18 (1H, m, H-6), 2.07 (1H, m, H-6), 7.07 (1H, d, H-2) ′), 6.80 (1H, d, H-5′), 6.97 (2H, d, H-6′), 7.58 (1H, d, H-7′), 6.28 (1H, d, H-8′) . ¹³ C-NMR (225 MHz, Methanol- d ₄ ) δ 74.9 (C-1), 37.6 (C-2), 70.6 (C-3), 72.3 (C-4), 70.1 (C-5), 36.9 (C-6), 175.7 (C-7), 126.4 (C-1′), 113.8 (C-2′), 145.4 (C-3′), 148.2 (C-4′), 115.1 (C-5) '), 121.6 (C-6'), 145.6 (C-7'), 113.9 (C-8'), 167.2 (C-9').

And as a result of analyzing the materials 1 to 4, as described in Table 1 below, two Bakkenolide compounds and two caffeoylquinic acid compounds were analyzed respectively (Material 1: Bakkenolide B, Material 2: Bakkenolide D, Material 3: 1 ,5-Dicaffeoylquinic acid, substance 4: 5- O- Caffeoylquinic acid).

Chemical structures of each compound are as shown in Formulas 1 and 2.

Experimental Example 2. Assay of neuraminidase inhibitory activity

Neuraminidase inhibitory activity analysis was performed on the four active compounds analyzed in Experimental Example 1. For neuraminidase, the bacterium neuraminidase derived from Clostridium perfringens was used. As a substrate, 4-methylumbelliferyl-N-acetyl-ad-neuraminidase acid sodium salt hydrate) was used, and the neuraminidase inhibitory activity analysis was confirmed by fluorescence analysis of its hydrolysis reaction. Quercetin was used as a positive control, and the enzymatic activity was calculated as in Equation 1 below.

As a result, as shown in FIG. 1 , it can be seen that all of substances 1 to 4 inhibit bacterial neuraminidase activity in a concentration-dependent manner. That is, in the case of materials 2 to 3, it can be seen that significant neuraminidase activity inhibitory activity is shown at _{IC 50 values in the range of 2.3-80.1 μM (IC 50} of material 2: 80.1 μM, IC ₅₀ of material 3: 2.3 μM _{, IC 50} of material 4: 22.6 μM). However, in the case of material 1, _{it can be seen that the IC 50} value exceeds 200 μM, thereby remarkably lowering the ability to inhibit neuraminidase activity. In particular, it can be seen that 1,5-Dicaffeoylquinic acid corresponding to substance 3 exhibits more than 10-fold inhibition of neuraminidase activity than quercetin, a positive control.

In addition, as shown in FIG. 2 , in the experiment in which the enzyme concentration and the concentration of the inhibitor (substance) are different, each change shows a linear equation and passes through the same intersection of the Y-axis, so it can be seen that the enzyme is a reversible enzyme inhibitor.

Experimental Example 3. Analysis of neuraminidase inhibition pattern

Among the four active compounds analyzed in Experimental Example 1, the dynamic parameters of the neuraminidase activity inhibitory ability of substances 2 to 4 were analyzed using a Lineweaver-Burk double-reciprocal-plot and dixon plot. All parameters were calculated using SigmaPlot (SPCC Inc., Chicago, IL, USA).

Specifically, using the results of Experimental Example 2, reactions occurring in reactants with various concentrations of substrates and inhibitors were analyzed as fluorescence emission values.

The change of 1/velocity (1/V) according to 1/[S] was analyzed using the Linewever-Burk plot, and the change of 1/V _maxapp according to the concentration of substances was analyzed using the Dixon plot.

Data analysis was performed with the gradient of the change in fluorescence value occurring for 20 minutes, and the change in fluorescence value in each section was measured every 20 and evaluated by Lineweaver-Burk plot and dixon plot.

As a result, the inhibition pattern was confirmed differently depending on the chemical structures of bakkenolide and caffeoylquinic acid. In the case of material 2, as shown in FIG. 3, the K _m value is constant, but the V _max value decreases, that is, the slope is different as the substrate concentration increases, but the same x-intercept is formed, so it can be seen that it is a non-competitive inhibitor. there is. Therefore, this substance can bind enzymes at sites other than the active site.

And in the case of materials 3 and 4, as shown in FIGS. 4 and 5, the V _max value was constant _{in the Lineweaver-Burk plot, but the K m} value was decreased, indicating that they were competitive inhibitors. This means that when substances 3 and 4 are treated with various concentrations of substrate, they can competitively affect the substrate-binding site to inhibit the activity of the enzyme. And substance 3 was identified as the most potent reversible inhibitor.

The Dixon plot is a widely accepted method for analyzing the type of enzyme inhibition and determining the Ki value for inhibitor binding, where Ki refers to the x-axis value (see the graph on the right of FIGS. 3 to 5 ). As shown in Table 1, K _i values were derived as 82.7 μM, 1.4 μM, and 16.1 μM. The K _i value represents the concentration required to produce half the maximum level of inhibition, and the _{inhibitor with a lower K i} value means a higher level of bacterial neuraminidase inhibitory activity, which has the ability to inhibit the disease more efficiently. suggests

On the other hand, the inhibitory activity of bacterial neuraminidase of the substances extracted from coltsfoot extract as above means that it is effective in improving sepsis (Paulson, JC, Kawasaki, N., 2011. Sialidase inhibitors DAMPen sepsis) (Nat. Biotechnol. 29, 406-407). Sepsis is one of the major causes of mortality among hospitalized patients, a disease that negatively affects vital tissues with extensive edema due to an overreaction of the immune system to bacterial pathogens.

Claims (3)

A food composition for preventing or improving sepsis, characterized in that it contains butterbur extract as an active ingredient.
A pharmaceutical composition for preventing or treating sepsis, characterized in that it contains butterbur extract as an active ingredient.
The method according to claim 1 or 2,
The composition, characterized in that the butterbur extract contains any one or more of Bakkenolide D, 5-O-caffeoylquinic acid, and 1,5-dicaffeoylquinic acid.

Images