KR102204452B1 - Method for producing rare saponins using saponins derived from platycodon grandiflorum - Google Patents

Abstract

The present invention relates to a method for manufacturing rare saponin using saponin derived from Platycodon grandiflorus, which manufactures de-apiosyl-xylosylated platicoside by treating and reacting Cytolase PCL5 from platycoside. Therefore, Cytolase PCL5 completely converted a platicoside substrate to a final product without further hydrolysis, indicating that the enzyme is effective in production of de-apiosyl-xylosylated platicoside D and de-apios-xylosylated polygalaxin D.

Description

Method for producing rare saponins using bellflower-derived saponins {Method for producing rare saponins using saponins derived from platycodon grandiflorum}

The present invention relates to a method for producing rare saponins using bellflower-derived saponins, and more particularly, to investigate the enzymatic conversion of platicoside, and Cytolase PCL5 to platicoside E and polygalacin D3 (polygalacin D3) The present invention relates to a method for producing rare saponins using bellflower-derived saponins that completely convert deapiose-xylosylated platicodin D and deapiose-xylosylated polygalaxin D.

Platycoside (platycoside) is a saponin with pharmaceutical activity present in the root of bellflower ( Platycodon grandiflorum ), and has been used as a medicinal plant in Northeast Asia [Non-Patent Documents 1,2]. These pharmacological properties include antioxidants [Non-Patent Literature 3,4], anti-inflammatory agents [Non-Patent Literature 5,6], anti-tumor [Non-Patent Literature 7-9], anti-allergic [Non-Patent Literature 10,11], It is known to have anti-obesity [Non-Patent Document 12-14] and immunomodulatory effect [Non-Patent Document 15-17].

Platicosides consist of a triterpenoid aglycone and two sugar chains located at C-3 and C-28. Most aglycones are platycodigenin-type (C-24 to CH ₂ OH) and polygalacic acid-type (C-24 to CH ₃ ), respectively, with total platicosides. It accounts for about 50% and 30% of [Non-Patent Documents 18 and 19] (see Fig. 1).

Deglycosylation of saponin increases biological activity because bioavailability and cell permeability are improved due to the smaller size [Non-Patent Documents 20 and 21]. Methods such as heating [Non-Patent Literature 22], acid treatment [Non-Patent Literature 23], fermentation [Non-Patent Literature 24], whole cell reaction [Non-Patent Literature 25], and enzyme conversion [Non-Patent Literature 26] It is known to hydrolyze the sugar moiety in glycosylated saponins. Among these methods, enzymatic conversion showed the highest yield, efficiency, and specificity. According to these results, various enzymes have been used to deglycosylate platicosides. Β-glycosidase derived from Caldicellulosiruptor bescii [Non-Patent Document 27], β-glycosidase derived from Caldicellulosiruptor owensensis [28], β- Glycosidase derived from Aspergillus usamii [Non-Patent Document 29], β-glycosidase derived from snailase [Non-Patent Document 30], β-glycosidase derived from laminarinase [Non-Patent Document 31], and β-glycosidase derived from cellulase [Non-Patent Document 32] contain platicoside E. As an intermediate, it was converted to platicodin D through platicodin D3. On the other hand, Dictyoglomus turgidum- derived enzymes were deglycosylated from platicoside E to deglycosylated platicodin D while passing through platicodin D3 and platicodin D as intermediates [Non-Patent Document 33]. Most of these enzymes hydrolyze the glucose molecule at the C-3 position of platicoside.

However, the crude enzyme of Aspergillus niger removed apiose and xylose portions from C-28 when reacting with Platicidine D [Non-Patent Document 34]. Nevertheless, deapiose-xylosylated platycodin D (deapiose-xylosylated platycodin D) has never been produced quantitatively, and other types of platycodin exhibited deapiosylation and dexylosylation. Said didn't exist.

