KR20240052343A - Anti-bacteria composition icluding Codonopsis ussuriensis extract as an effective element - Google Patents

Abstract

The present invention relates to an antibacterial composition containing Codonopsis ussuriensis extract as an active ingredient. The present invention demonstrated that MeOH extract of C. ussuriensis roots exhibits strong inhibitory activity against BNA. In addition, ussurienoside I and tangshenoside I were identified as the main components of C. ussuriensis root extract.

Description

Antibacterial composition including Codonopsis ussuriensis extract as an effective element {Anti-bacteria composition icluding Codonopsis ussuriensis extract as an effective element}

The present invention relates to an antibacterial composition containing Codonopsis ussuriensis extract as an active ingredient.

The present invention is a project result, and the project information is as follows.

1. Project number: GB-2022001

2. Name of Ministry: Gyeongsangbuk-do Bio and Life Industry Department

3. Name of project management (professional) organization: Gyeongbuk Bioindustry Research Institute

4. Research project name: Development and industrialization of high value-added convergence functional seasoning salt products utilizing forest life resources

5. Research project name: Nature Life Industry Technology Development Project

6. Name of project implementation organization: National Baekdudaegan Arboretum

7. Research period: 2022.03.01 ~ 2022.11.30

Codonopsis is a genus of the Campanulaceae family that includes three species found in Korea : Codonopsis lanceolata, C. pilosula, and C. ussuriensis . For hundreds of years, the fresh or dried roots of Deodeok species have been widely used in folk medicine to reduce blood pressure, appetite and stomach ulcers, improve poor gastrointestinal function, and strengthen the immune system [Non-patent 1]. It has been reported that Deodeok species have anti-tumor, anti-obesity, antioxidant, antibacterial and anti-mutagenic functions as well as cognitive enhancement and neuroprotective functions [Non-patent 2-5].

Different origins of plants lead to changes in their chemical composition. Chemical constituents isolated from the roots include alkaloids, phenylpropanoids, triterpenoids, polyacetylenes, flavones, organic acids, and polysaccharides [Non-patent 6, 7]. The main pharmacologically active compounds in Deodeok include water-insoluble polyacetylenes, including lovethiolinine and lovethiolin, and water-soluble phenylpropyl polyacetylenes, such as tangsenosides I, II, and IV. It is a noid. [Non-patent 1,8]

However, because C. ussuriensis grows in a limited area, only a few studies on its physicochemical composition and physiological activity have been reported. C. ussuriensis is a well-known source of triterpenoids, but is also known to contain phenylpropanoids [Non-Patent 9], making it a potential candidate for bacterial neuraminidase (BNA) inhibitors. .

Sialic acids constitute a family of nine-carbon acidic monosaccharides present at the ends of sugar chains in humans and other animals, the most widespread form being N-acetylneuraminic acid (Neu5Ac, NANA, and Sia). [Non-patent 10,11 ] Neuraminidase (commonly referred to as sialidase, EC 3.2.1.18) hydrolyzes terminal sialic acids in oligosaccharides, glycoproteins, glycolipids, and mucins, among other substrates. [Non-patent 12-14]

Many viruses and pathogenic bacteria that colonize mucus surfaces contain enzymes that contribute to their pathogenicity and virulence. Pathogenic bacteria secreting neuraminidase release sialic acid from various host sialoglycoconjugates along the scavenger pathway [Non-patent 15]. Neuraminidase also participates in bacterial cell metabolism, providing free sialic acid as an alternative carbon and energy source. [Non-patent 16,17] This enzyme enhances bacterial survival outside the microorganism, especially in environments containing sialic acid. [18]. These enzymes have been treated as inhibitor candidates in the development of antibacterial agents. [Non-patent 19-21]

Looking at patent documents among prior art documents, Republic of Korea Patent Publication No. 10-1753069 “Mesenchymal stem cells overexpressing hemagglutinin-neuraminidase and F protein and their uses” is described. Looking at claim 1, “Motor neuron disease involving adipose tissue-derived mesenchymal stem cells that overexpress hemagglutinin neuraminidase (HN) and F (fusion) protein “Pharmaceutical composition for prevention or treatment.”

In addition, Republic of Korea Patent Publication No. 10-1150179 describes “Deodeok fermentation method using an optimal culture medium of the lactic acid bacterium Lactobacillus bulgaricus for the purpose of enhancing the antibacterial activity of Deodeok.” This uses deodeok ( Codonopsis lanceolatae ) (identification number [0003]), and it can be seen that a method of fermenting deodeok using Lactobacillus bulgaricus has been registered (Claim 5).

