KR102102852B1 - Manufacturing method for platycodon saponin composition - Google Patents

Abstract

A platycodon saponin composition according to the present invention is prepared by bioconverting a platycodon extract containing platycodon saponin by an enzyme containing a lipase. By increasing the content of platycodin D, it is possible to improve an effect of inhibiting fat accumulation.

Description

Manufacturing method of platinum codon saponin composition {MANUFACTURING METHOD FOR PLATYCODON SAPONIN COMPOSITION}

The present invention relates to a bellflower extract, and more particularly, to a platinum codon saponin composition bioconverted by an enzyme to a platinum codon extract containing platinum codon saponin.

Platycodi-Radix (hereinafter, 'Platycodon') is the root of the Platycodon grandiflorum A. DC., A plant in the Campanulaceae family, and is known as Kilkyoung in Korea and Jigeng in China. ), Which is called Kikyo in Japan and has been used as a traditional oriental medicine. Platycodone is a herbal medicine that has been used in oriental medicine for respiratory diseases such as drainage, expectoration, bronchitis, asthma, and anti-inflammatory, and there are 273 cases of herbal prescriptions containing bellflowers, 49 cases in the drug combination. It is known to have pharmacological action. The main pharmacological component of platinum codon is platinum codon saponin, which contains about 2% of about 40 types of 5-cyclic oleanane-type triterpenoid saponin, in addition to inulin, Bettulin and stigmasterol are the main ingredients.

Platycodone saponin is a platycodine-based saponin that uses platycodigenin as a glycoside, and polygalacic acid as a glycoside, depending on the presence or absence of an alcohol group at the position 4 of the glycoside. Several platinum codon saponins have been reported to date, according to the type and number of sugars bound to carbons 3 and 28 of the saponins of the polaragalasin family (Lu Qiu et al., Journal of Asian Natural Products Research., (2018): Platycosides P and Q, two new triterpene saponins from Platycodon grandiflorum ). Among them, platycodin D, platycoside E, and polygalacin D are the main components of the longevity in content.

In particular, Platycodin D is a saponin that has the main pharmacological component of Platycodon grandiflorum A. DC., Anti-apoptosis, Republic of Korea Registration No. 10-0564927, anti-obesity, Republic of Korea Registration No. 10-0750333), anti-inflammation (anti-inflammation, Republic of Korea Registration No. 10-0696647) efficacy is known, a single component with a large pharmacological value (Hahn J., Nutr., 130, p .2760, 2000). Particularly, in order to maximize the effect of suppressing the differentiation of adipocytes from platicodine D, a method of bioconverting long-distance saponin fractions containing a large amount of platicodine D by enzymes has been developed (Patent Registration No. 1083045).

However, despite the excellent pharmacological effect and efficacy of platinum codon, the bellflower taste includes a combination of arithmetic, bitter and sweet tastes in addition to the structural texture and aroma. The old bellflower has good medicinal properties, but because it gives a sore throat like pain in the neck and tongue, it cannot be used as a tea or health drink, such as green tea or tangerine tea.

The present invention is intended to solve the above-described problem, and solves the limitations on the taste of platicone to increase the degree of preference, and at the same time, unlike the previously used enzyme, glycoside of carbon number 3 in the molecular structure of platinum codon saponin It is an object of the present invention to provide a platinum codon saponin composition and a method of manufacturing the same, which can sufficiently exert the pharmacological efficacy of platinum codon, by preserving the oligosaccharide linked to carbon 28 while cutting the bond.

The objects of the present invention are not limited to the objects mentioned above, and other objects not mentioned will be clearly understood from the following description.

The platinum codon saponin composition according to the present invention is characterized in that it is prepared by bioconversion of a platinum codon extract containing platinum codon saponin by an enzyme containing a lipase.

Here, the platinum codon extract may be an enhanced fraction of platinum. In addition, the platinum codon extract may be formed by hot extraction of platinum.