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Inhibitory Effect of the Saponins Derived from Roots of Platycodon grandiflorum on Carrageenan-Induced Inflammation. Biosci. Biotechnol. Biochem. 2006, 70, 858-864. Non-patent literature. Kim, M.; Hwang, I.G.; Kim, S.B.; Choi, A.J. Chemical Characterization of Balloon Flower (Platycodon grandiflorum) Sprout Extracts and Their Regulation of Inflammatory Activity in Lipopolysaccharide-Stimulated Raw 264.7 Murine Macrophage Cells. Food Sci. Nutr. 2020, 8, 246-256. Non-Patent Document 7. Yim, N.H.; Hwang, Y. H.; Liang, C.; Ma, J.Y. A Platycoside-Rich Fraction from the Root of Platycodon grandiflorum Enhances Cell Death in A549 Human Lung Carcinoma Cells Via Mainly Ampk/Mtor/Akt Signal-Mediated Autophagy Induction. J. Ethnopharmacol. 2016, 194, 1060-1068. Non-Patent Document 8. Kim, Y.S.; Kim, J.S.; Choi, S.U.; Kim, J.S.; Lee, H.S.; Roh, S.H.; Jeong, Y.C.; Kim, Y.K.; Ryu, S.Y. Isolation of a New Saponin and Cytotoxic Effect of Saponins from the Root of Platycodon grandiflorum on Human Tumor Cell Lines. Planta. Med. 2005, 71, 566-568. Non-Patent Document 9. Khan, M.; Maryam, A.; Zhang, H.; Mehmood, T.; Ma, T. Killing Cancer with Platycodin D through Multiple Mechanisms. J. Cell Mol. Med. 2016, 20, 389-402. Non-Patent Document 10. Halliwell, B. Oxidative Stress and Neurodegeneration: Where Are We Now? J. Neurochem. 2006, 97, 1634-1658. Non-Patent Document 11. Oh, Y.C.; Kang, O.H.; Choi, J.G.; Lee, Y.S.; Brice, O.O.; Jung, H.J.; Hong, S.H.; Lee, Y.M.; Shin, D.W.; Kim, Y.S.; et al. Anti-Allergic Activity of a Platycodon Root Ethanol Extract. Int. J. Mol. Sci 2010, 11, 2746-2758. Non-Patent Document 12. Han, L.K.; Zheng, Y. N.; Xu, B.J.; Okuda, H.; Kimura, Y. Saponins from Platycodi Radix Ameliorate High Fat Diet-Induced Obesity in Mice. J. Nutr. 2002, 132, 2241-2245. Non-Patent Document 13. Zhao, H.L.; Harding, S. V.; Marinangeli, C. P. F.; Kim, Y.S.; Jones, P.J.H. Hypocholesterolemic and Anti-Obesity Effects of Saponins from Platycodon grandiflorum in Hamsters Fed Atherogenic Diets. J. Food Sci. 2008, 73, H195-H200. Non-Patent Document 14. Hwang, K.A.; Hwang, Y.J.; Im, P.R.; Hwang, H.J.; Song, J.; Kim, Y.J. Platycodon grandiflorum Extract Reduces High-Fat Diet-Induced Obesity through Regulation of Adipogenesis and Lipogenesis Pathways in Mice. J. Med. Food 2019, 22, 993-999. Non-Patent Document 15. Xie, Y.; Ye, Y.P.; Sun, H.X.; Li, D. Contribution of the Glycidic Moieties to the Haemolytic and Adjuvant Activity of Platycodigenin-Type Saponins from the Root of Platycodon grandiflorum. Vaccine 2008, 26, 3452-3460. Non-Patent Document 16. Noh, E.M.; Kim, J.M.; Lee, H.Y.; Song, H.K.; Joung, S.O.; Yang, H.J.; Kim, M.J.; Kim, K.S.; Lee, Y.R. Immuno-Enhancement Effects of Platycodon grandiflorum Extracts in Splenocytes and a Cyclophosphamide-Induced Immunosuppressed Rat Model. BMC Complement. Altern. Med. 2019, 19, 322. 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The present invention has been devised in consideration of the above situation, and the problem to be solved in the present invention is to provide a new type of platicoside through deapiosylation or dexylosylation.