Republic of Korea Patent Publication No. 10-1753069 (registered on June 27, 2017) Republic of Korea Patent Publication No. 10-1150179 (registered on May 21, 2012)

1.He,J.Y. et al. “The genus Codonopsis (Campanulaceae): A review of phytochemistry, bioactivity and quality control.” J. Nat. Med. 2015, 69, 1. 2. Gao, S.M. et al. “Traditional uses, phytochemistry, pharmacology and toxicology of Codonopsis: A review.” J. Ethnopharmacol. 2018, 219, 50-70. 3.Song,S.Y. et al. “Standardization of diploid codonopsis laceolata root extract as an anti-hyperuricemic source.” Processes 2021, 9, 2065. 4.Han, A.Y. et al. “Codonopsis lanceolata extract prevents hypertension in rats.” Phytomedicine 2018, 39, 119-124. 5. Lee, J.S. et al. “Codonopsis lanceolata extract prevents diet-induced obesity in C57BL/6 mice.” Nutrients 2014, 6, 4663-4677. 6. He, J.Y. et al. “Quality evaluation of medicinally-used Codonopsis species and Codonopsis Radix based on the contents of pyrrolidine alkaloids, phenylpropanoid and polyacetylenes.” J. Nat. Med. 2014, 68, 326-339. 7.Du,Y.E. et al. “Chemical constituents of the roots of Codonopsis lanceolata.” Arch. Pharm. Res. 2018, 41, 1082-1091. 8. Hossen, M.J. et al. “Codonopsis lanceolata: A review of its therapeutic potentials.” Phyther. Res. 2016, 30, 347-356. 9. Lee, I.R. et al. “Isolation of triterpenoid and phenylpropanoid from Codonopsis ussuriensis.” Arch. Pharmacal Res. 1992, 15, 289-291. 10. Angata, T. et al. “A. Chemical diversity in the sialic acids and related α-keto acids: An evolutionary perspective.” Chem. Rev. 2002, 102, 439-469. 11. Wang, B. et al. “The role and potential of sialic acid in human nutrition.” Eur. J. Clin. Nutr. 2003, 57, 1351-1369. 12. Hunter, C.D. et al. “Human neuraminidases have reduced activity towards modified sialic acids on glycoproteins.” Carbohydr. Res. 2020, 497, 108139. 13.Achyuthan, K.E. et al. “Comparative enzymology, biochemistry and pathophysiology of human exo-α-sialidases (neuraminidases).” Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 2001, 129, 29-64. 14. Miyagi, T. et al. “Biological and pathological roles of ganglioside sialidases.” Prog. Mol. Biol. Transl. Sci. 2018, 156, 121-150. 15. Lewis, A.L. et al. “Host sialoglycans and bacterial sialidases: A mucosal perspective.” Cell. Microbiol. 2012, 14, 1174-1182. 16. Sudhakara, P. et al. ""Bacterial sialoglycosidases in virulence and pathogenesis. Pathogens 2019, 8, 39. 17. Glanz, V.Y. et al. “Sialidase activity in human pathologies.” Eur. J. Pharmacol. 2019, 842, 345-350. 18. Vimr, E.R. et al. “Diversity of microbial sialic acid metabolism.” Microbiol. Mol. Biol. Rev. 2004, 68, 132. 19.Wang, Y.H. "Sialidases from clostridium perfringens and their inhibitors." Front. Cell. Infect. Microbiol. 2020, 9, 462. 20. Choi, H.M. et al. "Molecules effectiveness of prenyl group on flavonoids from epimedium koreanum nakai on bacterial neuraminidase inhibition." Molecules 2019, 24, 317. 21.Wang, Y. et al. “Highly potent bacterial neuraminidase inhibitors, chromenone derivatives from Flemingia philippinensis.” Int. J. Biol. Macromol. 2019, 128, 149-157. 22.Woo, H.S. et al. “Bakkenolides and caffeoylquinic acids from the aerial portion of petasites japonicus and their bacterial neuraminidase inhibition ability.” Biomolecules 2020, 10, 888. 23. Ochs, R.S. “Understanding enzyme inhibition.” J. Chem. Education. 2000, 77, 1453-1456. 24. Ren, J. et al. "Tangshenosides from Codonopsis lanceolata roots." Phytochem. Lett. 2013, 6, 567-569. 25. De La Luz Cadiz-Gurrea, M. et al. “LC-MS and spectrophotometric approaches for evaluation of bioactive compounds from peru cocoa by-products for commercial applications.” Molecules 2020, 25, 3177.