In another aspect, the present invention may be embodied as a pharmaceutical composition or a health functional food, using the aforementioned platinum codon saponin composition as an active ingredient.

In another aspect, a method of preparing a platinum codon saponin composition according to the present invention comprises: a first step of obtaining a platinum codon extract containing platinum codon saponin from platinum; And a second step of reacting the platinum codon extract with an enzyme containing lipase; wherein the platinum codon saponin contained in the platinum codon extract is bioconverted by the lipase.

When lipase was treated as an enzyme to platinum codon saponin (long-chain saponin), it was confirmed that the content of platicodine D and polygalacin D was increased by sugar hydrolysis. When platase is used to enzymatically treat platinum codon saponins, only the sugar chains located at carbon 3 are selectively cleaved while maintaining the sugar chain located at carbon 28. As a result, when using the platinum codon saponin composition enzymatically treated with lipase, it is possible to improve the fat accumulation inhibitory effect through an increase in the content of platicodine D, and at the same time, improve the bitter and arin tastes due to simplification of the sugar chain.

1 shows a chromatogram analyzed by HPLC of a platinum codon saponin composition having a different lipase content.
Figure 2 shows the HPLC-ELSD chromatogram confirming the bioconversion effect of the enzymes used in the examples and the enzymes used in the comparative examples with respect to the standard product of platicodine D.
Figure 3 shows the HPLC-ELSD chromatogram confirming the bioconversion effect of the enzyme used in the examples and the enzyme used in the comparative examples for the standard product of polygalacin D.
FIG. 4 shows the results of ESI EIC using a mass spectrometer for a platicodone saponin composition sufficiently bioconverted by Examples and Comparative Examples, in comparison with a standard platicodine D.
FIG. 5 shows the results of ESI EIC using a mass spectrometer for a platinum codon saponin composition sufficiently bioconverted by Examples and Comparative Examples in comparison with the standard polygalacin D.
6 is a view schematically illustrating the process of enzymatic conversion of saponins of the platinum-type family.
7 is a graph showing the effect of inhibiting the differentiation of adipocyte differentiation of PD, PD3 and PE based on the control group.
FIG. 8 shows images taken with an optical microscope after treating 1.25 μM of Platycodin D and Diafi-Platinodine D, respectively, for mast cells.
Figure 9 shows the sensory evaluation results for the enzyme-treated Dorajicheong (Sample 1) and lipase enzyme-treated Dorajicheong (Sample 2).

The inventors of the present invention have developed a method of bioconverting long-distance saponin fractions containing a large amount of platicodine D by enzymes in order to maximize the effect of suppressing the adipocyte differentiation of platicodine D (Korea Patent Registration) 1083045). In this method, as an enzyme for the bioconversion of the extract, the glucosidase, Cellulase, Lactase, Mannosidase, Glycosidase, Galatosidase, Galactosidase ), Naringinase, Hesperidianase, or Glucuronidase.

However, among the enzymes used in the related art, it was found that as the cellulase decomposes the sugar chain bound to carbon 28, the content of platicodine D decreases as the enzymatic reaction proceeds. In addition, it was confirmed that other enzymes had low sugar chain cleavage activity located at carbons 3 or 28.

The inventors of the present invention, as a result of repeated research on long saponins, the inventors of the present invention, when using a lipase as an enzyme for the bioconversion of saponins, while increasing the content of platinum codon saponins with excellent pharmacological efficacy, 3 It was found that sugar molecules bound to carbon once can be selectively removed to improve the taste of bellflower. Through this, a platinum codon saponin composition according to the present invention to be described below and a method of manufacturing the same were developed.