In addition, the problem to be solved in the present invention is to provide a new enzyme that can contribute to the deglycosylation of platicodin.

The method for producing rare saponins using bellflower-derived saponins according to the present invention for solving the above problems is deapiosyl-xylosylated platicoside by treating and reacting Cytolase PCL5 from platicoside. It is characterized by a technical feature to prepare xylosylated platycoside).

The platicoside of the present invention for solving the above problems is platicoside E or polygalacin D3, and the deapiosyl-xylosylated platicoside is It is technically characterized in that it is deapiose-xylosylated platycodin D (deapiose-xylosylated platycodin D) or deapiose-xylosylated polygalacin D (deapiose-xylosylated polygalacin D).

The deapiose-xylosylated platicodin D of the present invention for solving the above problems is a technical feature that is prepared through the following route; Platicoside E → Platicodin D3 → Platicodin D → Deapiosylated Platicodin D → Deapios-xylated Platicodin D.

The deapiose-xylosylated polygalaxin D of the present invention for solving the above problems is characterized in that it is produced through the following route; Polygalaxine D3 → Polygalaxine D → Deapiosylated polygalaxine D → Deapiose-xylosylated polygalaxine D.

The reaction conditions of the present invention for solving the above problems are characterized by a pH of 4.5 to 5.5 and a temperature of 45 to 55 ℃.

The platicoside of the present invention for solving the above problems is a technical feature that is extracted from the root of bellflower ( Platycodon grandiflorum ).

In the method for preparing rare saponins using bellflower-derived saponins according to the present invention, it was confirmed that deglycosylated saponins exhibit higher biological activity than glycosylated saponins.

In addition, in the method for producing rare saponins using bellflower-derived saponins according to the present invention, Cytolase PCL5 completely converted the platicoside substrate into the final product substrate without further hydrolysis, and this enzyme is deapiose-xylosylated. It means that it is effective in the production of platicodin D and deapiose-xylosylated polygalaxin D.

1 is ^® platinum coordination Janine found in the roots of Platycodon grandiflorum - type (platycodigenin ^® -type) Atlantico Playa side and go to Leshan poly-type (polygalacic acid-type) of the chemical structure Plata Tico side. Platicosides contain glycosides at positions C-3 and C-28. The glycosides of C-3 are Glc, Glc-Glc and Glc-Glc-Glc. The glycosides of C-28 are Arap-Rha-Xyl and Arap-Rha-Xyl-Api. Glc, β-D-glucopyranosyl-; Arap, α-L-arabinopyranosyl-; Rha, α-L-rhamnopyranosyl-; Xyl, β-D-xylopyranosyl-; And Api, β-D-apiofuranosyl-
Figure 2 is an HPLC profile showing an unknown product obtained after the catalyst of (a) Platicoside E and (b) polygalaxin D3 by Cytolase PCL5. Standards used in the assay include Platicoside E, Platicodin D3, Platicodin D, Deapiosylated Platicodin D, Polygalaxin D3 and Polygalaxin D (1, 2, 3, 4, 5 and 6) was included. Red 1', 2', and 3'represent unknown products in Platicoside E and Polygalaxin D3, respectively.
Figure 3 is the effect of temperature and pH on the activity of Cytolase PCL5 on Platicoside E. (a) Effect of pH. The reaction was carried out for 10 minutes with 0.4mM Platicoside E in 50mM citrate/phosphate buffer (pH 4.0-7.0). (b) The effect of temperature. The reaction was carried out at 40-65° C. in 50 mM citrate/phosphate buffer (pH 5.0) with 0.4 mM Platicoside E for 10 minutes. Data are expressed as the mean of 3 experiments and error bars represent standard deviation.
FIG. 4 is a bioconversion of platicoside E to deapios-xylated platicodin D via platicodin D3, platicodin D and deapiosylated platicodin D. FIG.
Figure 5 is a biotransformation of platicogenin-type and polygalaxane-type platicoside by Cytolase PCL5. (a) Platicoside E (closed circle) is deapiosylated via platicodin D3 (closed triangle), platicodine D (closed square) and deapiosylated platicodine D (open triangle). Biotransformation to Platicodin D (open diamond). (b) Polygalaxin D3 (closed triangle) through polygalaxin D (closed diamond) and deapiosylated polygalaxin D (open triangle), deapios-xyloxylated polygalaxin D (open square) ) To biotransformation. Data are presented as the mean of 3 experiments and error bars represent standard deviation.
6 is an LCMS/HRMS analysis of Deapi-dexyl-platycodin D (1′)
7 is an LCMS/HRMS analysis of deapi-patycodin D (2′)
8 is an LCMS/HRMS analysis of Deapi-dexyl-platycodin D (3′)