The problem is to provide an antibacterial composition, an antibacterial agent, and a bacterial neuraminidase inhibitor containing Codonopsis ussuriensis extract as an active ingredient.

The problem is to provide an antibacterial composition, an antibacterial agent, and a bacterial neuraminidase inhibitor containing compounds derived from Codonopsis ussuriensis as active ingredients.

The solution is an antibacterial composition containing Codonopsis ussuriensis root extract as an active ingredient.

The solution is an antibacterial agent containing Codonopsis ussuriensis root extract as an active ingredient.

The solution is a bacterial neuraminidase inhibitor containing Codonopsis ussuriensis root extract as an active ingredient.

The solution is an antibacterial composition containing one or more of the following Compound 1 and Compound 2 as an active ingredient.

The solution is an antibacterial agent containing one or more of the following Compound 1 and Compound 2 as an active ingredient.

The solution is a bacterial neuraminidase inhibitor containing one or more of the following Compound 1 and Compound 2 as an active ingredient.

The present invention demonstrated that MeOH extract of C. ussuriensis roots exhibits strong inhibitory activity against BNA. In addition, ussurienoside I and tangshenoside I were identified as the main components of C. ussuriensis root extract.

The main compounds showed significant inhibitory activity with IC50 values of 56.0 μM and 203.3 μM, respectively.

1 is a diagram showing the structural formulas of Compounds 1 and 2 (Compound 1 and 2).
Figure 2 is a graph of HPLC analysis results of compounds 1 and 2.
Figure 3 is a graph showing the results of analysis of the BNA inhibitory ability and mechanism of compounds 1 and 2.
Figure 4 is a graph of LC-QTOF-MS chromatograms results.
Figure 5 is a table showing the BNA inhibitory effect of compounds 1 and 2.
Figure 6 is a table of LC-QTOF-MS chromatograms results.
Figures 7a and 7b are tables of spectral analysis results of compounds 1 and 2 in MeOD.
Figures 8a, 8b and 8c are graphs of LC-QTOF-MS results.

Terms or words used in this specification and claims should not be construed as limited to their ordinary or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain his or her invention in the best way. It must be interpreted as meaning and concept consistent with the technical idea of the present invention based on principles.

Therefore, the matters described in the experimental examples, examples, and drawings described in this specification are only the most preferred examples of the present invention and do not represent the entire technical idea of the present invention, so they can be replaced at the time of filing the present application. It should be understood that various equivalents and variations may exist.

This specification has been written based on an English paper, which is an exception to the public notice, and if the translation vocabulary for the experimental method and experimental results is unclear, the details in this specification can be interpreted more clearly by cross-checking with the English paper. .

In the present invention, we aimed to examine and characterize the main components of C. ussuriensis roots. The inventors isolated a potential bacterial neuraminidase (BNA) inhibitor from the roots of C. ussuriensis using bioactivity-guided fractionation. Compounds were identified and investigated using spectroscopic methods for their ability to reduce bacterial neuraminidase activity. Lineweave-Burke and Dixon plots were used to determine the mechanism of inhibition. Additionally, chemical compounds in the root extract were identified using ultra-performance liquid chromatography-quadpolarization-overnight mass spectrometry (UPLC-Q-TOF/MS).

Experimental Example 1. Materials and Methods

(1) Chemicals and Instruments

Thin layer chromatography (TLC) experiments were performed using solid phases of Kieselgel 60 F254 (Merck, Kenilworth, NJ, USA) and RP-18 F254 (Merck, Kenilworth, NJ, USA). Ultraviolet (UV) light from a Spectroline ENF-240 C/F from Spectronics, Melville, NY, USA, or 10% aqueous H2SO4 heated and sprayed onto a TLC plate were used to detect stains. The Biotage Isolera One Specktra Flash Puritation System with prepackaged cartridges of SNAP Ultra C18 (400, 120, 30 g) and SNAP Ultra (25 and 10 g) was used for flash chromatography (FC) (Biotage, Uppsala, Sweden). did. FC was performed using manually packaged Biotage SNAP dry material cartridges and Sephadex LH-20 (Millipore Sigma, St. Louis, MO, USA) (340, 100, and 10 g scales). A Bruker 700 spectrometer was used to record nuclear magnetic resonance (NMR) spectra (700 MHz; Bruker, Karlsruhe, Germany). A Waters Xevo G2-S series instrument (Waters Corporation, Milford, MA, USA) was used for UPC-Q-TOF/MS analysis with negative ion mode selected. A Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, UK) was used to collect infrared (IR) spectra.