Specifically, the polaricodon saponin composition according to the present invention is characterized in that it is prepared by bioconverting a platinum-containing extract containing platinum codon saponin by an enzyme containing lipase. In addition, the platinum codon extract may be formed by extraction methods such as cold needle extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, and heating extraction, and preferably hot water extraction. When using the hot water extraction method, water, ethanol, methanol, and the like can be used as a solvent. In addition, the platinum codon extract may use an enhanced fraction containing platinum codon saponin. The platinum codon saponin composition thus prepared may be used as a pharmaceutical composition or a health functional food containing it as an active ingredient.

Hereinafter, the present invention will be described in detail.

[I. Preparation of platinum codon extract]

1. Preparation of hot water extract

1 kg of chopped and dried 2-year-old roads are placed in a 5 L reaction tank, 3 L of filtered drinking water is added thereto, heated at 90 ° C. for 5 hours to extract, and the filtrate obtained by filtration using a paper filter is used under a reduced pressure distiller. And concentrated in vacuo to obtain a concentrate. In this way, as a hot water extract of Gilkyung, 200 g of a concentrate having a sugar content of 60 brix was obtained.

2. Preparation of reinforced fractions

1 kg of the shredded and dried 2-year-old road was extracted with methanol, and the obtained methanol extract was dried under reduced pressure, and then dissolved in distilled water to adsorb on a column (100 cm x 10 cm) filled with a polymer synthetic resin (Diaion-HP20), Water-soluble substances including polysaccharides were removed through chromatography using a water: methanol solvent system. The fraction obtained through this process was concentrated under reduced pressure using a vacuum distiller and a lyophilizer to obtain 3.5 g of a powdered powder containing a large amount of long saponin.

[II. Preparation of enzyme-treated platinum codon saponin composition]

[Example 1]

After diluting 60 mL of platycodone concentrate of 60brix obtained from I-1 with 25 mL of drinking water at pH 5.5, lipase was added as an enzyme, followed by enzymatic reaction at a temperature of 55 ° C. for 24 hours in an incubator. For enzyme inactivation, the reaction was performed in a water bath at 80 ° C. for 3 minutes to obtain a final platinum codon saponin composition. For reference, the lipase used herein is a powder-type enzyme extracted from Aspergillus niger purchased from SIGMA-ALDRICH and has an activity of 165.4 units per gram.

[Example 2]

After dissolving 10 mg of the platinum-enriched fraction obtained in I-2 in 5 mL of distilled water at pH 5.5, lipase was added, followed by enzymatic reaction at 55 ° C. for 24 hours in an incubator, followed by 80 ° C. for enzyme inactivation. The reaction was carried out in a water bath for 3 minutes to obtain a final platinum codon saponin composition.

[Comparative Example 1]

In the same manner as in Example 1, a long-cured saponin composition was prepared, but in order to compare the efficacy with lipase used as an enzyme in Example 1, glucanase and xylanase, enzymes used in the prior art, Comparative Example 1 was prepared using Viscozyme L as an enzyme including Xylanase, Cellulase, and Hemicellulase.

[Comparative Example 2]

In the same manner as in Example 2, a long-cured saponin composition was prepared, but in order to compare the efficacy with lipase used as an enzyme in Example 2, glucanase, xlucanase, which are enzymes used in the prior art ( Comparative Example 2 was prepared using Viscozyme L as an enzyme including Xylanase), cellulase, and hemicellulase.

[III. Analysis]

[One. Analysis of the components of platinum codon saponin according to enzyme treatment]

High performance liquid chromatography (HPLC; High Performance Liquid Chromatography) was performed to analyze the components of platinum codon saponin. As the analysis equipment, an Agilent 1100 series was used, and all reagents used in HPLC analysis were purchased from J.T.Baker (USA) for HPLC. As a solvent, water (0.1% formic acid) and acetonitrile (0.1% formic acid) are used, but the content of acetonitrile is changed from 20% to 100% for 60 minutes of retention time in a gradient method. While flowing at 0.9 mL / min, an HPLC analysis was performed at room temperature using an INNO C18 column (250 mm, 4.6 mm id, particle size 5 μm) as an analysis column. As a detector, a vapor light scattering detector (ELSD; Evaporative Light Scattering Detector, Sedex 60LT, France) was used, and the ELSD system conditions were set to 60 ° C, 12.0 gain, and a nebulizer N2 gas 2.5 bar.