Terms and words used in the 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 terms in order to describe his or her own invention in the best way." It should be construed as a meaning and concept, but not limited to a conventional or dictionary meaning.

Therefore, the embodiments described in the present 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, and thus various equivalents that can replace them at the time of application It should be understood that there may be water and variations.

The experiments to be carried out below were performed according to each of the following experimental methods.

Experimental method 1. Material preparation

Cytolase PCL5 was purchased from DSM Food Specialties (Heerlen, Netherlands). Platicoside standard substances (platicoside E, platicodin D3, platicodin D, deapiosylated platicodin D, and polygalaxin D3 (polygalaxin D3)) are from Ambo Laboratories (Daejeon, Republic of Korea). ). Polygalaxin D was provided by Dr. Dae-Young Lee of the National Institute of Horticultural and Herbal Science in Eumseong, Korea. From the reaction product catalyzed by Cytolase PCL5, deapios-xylosylated platicodin D, deapiosylated polygalaxin D, and deapios-xylosylated polygalaxin D were purified. Platicoside was prepared by using a Hydrosphere C18 prep column (10 × 250 mm, 5 μm particle size; YMC, Kyoto, Japan) for preparative high performance liquid chromatography (Prep-HPLC) (Agilent 1260; Agilent, Santa Clara, USA, CA). Prepared by). The product was detected by eluting the column with water at a flow rate of 4.7 mL/min at 30° C. and measuring the absorbance at 203 nm. The thus prepared platicoside was used as a standard for quantitative and qualitative analysis.

Experimental method 2. Analysis of hydrolysis activity

Unless otherwise stated, reactions were carried out at 50° C. for 10 minutes in 50 mM citrate/phosphate buffer (pH 5.0) containing 0.05 mg/mL Cytolase PCL5 and 0.2 mg/mL platicoside. Platicoside E, Platicodin D3, Platicodin D, Deapiosylated Platicodin D, Deapios-xylated Platicodin D, Polygalaxin D3, Polygalaxin D, Deapiosylated Polygal The specific activity of Cytolase PCL5 against Laxin D and deapios-xylosylated polygalaxin D was at 50° C. and pH at various concentrations of enzymes (0.005-0.5 mg/mL) and 0.4 mg/mL of each platicoside. It was evaluated for 10 minutes under the condition of 5.0. The effect of pH and temperature on the activity of Cytolase PCL5 was confirmed by changing the pH to 4.0 to 7.5 at 50°C and changing the temperature to 40 to 65°C at pH 5.0, respectively.

Experimental method 3 biotransformation

The biotransformation from platicoside E to deapiose-xylosylated platicodin D and polygalaxin D3 to deapiose-xylosylated polygalaxin D was 0.5 mg/mL Cytolase PCL5 and 1 mg/ mL of a 50 mM citrate/phosphate buffer (pH 5.0) containing Platicoside E and Polygalaxine D3, respectively, was carried out at 50° C. for 15 and 18 hours.