(2) Plant Materials

C. ussuriensis roots were collected from 2-year-old plants grown at the experimental site of the National Institute of Horticultural and Herbal Science in Korea in October 2021. The specimen (voucher number MPS006563) was stored in the Herbarium of Oriental Medicine (NIHHS) and authenticated by a plant taxonomy expert.

(3) Extract Preparation and Fractionation

60 g of C. ussuriensis roots were dried and powdered, and an extract was prepared using 70% v/v methanol (MeOH; 1.5 L × 3) for 24 hours at room temperature. The extract was evaporated under reduced pressure at 45°C and filtered through filter paper to produce 16.4 g of extract.

43 fractions (F1-F43) were obtained from the EtOH extract (10 g) using an FC system equipped with a SNAP Ultra C18 cartridge (400 g, water: ACN, 95:5-0:100). Six fractions (F23a-F23f) were obtained from original F23 (620 mg) using a SNAP Ultra C18 cartridge (120 g, water: MeOH, 85:15).

76 mg of F23e was separated into Compound 1 (31 mg) using a Sephadex LH-20 and SNAP Ultra cartridge (25 g, CHCl3:MeOH:water, 90:10:1).

Compound 2, along with three fractions (F23b1-F23b3), was obtained from F23b (65 mg) using a SNAP Ultra C18 cartridge (30 g, water: MeOH, 50:50). Fraction F23b1 (39 mg) was subjected to numerous chromatographs using FCS with manually packaged Sephadex LH-20 (100 g scale 2, water: MeOH, 50:50) cartridge to yield 17 mg of Compound 2. It went through a graph separation process.

(4) BNA Inhibition Assay

Compounds were tested for their ability to inhibit Clostridium perfringens neuraminidase (EC 3.2.1.18) using the following protocol, with quercetin serving as a positive control. (quercetin).

We performed an evaluation of all previously used methods [Non-Patent 22]. The reaction was performed by adding Tris buffer (pH 7.5) to 90 μL of 4-methylrumbelliferyl-a-D-N-acetyl-neuraminic acid (MU-Neu5Ac) substrate (0.1 mM) at room temperature. Next, neuraminidase (0.2 units/mL) and sample solution (10 microliters each) were added to each well of a 96-well microplate (SPL Life Sciences, Korea, Pocheon). This mixture was captured using a Spectra Max M3 at specific wavelengths of 365 nm and 450 nm, respectively (Molecular Device, Sunnyvale, CA, USA). In this experimental method, a 50% inhibitory concentration (IC50) of 21.5 μM of quercetin was used as a positive control. Dose-response curves were used to calculate the IC50 for the enzymatic activity of neuraminidase. Each test was run three times.

Enzyme activity (%) = [1 - ([I]/IC ₅₀ )] x 100 (Equation 1)

(5) Enzyme Kinetic Assay and Progress Linear Determination

The kinetics of enzyme inhibition by the isolated compounds were contrasted with data obtained in the absence of inhibitors using Rheinweaver-Burk plots. For neuraminidase, steady-state rates were obtained at various inhibitor doses and variable substrate concentrations to establish kinetic parameters related to the inhibition mechanism. Straight or vertical sections ( , respectively) for the slope of the secondary plots vs. Using the inhibitor concentration, two constants, K _I or K _IS , for inhibitor binding to free or enzyme-substrate complexes were determined. (Secondary plots of the slopes of the straight lines or the vertical intercept( , respectively) vs. the concentration of inhibitors were used to determine the two constants, KI or KIS , for inhibitor binding with either free or enzyme-substrate complexes.) K _I and K _IS are respectively expressed as (Equation 2) and (Equation 4) .[Non-patent 23]:

1/V = Km / Vmax(1+[I]/K _I )1/S + 1/Vmax (Equation 2)

Slope = Km/K _I Vmax[I] + Km/Vmax (Equation 3)

Intercept = 1/V _IS Vmax[I] + /Vmax (Equation 4)

Kinetic parameters were obtained by increasing substrate and inhibitor concentrations using Rheinweaver-Burke double reciprocal plots and Dixon plots. Dixon plot was used to calculate the inhibition constant (Ki). All parameters were determined by constructing a sigma plot (SPCC Inc., Chicago, IL, USA).

(6) Identification of Chemical Constituents of C. ussuriensis

UPLC was performed with a Waters UPLC I-Class system (Waters Corporation, Milford, USA) with an ACQUITY HSS T3 column (2.1 × 100 mm, 1.7 μm).