1 shows a chromatogram analyzed by HPLC of a platinum codon saponin composition having a different lipase content according to Example 1. 1, the amount of lipase was changed to 0 mg (A), 50 mg (B), 100 mg (C), 150 mg (D) and 200 mg (E) with respect to 5 g (60 brix) of platinum codon concentrate prepared by hot water extraction. The results of the analysis are shown. As shown in FIG. 1, as the weight of the lipase used for the enzymatic reaction increases, under the given enzymatic reaction conditions (pH 5.5, 55 ° C., 24 hours), the retention time between A and D is between 10 and 20 minutes. It was confirmed that the saponin components were not completely bioconverted based on the remaining peaks (areas indicated by dotted boxes). On the other hand, in the case of E, it can be seen that all of the peaks (areas indicated by the dotted box) between the retention time 10 minutes and 20 minutes disappeared, and thus it can be seen that enzymatic conversion was sufficiently performed.

On the other hand, in order to confirm the difference between Example 2 and Comparative Example 2, HPLC analysis was performed using a reference standard of Platycodin D and Polygalacin D. Here, the standard used in the analysis was separated by countercurrent chromatography (Countercurrent Chromatography).

Figure 2 shows a chromatogram confirming the bioconversion effect of the enzyme used in Example 2 and the enzyme used in Comparative Example 2 with respect to the standard product of platicodine D. As shown in Figure 2, in the case of the enzyme treatment with lipase as in Example 2 (B), in light of the peak (1) corresponding to the platicoidin D with a molecular weight (Molecular weight) of 1224.5 is maintained, the plasmid by lipase It can be seen that the enzymatic conversion of thycodine D does not occur. On the other hand, when treated with Biscozyme L as in Comparative Example 2 (C), it was confirmed that the peaks (1) corresponding to Platycodin D were completely eliminated and new peaks 2, 3, and 4 were generated. Among them, the peak 3 was confirmed by using a mass spectrometer that Diapio-Platinoid D, which is a truncated form of the terminal apioz among the oligosaccharides attached to carbon 28. Accordingly, it can be seen that when biscozyme L was treated with respect to platicodine D, glycolysis was performed and split into various peaks. For reference, A in FIG. 2 shows a chromatogram for the Platydin D standard.

Similarly, as shown in Figure 3, through a chromatogram confirming the bioconversion effect of the enzyme used in Example 2 and the enzyme used in Comparative Example 2 with respect to the standard product of polygalacin D, the enzyme with lipase as in Example 2 In the case of treatment (B), in view of the peak 1 corresponding to polygalacin D having a molecular weight of 1208.6 is maintained, it can be seen that enzymatic conversion of polygalacin D by lipase is not performed. On the other hand, when treated with Biscozyme L as in Comparative Example 2 (C), it was confirmed that peak 1 corresponding to polygalacin D was completely eliminated and new peaks including peak 2 were generated. Accordingly, it can be seen that even when biscozyme L is treated with respect to polygalacin D, glycolysis is performed to split into various peaks. For reference, FIG. 3A shows a chromatogram for the polygalacin D standard.

2 and 3, when enzymatically treated with lipase, the enzymatic conversion of platicodine D and polygalacin D does not occur, whereas in the case of enzymatic treatment with biscozyme L, platinumicodine D and It can be seen that polygalacin D is enzymatically converted. This can be understood because, when enzymatically treated with biskozyme L, the sugar chains bound to carbon 28 of platicodine D or polygalacin D are decomposed and split into various peaks.