Experimental method 4. HPLC analysis

The same amount of n-butanol was added to the reaction mixture to stop the reaction and the platicoside was extracted. This separated the reaction into an aqueous and n-butanol soluble fraction. The n-butanol soluble fraction of the extract was dried to completely evaporate butanol. Subsequently, the dried residue containing platicoside was dissolved in methanol, and an HPLC system (Agilent 1100) was performed at a wavelength of 203 nm with a hydrosphere C18 column (4.6×150 mm, 5 μm particle size; YMC, Kyoto, Japan). Analyzed using. The column was eluted with solvent A (acetonitrile) and solvent B (water) at a temperature of 30° C., and the ratio and elution time were 10:90 to 40:60 for 30 minutes, 40:60 to 15 minutes at a ratio of 90:10, 5 minutes at a ratio of 90:10 to 10:90, and 10 min at a constant ratio of 10:90, and the flow rate was 1 mL/min. A linear calibration curve for the logarithmic value of the peak area and the concentration of platicoside was constructed using a standard platicoside solution (0.2 to 1.0 mM), and the curve was used to determine the concentration of platicoside. Was used.

Experimental Method 5. Platicoside liquid chromatography-mass spectrometry

Platicoside was further analyzed using Waters SYNAPT G2-Si HDMS instrument (Waters Co. Taunton, MD, USA). The system consists of a Waters Acquity UPLC system connected to a quadrupole time-of-flight mass spectrometer. Samples were eluted using a Waters Acquity BEH C18 column (100 mm×2.1 mm, 1.7 mm) set at 40°C. All solvents used were of LC-MS grade and ultrapure 18.2 MΩ water was used in each step. Mobile phase A consisted of water+0.1% formic acid, whereas mobile phase B was acetonitrile+0.1% formic acid. Gradient elution is 100% A (0.00 min), 95% A + 5% B (0.00-1.00 min), 5% A + 95% B (1.00-9.00 min), 100% B (9.00-10.50 min), 100 % A (10.50-11.00 min), 100% A (11.00-12.50 min), the flow rate was 0.4 ml/min, and the injection volume was 3 μL for positive mode and 2 μL for cathode mode.

Mass spectrometry was performed through electrospray ionization using MSE (Mass Spectrometry Elevated Energy) centered experiments, and the MSE was performed in both positive and negative modes, and the analyzer was performed at full with at half maximum (FWHM) resolution. The m/z scan range of 50-1500 Da was screened with the mode set. Scanning was performed every 0.5 sec. The collision energy was set with two functions: one low energy function with no collision energy applied, voltage 2.00 kV, sampling cone 10 V, source temperature 100°C, desolvation temperature 400. Both high-energy functions with ℃ and 700 L/h of desolvation gas applied. Prior to sample analysis by direct injection of sodium iodide calibrant solution, the correct mass was initially calibrated. In addition, leucine encephalin lock mass solution (2 ng/μL) was injected at 5 μl/min parallel to the flow rate of the mobile phase, and scanned and automatically corrected to check the exact mass, and the scan range in the submitted sequence This ensures full mass accuracy (<5 ppm). The instrument was controlled and data analysis was performed using Masslynx v.41 software.

Each of the following experimental examples was performed based on the above experimental methods, and the results will be described.

Experimental Example 1. Identification of the product obtained by the action of Cytolase PCL5 on Platicoside E and Polygalaxin D3

Cytolase PCL5 was used in the present invention to promote biotransformation of platicoside E and polygalaxin D3. The product was prepared by HPLC using a reversed-phase hydrosphere C18 column and reference standards such as Platicoside E, Platicodin D3, Platicodin D, Deapiosylated Platicodin D, Polygalaxin D3, and Polygalaxin D. It was analyzed (see Fig. 2). The results of the present invention show that the enzyme converts Platicoside E to a product having the same retention time as the deapiosylated platicodine (26.3 min), while the retention time of the other product is 25.7 min. Polygalaxine D3 was catalyzed with two unknown products with retention times (2', 27.0 min and 3', 26.7 min) that did not match the standard.

Three unknown products were characterized using LC/TOF-MS in positive and negative modes. Its theoretical mass, molecular formula and observed mass are summarized in Table 1. The total ion chromatogram (TIC) of the three unknown products is shown in Figures S1-S3. For compound 1'of ESI+, [M+H] ⁺ pseudomolecular ions are presented in m/z 961.4966, which corresponds to the molecular formula of C ₄₇ H ₇₆ O ₂₀ . In ESI-, [M + HCOO] ^- adduct was detected in the compounds 2 'and 3'. Due to the high resolution, compound identification was based on the exact mass of each molecule. As a theoretical value, the mass accuracy of the experimental mass data was less than 5 ppm. According to HRMS (High-Resolution Mass Spectrometry) data, the three unknown products were deapiosylated platicodine D (1'), deapiosylated polygalaxine (3') and deapiosylated platicodine D ( 2').