The mobile phase consisted of water containing 0.1% formic acid (v/v) (A) and 0.1% formic acid (v/v) in acetonitrile (B). For the elution gradient, 0 to 1 minute, 10% B was used, 1 to 18 minutes, 10% B was used, 18 to 22 minutes, 40% B was used, and 22 to 22.5 minutes, 100% B was used. used. The injection volume for each run was 2 L and the flow rate was 0.4 mL/min.

For qualitative analysis, UPLC-Q-TOF-MS was performed on a Waters Xevo G2-XS.

A QTOF mass spectrometer equipped with a photodiode array (PDA) detector and electrospray interface (ESI) was used (Waters Corporation, Milford, MA, USA). The mass spectrometer was run in negative ionization mode, and the scan range was set from 50 to 1800 m/z. The source temperature was 120°C, the desorption temperature was 600°C, the detachment gas flow was 800 L per hour, the cone gas flow was 50 L per hour, and the collision energy was 30 eV, which were typical conditions for ionization. Typical ionization source conditions are: for negative ion mode, capillary voltage of 2.5 kV, cone voltage of 40 V, source temperature of 120 °C, desorption temperature of 600 °C, desorption gas of 800 L/h, desorption gas of 50 L/Hr. Cone gas flow, collision energy of 30 eV. The instrument is MssLynx v. Controlled using 4.2. Data collection and analysis were performed using UNIFI software (v. 1.9.4; Water, UK). 1 mg of sample was dissolved in 1 mL of MeOH to prepare a 1 mg/mL working solution, which was stored at 4°C.

(7) Data Processing and Statistical Analysis

Each experiment was performed three times. Results were analyzed by analysis of variance using Sigma Plot version 14.0. (Systat Software, Inc., San Jose, CA, USA) Statistically significant differences were defined at p < 0.05.

Experimental Example 2. Experimental results

(1) Compound 1 and 2 analysis results

The inhibitory activity of crude methanol extract of C. ussuriensis roots against bacterial neuraminidase was tested. To evaluate the inhibitory ability of the extract, column chromatography on octadecyl silica gel was performed, followed by purification through repeated preparative high-performance liquid chromatography (Prp-HPLC) and gel filtration on Sephadex LH-20. and two phenylpropanoids were produced. By comparing their spectroscopic data ( ¹ H- and ¹³ C-NMR, UV, LC-QTOF/MS) with those of [Non-patent 9,24], two phenylpropanoids were identified: ussurienoside I (compound 1) and tanshenoside I (compound 2).

Compound 1 was obtained as a colorless gum and showed an olive-green color on TLC after spraying and heating with 10% H2SO4. High-resolution electrospray ionization MS revealed _the molecular formula of C42H46O20 ( _C42H46O20 ) from the pseudomolecular ion at m/z ₅₁₅ [MH] ^- . IR (v max 3368, 2938, 1716, 1586, and 1505 cm ^-1 ) and UV (λ max 220 and 267 nm) spectra revealed hydroxyl, aromatic ring, carbonyl, and α,β-unsaturated absorption systems.

¹ H- and ¹³ C-NMR spectra (Figures 7a and 7b, Table S1) show a 1,3,4,5-tetra substituted benzene ring (one aromatic proton at one glucose moiety with δH 6.78), two methoxyl groups group (six proton singlets at δH 3.88), a pair of olefinic signals at δH 6.32 and δH 6.58, and a 3-hydroxy-3-methyl-glutarate moiety (two pairs of methylene signals at δH 2.68 and 2.69, a It showed characteristic signals that could be attributed to the methyl signal at δH 1.41, one acid signal at δC 171.0, and one ester carbonyl signal at δC 173.6), and the sugar portion (anomeric proton signals at δH 4.90). The overall NMR properties suggested that compound 1 had a phenylpropanoid skeleton. Spin-spin coupling and chemical signals were evaluated through 1H-1H correlation spectroscopy, heteronuclear multiple quantum coherence and heteronuclear multiple bond correlation experiments. Figures 7a and 7b (Table S1) reveal the presence of one β-glucopyranosyl unit.

Compound 1 was found to be the 3-hydroxy-3-methyl-glutarate ester of phenylpropanoid glycoside. This structure is a typical tangsenoside skeleton. Through gradient heteronuclear multiple bond correlation (gHMBC) spectra between the anomeric proton signal (H4.90(H-1")) and the oxygenated quaternary carbon signal (C134.7(C-4)) of the aglycone. A long-range correlation was observed, indicating that glucopyranose is linked to the hydroxyl group at C-4 (see Figure 1). Furthermore, the methoxy proton signal (H 3.88(3,5-) The correlation between 2OCH3)) and the oxygenated quaternary carbon signal (C 152.9 (C-3,5)) showed that the methoxy group was associated with C-3,5, and the structure of compound 1 was ussurienoside I. (Figure 1: The chemical structures of compounds 1 and 2, and the gHMBC correlation of compound 1, which is shown by arrows.)