In addition, the results of performing mass spectrometry (Mass Spectrometry; ESI-EIC, Negative mode, ionization energy 180v) for the platinum codon saponin composition in the state where the enzymatic reaction was moderately advanced by Example 2 and Comparative Example 2 And FIG. 5. Here, A in FIGS. 4 and 5 shows chromatograms before the enzymatic reaction of Platycodin D and Polygalacin D extracted from the enriched fraction, B shows the case of enzymatic treatment with lipase, and C is the enzyme with Biscozyme L It shows the case of processing. It can be seen that in the case of the enzyme treatment with lipase (B), the signals for both platicodine D and polygalacin D are increased compared to the case where the enzyme was not treated (A). On the other hand, in the case of enzymatic treatment with biskozyme L (C), compared to the case with no enzymatic treatment (A), the signals for platicodine D and polygalacin D increased, but when enzymatically treated with lipase ( Compared to B), it can be seen that the signal is relatively low. This is interpreted to be due to the further degradation of biscozyme L by platicodine D (or polygalacin D). For reference, in Figs. 4 and 5, the numerical values described in each chromatogram represent the area values of the corresponding peaks.

Referring to FIG. 6, when enzymatically treated with lipase, the distribution of glucose bound to carbon number 3 of platicodine E, platicodine D3, and platicodine D2, which are platinum-type saponins This is because the (Glycosidic bond) is cut, and finally, it is bioconverted to Platycodine, which is a molecule of glucose bound to carbon number 3. At this time, even if it is sufficiently bioconverted by lipase, the sugar chains of Arabinose (Ara; Arabinose), Rha (Rhamnose), Xyl (Xylose), and Apiose (Apiose) bound to carbon 28 As it remains, Platycodin D does not undergo enzymatic conversion. For this reason, when enzymatically treated with lipase, as all of the platinum-based saponins present in the hydrothermal extract of the longitude are enzymatically converted to platinumicodin D, when sufficiently enzymatic reaction is performed, platinumicodine D present in the final composition The content of is increased. However, when enzymatically treated with Biscozyme L, the sugar chain attached to carbon 28 as well as the sugar bound to carbon 3 of the platinum-based saponin is cleaved. That is, when sufficiently enzymatically treated with Biscozyme L, the apiose molecule present on carbon 28 of Platycodin D is cleaved to pass sugars other than Apios through Diapi-Platycodin D. The molecules, i.e. xylose, arabinose, rhamnose, are also cleaved, causing the platycodin D to disappear from the final composition.

[2. Fat accumulation inhibitory effect test result]

Oil red O staining was performed to confirm the effect of inhibiting liposynthesis according to the platinum codon saponins. Among the derivatives of gilkyung compounds, the primary screening for fat synthesis in adipocytes was targeted by morphologically confirming that the accumulation of physiological activity at low concentrations was targeted to the primary synthesis for fat synthesis in adipocytes. First, after treatment of platicodone in adipocytes, the expression levels of PPARγ and cEBPα, proteins related to fat synthesis, were confirmed by Western blotting. In addition, for comparison, the effect of inhibiting fat accumulation was confirmed in the same manner for platicodine D3 and platicoside E.

In FIG. 7, after treatment with 5 μM of platicodine D (PD), platicodine D3 (PD3), and platicoside E (PE) for mast cells treated with adipocyte-inducing substances (control), the control group ( Based on the fat level of DM) as 100%, it is a graph showing numerically the inhibitory effect of adipocyte differentiation according to the oil red O staining results of PD, PD3 and PE. As shown in FIG. 7, it can be seen that in the case of PD, the inhibitory effect of adipocyte differentiation is excellent.

In addition, FIG. 8 shows an image photographed with an optical microscope after treating 1.25 μM of Platycodin D and Diafio-Platinodine D, respectively, for mast cells. As shown in FIG. 8, it can be seen that the effect of inhibiting adipocyte differentiation on the control group (adipocyte) is superior to that of diapi-platincodin D.