Experimental Example 2. Effect of pH and temperature on the hydrolytic activity of Cytolase PCL5

Optimal conditions for Cytolase PCL5 activity have been reported in a wide range of pH (2.5-5.0) and temperature (10-55°C) [Non-Patent Document 37]. In the present invention, using Platicoside E as a substrate, the hydrolytic activity of Cytolase PCL5 was analyzed at 40-65°C over pH 4.0-7.0. Maximum activity was observed at pH 5.0 (Fig. 3A). The enzyme activity at pH 4.0 was less than 25% of the maximum activity, indicating that Platicoside E was sensitive to pH changes when used as a substrate. Next, as a result of examining the effect of temperature on the enzyme activity, the maximum activity was observed at 50°C (see FIG. 3B).

In addition, flavanone rutinoside [Non-Patent Document 35] (pH 4.0 and 60°C), white ginseng extract [Non-Patent Document 36] (pH 4.3 and 55°C) and red ginseng powder [Non-Patent Document 38] (pH 5.0 and 45[deg.] C.), the optimum pH and temperature of Cytolase PCL5) was similar to the substrate regardless of glycosides.

Example 3. Substrate specificity of Cytolase PCL5 for platicoside

The substrate specificity of Cytolase PCL5 was investigated using platycodigenin-type and polygalaxane-type platicoside as substrates (see Table 2). The specific activity of the enzyme was observed in the following order: Platicoside E> Platicodin D3> Platicodin D> Deapiosylated Platicodin D, and Polygalaxin D3> Polygalaxin D> Deapiosylated Poly Galaxin D. These results show that Cytolase PCL5 acts more efficiently in the external glucose moiety (C-3) than glycosides in C-28, and acts more efficiently in apiose in C-28 than in xylose in C-28. Show. The hydrolysis of glucose of C-3 and apiose of C-28 was 3.1-1.3 times higher in polygalaxane-type than platicodigenin-type platicoside. In contrast, the hydrolysis of xylose of C-28 was 1.2 times higher in the platicodigenin-type than in the polygalaxane-type.

Based on the substrate specificity results, the biotransformation pathway of Platicoside E by Cytolase PCL5 was determined as follows: Platicoside E → Platicodin D3 → Platicodin D → Deapiosylated Platicodin D → De Apiose-xylosylated Platicodin D (see Figure 4). The biotransformation pathway of polygalaxin D3 was similar to the transformation sequence of platicodin D3 as follows: polygalaxin D3 → polygalaxin D → deapiosylated polygalaxin D → deapios-xylated poly Galaxin D. The pathway from platicoside E to deapiose-xylosylated platicodin D and from polygalaxin D3 to deapiose-xylosylated polygalaxin D have not yet been reported. This is the first report on piosylated polygalaxin D and deapios-xylosylated polygalaxin.

Experimental Example 4. Biotransformation of Platicoside E and Polygalaxin D3 to Deapios-Xylosylated Platicoside by Cytolase PCL5

Biotransformation of platicoside E and polygalaxin D3 into deapiose-xylosylated platicodin D and deapiose-xylosylated polygalaxin D was performed as 0.5 mg/mL of enzyme and substrate. It was carried out with 1 mg/mL of platicoside. The enzymes converted platicoside E and polygalaxin D3 into deapiose-xylosylated platicodin D and deapiose-xylosylated polygalaxin D within 12 and 15 hours, respectively, wherein the concentration was They were 0.62 and 0.69 g/L, respectively, and productivity was 51.70 and 45.95 mg/L/h, respectively (see FIG. 5). Because the sugar portion of the substrate was removed from the substrate, the concentration of deapiose-xylosylated platicoside was lower than that of the substrate. Platicosides such as Platicoside E, Platicodin D3 and Polygalaxin D3 were not detected after 5 minutes due to rapid conversion. The use of commercially available food-grade enzymes for the production of small amounts of platicoside is economically advantageous due to high enzyme activity and stability. In addition, the enzyme can be used as an industrial biocatalyst through an additional process such as immobilization through improvement of stability and biocatalytic functionality [Non-Patent Document 39-42].