Identification of compound 2 as tangsenoside I was made based on comparison of the NMR and MS data of compound 2 with the data reported in [Non-Patent 24 Figure 1]. The calculated purity of each compound was more than 95% as measured using HPLC analysis (Figure 2: (a) High-performance liquid chromatography-photodiode array (HPLC-PDA) chromatogram (detection wavelength, 280 nm) of 50% (v/v) aqueous MeOH extract of dried roots of C. ussuriensis (b,c) HPLC chromatogram and UV-Vis spectrum of the major compounds (tang-shenoside I and ussurienoside I with retention of 46.4 min and 53.6 min. ) with a maximum absorption at 220 nm wavelength.)

(2) Results of bacterial neuraminidase (BNA) inhibition assay

Enzyme analysis was performed based on an already reported phenomenon, which is an increase in fluorescence after hydrolysis of MU-Neu5Ac. [Non-patent 21]

Figure 3 is a graph showing the results of analysis of the BNA inhibitory ability and mechanism of compounds 1 and 2. (Figure 3: Inhibitory activity and mechanism of action of compounds against bacterial neuraminidase (BNA). (a) Dose-dependent inhibition of BNA by compound 1, compound 2, and quercetin (positive control). (b) Reversibility of the inhibitory effect of compound 1. (c) Lineweaver-Burk plots for BNA inhibition by compound 1. (d) Dixon plots of BNA inhibition by compound 1. (Inset) Tendencies of the intercept and slope from each straight line of the Lineweaver-Burk plot. )

Figure 5 is a table showing the BNA inhibitory effect of compounds 1 and 2. (Figure 5: Table 1. Inhibitory effects of isolated compounds on bacterial neuraminidase (BNA) activity. All compounds were examined by repeating the experiments thrice. a: IC50 values of the compounds represent the concentration responsible for 50% loss in enzymatic activity. b : Value of the inhibition constant; c : NT denotes not tested; d : Quercetin was a positive control.)

As shown in Figure 3a and Figure 5 (Table 1), compounds 1 and 2 showed significant neuraminidase inhibition in a dose-dependent manner with IC50 values of 56.0 μM and 203.3 μM, respectively. The IC50 value suggested that compound 1 was four times more active than compound 2. The IC50 of the positive control (quercetin) was 21.0 μM under the same conditions.

Inhibition of neuraminidase by compound 1 is shown in Figure 3b. When we plotted the initial rate against the concentration of the enzyme in the presence of various concentrations of Compound 1, we obtained a family of straight lines with all axes at the origin. As the inhibitor concentration increased, the line slope decreased, demonstrating that compounds 1 and 2 were reversible inhibitors.

Our data reveal an interesting aspect of the structure-activity relationship of the compounds. Better inhibition was observed when a hydroxy group was present at C-3', as seen by comparing the C-3 hydroxy substituted compound 1 (IC50 = 56.0 μM) with the glucosyl analogue of compound 2 (IC50 = 203.3 μM). there was. As shown in Figure 3c, d, the inhibition kinetics analyzed using Dixon and Lineweaver-Burk plots showed that compound 1 (Ki = 28.4 μM) was a type I mixed inhibitor. This is because increasing the concentration of the substrate created a family of lines that intersected at non-zero points on both the x and y axes (Figure 3d).

As a result of the mixed nature of the inhibitors, the inhibitors had different degrees of affinity for substrate-bound and free enzymes. Because inhibitors can bind to free enzyme or enzyme-substrate complexes, efforts have been made to separate inhibition constants for these two states.

Type I inhibitors occurred when the inhibitor was bound to the free enzyme, and type II inhibitors occurred when the inhibitor was bound to the enzyme-substrate complex. Both free enzyme and enzyme-substrate complex states are referred to as enzyme states. To conduct this analysis, the concentrations of inhibitor and substrate were changed based on Equation 2 to Equation 4. The equilibrium constants for the two distinct binding events, inhibitor binding to free enzyme (KI) and inhibitor binding to enzyme-substrate (KIS), are given by the quadratic plot of Km/Vmax and 1/Vmax against the concentration of compound 1, respectively. It was measured using

Therefore, we set the following constants for compound 1: KI = 27.79 μM and KIS = 92.0 μM (Figure 3 (insets)). These results show that the affinity of the inhibitor for the free enzyme was only slightly greater than the affinity for the enzyme-substrate complex. Accordingly, compound 1 was classified as a type I mixed inhibitor .