From these experimental results, it can be seen that when all of the platinum-based saponins are enzymatically converted to platinum-D by lipase, the activity increases as sugar molecules bound to carbon 3 are removed. Furthermore, it can be seen that when the apiose located at the end of the sugar chain bound to the carbon No. 28 is cleaved by Biscozyme L, the fat suppressing effect is significantly reduced.

[3. Sensory evaluation of platinum codon saponin composition]

After diluting the hot water extract with 20brix to make 500ml, treat 20 parts by weight of lipase with respect to 100 parts by weight of the diluted solution, stir for 24 hours at 55 ° C, inactivate the enzyme for 10 minutes at 80 ° C, and filter it for final sensory evaluation Samples for preparation were prepared. Sensory evaluation was performed for the taste of the platinum codon saponin composition through "Food Environment Research Center". The sensory test method used a 9-point rating method (where 1 point means "very weak", 9 point means "very strong", and graded in 1-point increments). It was tested by dividing it into sweet taste, bitter taste and acrid taste. In particular, the evaluation of "Arin" was made to evaluate the condition felt after 6 to 8 seconds after swallowing the sample, and it prevented communication between the panels during the inspection to induce objective evaluation. Samples can be evaluated.

9 shows the sensory evaluation results of the enzyme-treated Dorajicheong (Sample 1) and the enzyme-treated Dorajicheong (Sample 2). As a result of the evaluation, it can be seen that when compared with Sample 1, Sample 2 further improves the intensity of sweetness while simultaneously decreasing the intensity of bitterness and astringency. For reference, in the sensory evaluation of FIG. 9, after one-way variance analysis using the PASWStatistics18 program, Duncan's post-test was performed, and the confidence level was 95% (p <0.05), where the probability value p was less than 0.05. Small means there is a significant difference.

As described above, when lipase was treated as an enzyme to platinum codon saponin (long-strength saponin), it was confirmed that the content of platicodine D and polygalacin D was increased by sugar hydrolysis. On the other hand, glucanase, xylanase, cellulase, and hemicellulase, which are conventionally used for enzymatic conversion, are sugar chains attached to carbon 28 as well as sugars located on carbon 3 of gil Gyeongsaponin. Cut. On the other hand, in the case of lipase, the glycosidic bond of the sugar molecules located at carbon 3 is cut and finally only one glucose molecule remains, but at the same time, the sugar chain bound to carbon 28 is not affected. That is, unlike conventional enzymes, when platase is used to enzymatically treat platinum codon saponins, only the sugar chains located on carbon 3 are selectively cleaved while maintaining the sugar chains located on carbon 28. As a result, when using the platinum codon saponin composition enzymatically treated with lipase, the fat accumulation inhibitory effect is improved through an increase in the content of platicodine D, and at the same time, the bitter and arin tastes are improved along with the improvement of sweetness by simplification of the sugar chain It has the effect of improving.

The preferred embodiments of the present invention have been described so far, but those skilled in the art to which the present invention pertains may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the embodiments of the present invention described herein should be considered from a descriptive point of view rather than a limiting point of view, and the scope of the present invention is shown in the claims rather than the above description, and all differences within the equivalent ranges of the present invention It should be interpreted as being included in.

Claims (8)

delete delete delete delete delete A first step of obtaining a platicone extract comprising platicone saponin from the platicone; And
The reaction chain of the platinum codon extract is reacted with an enzyme containing lipase, so that the chain of platinum-based saponins containing at least one of platicoside E, platicodine D3, and platicodine D2 is bound to carbon 28. The second step of enzymatic conversion to Platycodin D by selectively removing only the oligosaccharide bound to carbon 3 maintained and characterized in that it comprises a method for producing a platinum codon saponin composition.
The method of claim 6,
In the second step, the platinum codon extract is characterized in that the reinforced fraction of platinum codon, the production method of the platinum codon saponin composition.
The method of claim 6,
In the first step, the platinum codon extract is characterized in that it is obtained by hot extraction of platinum codon, a method for producing a platinum codon saponin composition.

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