Platycoside, a saponin abundantly contained in Gilkyung (the root of Platycodon grandiflorum ), has various pharmacological activities and is used as a food supplement. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are being made to convert glycosylated platicosides to deglycosylated platicosides using enzymes; However, the lack of diversity and specificity of these enzymes limited the types of platicosides that could be deglycosylated. In the present invention, the enzymatic conversion of platicoside was investigated, and Cytolase PCL5 converts platicoside E and polygalacin D3 to deapios-xylosylated platicodin D and deapios-xylosylation. Complete conversion to polygalaxin D, which was confirmed by LC-MS analysis. The platicoside substrate was hydrolyzed through the following novel hydrolysis pathway: Platicoside E → Platicodin D3 → Platicodin D → Deapiosylated Platicodin D → Deapios-xylated plaque. Thycodin D; And polygalaxine D3 → polygalaxine D → deapiosylated polygalaxine D → deapios-xylosylated polygalaxine D.

In the present invention, it was shown that Cytolase PCL5 exhibited new substrate specificity by hydrolyzing xylose and apiose at C-28 as well as the external glucose moiety at C-3 of Platicoside E or Polygalaxin D3, respectively. Cyotolase PCL5 is an enzyme blend rather than an individual enzyme, so it can be specific to a variety of sugars. The chemical structure of the unknown product obtained by the catalysis of platicoside was confirmed by LC-MS, and the deapiose-xylosylated platicodin D and the deapiose-xylosylated polygalaxin D were final It was identified as a product.

Cytolase PCL5 completely converted the platicoside substrate into the final product without further hydrolysis, and this enzyme is responsible for the production of deapios-xylosylated platicodin D and deapios-xylosylated polygalaxin D. Showed effective. Food grade commercial enzymes are economically suitable for industrial applications through additional immobilization techniques. The present invention is the first invention to report the quantitative production of deapiose-xylosylated platicodine D and deapiose-xylosylated polygalaxin D.

The results of the present invention show that Cytolase PCL5 can play a potential role in the development of biologically activated platicosides that can be used for various pharmacological activities.

Claims (6)

Deapiose-xylosylated platycoside was prepared by treating and reacting Cytolase PCL5 from platycoside,
The platicoside is platicoside E or polygalacin D3,
The deapiosyl-xylosylated platicoside is deapiose-xylosylated platycodin D (deapiose-xylosylated platycodin D) or deapiose-xylosylated polygalacin D (deapiose-xylosylated polygalacin D). Characterized in that,
Bellflower-derived method for producing rare saponins using saponins.
delete The method according to claim 1,
The method of producing rare saponins using bellflower-derived saponins, characterized in that the deapios-xylated platicodin D is prepared through the following route;
Platicoside E → Platicodin D3 → Platicodin D → Deapiosylated Platicodin D → Deapios-xylated Platicodin D.
The method according to claim 1,
The method for producing rare saponins using bellflower-derived saponins, characterized in that the deapios-xylosylated polygalaxin D is prepared through the following route.
Polygalaxine D3 → Polygalaxine D → Deapiosylated polygalaxine D → Deapiose-xylosylated polygalaxine D.
The method according to any one of claims 1, 3 and 4,
The reaction is a method for producing rare saponins using bellflower-derived saponins, characterized in that the reaction is carried out under conditions of pH 4.5 to 5.5 and a temperature of 45 to 55°C.
The method according to any one of claims 1, 3 and 4,
The method for producing rare saponins using bellflower-derived saponins, wherein the platicoside is extracted from the roots of bellflower ( Platycodon grandiflorum ).

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