(3) Chromatogram analysis results

LC/Q-TOF/MS technology is useful for identifying constituents of plant extracts. The chemical composition of C.ussuriensis roots extracted using 50% MeOH was identified using LC/Q-TOF/MS method applied in negative ion mode.

Figure 4 is a graph of LC-QTOF-MS chromatograms results. (Figure 4: LC-QTOF-MS chromatograms of compounds of C. ussuriensis root extract. (a) PDA spectra; (b) MS spectra; (c) Tangshenoside I; (d) Ussurienoside I.) Figure 6 is LC-QTOF -MS chromatograms result table.

Figure 4 shows a representative Total Ion Chromatogram (TIC) with labeled peaks, indicating that ion mode was used. As shown in Figure 6 (Table 2), eight peaks were identified, including two for unknown compounds. Their chemical structures are detailed in Figure 8. (FIGS. 8a, 8b, and 8c are graphs of LC-QTOF-MS results. LC-Q-TOF-Ms (a) PDA analysis spectra (FIG. 8a) (b) MS spectra analysis of C. ussuriensis . (FIGS. 8b and 8c )

As a result of the removal of CO2 group (-44Da) and H2O molecule (-18Da) from the deprotonated precursor ion, peak 1 (m/z 191.0198, C6 H8O7-) is [MH-CO2] in negative ionization mode. Following -, the fragmentation pattern of [MH-H2O2]- was shown. (FIG. 8a, b, c) The compound was identified as citric acid ( peak 1 ), and the MS/MS spectrum was previously reported [Non-patent 25]. Peak 2 (m/z 203.0827, C11H12N2O2-) initially produced an ion at m/z 159 due to loss of CO2 molecule (-44 Da), followed by dissociation of NH3 and CO2 molecules to produce fragment ion at m/z 142. did. The presence of a fragment ion at m/z 116, similar to the molecular weight of indole (116 g/mol), suggested that this chemical was tryptophan. In negative ionization mode, peak 3 was identified as tangsenoside I (m/z 677 [MH]- and m/z 1355 [2M-H]-, C29H42O18- and C58H84O36-). The precursor ion (MH)- generated fragments at m/z 497 [C23H29O12]-, m/z 261 [C11H17O7]-, and m/z 99 [C5H7O2]-, which are typical fragmentation patterns of phenylpropanoid glycosides. Peak 5 showed molecular ions at m/z 515.1777 [MH]- and m/z 1030 [2M-H] in negative ion mode, with molecular formula C22H33O55, and MS2 spectrum at m/z 471.1 [MH-CO2]-, Ions are shown at 371 (C5H7O2-) and 16 (C6H10O5-). Based on the fragmentation information, peak 5 was tentatively identified as ussurienoside I. In negative ion mode, peak 6 represents the format adducted molecular ion [M+HCOO]- at m/z 469.1355 and was generated at m/z 325.0, m/z 225.0, and 163.0. The discovered diterpenoid trilactone was identified as ginkgolide B. For peak 8 , in negative ion mode, the additional molecular ion m/z 441.1768 [C44H44O44]-, molecular ion m/z 395.1705 [MH]-, fragment ion m/z 305 [MH-C7 H6]-, and m/z 179 [MH-C14H16O2]- was observed. Peak 8 was tentatively characterized as lobetyolin.

(4) General

The present invention demonstrated that MeOH extract of C. ussuriensis roots exhibits strong inhibitory activity against BNA. In addition, ussurienoside I and tangshenoside I were identified as the main components of C. ussuriensis root extract.

The main compounds showed significant inhibitory activity with IC50 values of 56.0 μM and 203.3 μM, respectively. All compounds showed mixed type I inhibitory activity. In studies to date, ussurienoside I has not been associated with antibacterial activity.

Furthermore, phytochemical compounds present in the extract were confirmed through UPLC-Q-TOF/MS. Two compounds exist in this plant and can be used to produce anti-bacterial drugs.

Example 1. Codonopsis usuriensis ( Codonopsis ussuriensis ) Antibacterial composition containing root extract as an active ingredient

This Example 1 includes C. ussuriensis root extract as an active ingredient in the composition based on Experimental Examples 1 and 2 above. As an example, methanol extract of C. ussuriensis roots can be used.

The present invention can provide an antibacterial preparation formulated in a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient, or diluent to the active ingredient. Here, carriers, excipients, and diluents include sorbitol, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, and not yet determined. Examples include quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Additionally, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate combination. Additionally, when formulating the active ingredient, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, the above pharmaceutical dosage forms can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can.

The solid preparation for oral administration may be prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. The non-aqueous solvent and suspension may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

The preferred dosage of the extract of the present invention varies depending on the patient's condition and weight, degree of disease, age, gender, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, the extract of the present invention is preferably administered at 0.001 to 300 mg/kg, and may be administered once a day or in divided doses. The above dosage does not limit the scope of the present invention in any way.

The extract of the present invention can be administered through various routes to mammals such as rats, mice, livestock, and humans. All modes of administration are contemplated, for example, it may be administered orally, rectally or by intravenous, intramuscular, or subcutaneous injection.

A health functional food composition for antibacterial immunity can be provided by adding a food supplement to the active ingredient of the present invention.

Foods to which the above active ingredients can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc.

The amount of the active ingredient in the food or beverage may be 0.01 to 20% by weight of the total weight of the food or beverage, and the health drink composition may be added at a rate of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. there is.

The health functional beverage composition of the present invention has no particular restrictions on other ingredients other than containing the above-mentioned active ingredients, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose; Common sugars such as disaccharides, such as maltose, sucrose, etc., and polysaccharides, such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above-described flavoring agents, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, organic acids, and protection. It may contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages.

In addition, the composition of the present invention may contain pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

It can also be provided as an antibacterial cosmetic composition containing the active ingredient of the present invention.

The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing products. , oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray, etc., but is not limited thereto. More specifically, it can be manufactured in the form of softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier component. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide ether. Sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it can be applied to the skin and then wiped off, removed, or washed with water. As a specific example, the soap is liquid soap, powdered soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is cleansing foam, cleansing water, cleansing towel, and cleansing pack, and the surfactant-free cleansing formulation is cleansing cream. , cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.

Example 2. Codonopsis usuriensis ( Codonopsis ussuriensis ) Antibacterial preparation containing root extract as an active ingredient

This Example 2 can provide C. ussuriensis root extract as an antibacterial agent based on Experimental Examples 1 and 2 above. As an example, methanol extract of C. ussuriensis roots can be used. Antibacterial agents can also be used as additives to various compositions for antibacterial purposes.

Example 3. Codonopsis usuriensis ( Codonopsis ussuriensis ) Bacterial neuraminidase inhibitor containing root extract as an active ingredient

Based on Experimental Examples 1 and 2 above , C. ussuriensis root extract can be prepared and provided as a bacterial neuraminidase inhibitor. Similar to that described in Example 1, it can be provided as a composition for various purposes.

Example 4. Codonopsis usuriensis ( Codonopsis ussuriensis ) Antibacterial composition containing one or more of root extract-derived compounds 1 and 2 as an active ingredient

Based on Experimental Examples 1 and 2, an antibacterial composition containing one or more of Compounds 1 and 2 as an active ingredient can be provided. As described in Example 1, the composition may be provided as a pharmaceutical, food, or cosmetic composition.

Example 5. Codonopsis usuriensis ( Codonopsis ussuriensis ) Antibacterial preparation containing one or more of root extract-derived compounds 1 and 2 as an active ingredient

Based on Experimental Examples 1 and 2 above, an antibacterial preparation containing one or more of C. ussuriensis root extract-derived compounds 1 and 2 as an active ingredient can be provided.

Example 6. Codonopsis usuriensis ( Codonopsis ussuriensis ) Bacterial neuraminidase inhibitor containing one or more of root extract-derived compounds 1 and 2 as an active ingredient

Example 7. Antibacterial composition, antibacterial agent, or bacterial neuraminidase inhibitor comprising one or more of compounds 1 and 2 shown in Figure 1 as an active ingredient

Claims (7)

An antibacterial composition comprising Codonopsis ussuriensis root extract as an active ingredient. An antibacterial preparation containing Codonopsis ussuriensis root extract as an active ingredient. A bacterial neuraminidase inhibitor containing Codonopsis ussuriensis root extract as an active ingredient. An antibacterial composition containing one or more of the following Compound 1 and Compound 2 as an active ingredient.
An antibacterial preparation containing one or more of the following Compound 1 and Compound 2 as an active ingredient.
A bacterial neuraminidase inhibitor containing one or more of the following Compound 1 and Compound 2 as an active ingredient.
In claim 4,
An antibacterial composition characterized in that the compound 1 and compound 2 are derived from the root extract of Codonopsis ussuriensis.