KR20190030850A - Platycodon radix composition containing high protocatechuic acid, epicatechin, and oleic acid and enhanced inhibition activities of alpha-glucosidase and pancreatic lipase, and preparation method thereof - Google Patents

Abstract

Disclosed are Platycodon grandiflorum fortified with protocatechuic acid, epicatechin, and oleic acid and having improved inhibitory activities of alpha-glucosidase and pancreatic lipase, and a method for manufacturing the same. The Platycodon grandiflorum according to the present invention has remarkably increased content of protocatechuic acid, epicatechin, and oleic acid, and also has excellent inhibitory activities of alpha-glucosidase and pancreatic lipase so as to have excellent antioxidant, antidiabetic, and anti-obesity effects, thereby being used as a functional food and pharmaceutical ingredient.

Description

[0002] Platycodon radix compositions containing high protocatechuic acid, epicatechin, and oleic acid and enhanced inhibition activities of alpha-glucosidase and pancreatic lipase inhibitory activity, glucosidase and pancreatic lipase, and preparation method thereof}

The present invention relates to platycetic acid, epicatechin and alpha- glucosidase with enhanced and enhanced oleic acid and platelets having pancreatic lipase inhibitory activity, and a process for producing the same. More particularly, the present invention relates to a process for producing a fermentation broth, which comprises fermenting bellflower with a mixed seed of Lactobacillus plantai P1201 strain and Lactobacillus brevis BMK184, and cultivating and aging the protocatechuic acid, epicatechin and oleic acid enhanced and enhanced alpha-glucosidase, The present invention relates to a bellflower having pancreatic lipase inhibitory activity and a method for producing the same.

Platycodon grandiflorum ) is called Gil Kyung, Gil-gyeonggi, jeongyeong, or mountain bellflower. It grows in mountains and fields. Its roots are thick, stem is straight, and its height is 40 ~ 100cm. Platycodon contains about 90% or more carbohydrate, about 2.5% protein, and about 0.1% lipid.

The bellflower contains saponins of latycodin D, platycodin D3, polygalacin D and deapioplatycodin D, and these saponins are known to exhibit anticancer, neuroprotective and antioxidant activities. As described above, saponins are mainly studied in relation to the active ingredient of platycodon, and other active ingredients are not well known (Patent No. 10-1732036, No. 10-0915860). Therefore, there is a great need to develop a method for strengthening the active ingredient of the plant material other than saponin.

Protocatechuic acid is a major polyphenol with antioxidant activity and anti-inflammatory activity and is known to have anticancer effect. Epicatechin is a natural phenol and a major flavonol with antioxidant activity. It is known that oleic acid is an unsaturated fatty acid and has activity to lower blood pressure and reduce the incidence of breast cancer.

However, there is no known method for producing bellflower having enhanced active ingredients such as protocatechuic acid, epicatechin and oleic acid.

Accordingly, the present inventors have continued their studies to meet the needs of the prior art, and as a result, they have found that the addition of lactic acid bacteria, specific lactic acid bacterial fermentation, and high temperature aging technology to enhance protocatechuic acid, epicatechin and oleic acid and simultaneously inhibit alpha-glucosidase And pancreatic lipase inhibitory activity were improved, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a method for preparing a broth having an inhibitory activity of alpha-glucosidase and an activity of inhibiting pancreatic lipase, which are enhanced and enhanced by protocatechuic acid, epicatechin and oleic acid.

Yet another object of the present invention is to provide platycetic acid, epicatechin and oleic acid, which are enhanced and enhanced by alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity, which are produced by the above-described method.

It is still another object of the present invention to provide a functional food having antioxidant activity, antidiabetic activity and anti-obesity activity, which comprises the abovementioned bellflower as an active ingredient.

In order to accomplish the above object, the present invention provides a method for preparing a bellflower having the activity of inhibiting alpha-glucosidase and the activity of inhibiting pancreatic lipase, which is enhanced and enhanced by protocatechy acid, epicatechin and oleic acid, :

I) steaming the bellflower;

Ii) fermenting the cooked bellflower with a mixed seed of Lactobacillus plantai P1201 strain and Lactobacillus brevis BMK184 strain; And

Iii) aging the fermented bellflower at a high temperature.

Step i): Steam

Steaming the bellflower.

In the present invention, the bellflower is used for two years or more. Wash the bellflower three times in flowing water, remove the water and cook.

The steaming may be carried out using a steam boiler such as a conventional steamer, and is carried out at 100 ° C for 30 to 120 minutes.

If the steaming time is less than 30 minutes, sufficient steaming does not proceed and subsequent steps such as fermentation or aging may not be smooth and contamination of germs may occur. If it is cooked more than 120 minutes, the nutritional ingredient of the bellflower may be destroyed by long heat treatment.

Step ii): Mixed seed fermentation

The matured bellflower is fermented into mixed seeds.

In the production method of the present invention, the mixed seed is used by mixing Lactobacillus plantai P1201 strain and Lactobacillus brevis BMK184 strain.

The strain Lactobacillus plantai P1201, which constitutes a mixed strain in the present invention, is a strain deposited with the National Institute of Agricultural Science and Technology (KACC) on Aug. 13, 2013 KACC91848P), which has excellent probiotics and high productivity of physiologically active substances, but lactic acid bacteria of homo-lactic acid fermentation were required to be excreted excessively during fermentation alone.

The strain Lactobacillus BMK184, another strain that constitutes the mixed seeds in the present invention, was also isolated from the strain (KACC 92156P, Accession No. KACC 92156P) deposited on Dec. 12, 2016 at the National Institute of Agricultural Science and Technology GABA productivity and productivity of physiologically active substances were excellent. However, lactic acid fermentation with hetero-lactic acid fermentation did not produce lactic acid during fermentation alone. However, when lacto-Bacillus plantai P1201 was used as a mixed germ , The fermentation characteristics of the two strains were complementary to each other and exhibited a synergistic action (Patent Application No. 2017-0068747).

Lactobacillus plantai P1201 and Lactobacillus brevis BMK184 are cultivated as seeds in a malt extract liquid medium and mixed at a ratio of 1: 1 before or after culturing to produce mixed seeds.

The fermentation can be carried out by inoculating the mixed seed culture with the fermented broth at a concentration of 2 to 10% (v / w) and fermenting at 25 to 40 ° C for 2 to 7 days.

If the inoculum is less than 2% (v / w), the fermentation rate may be delayed. If the fermentation temperature is less than 25 ° C, the fermentation period is prolonged to cause contamination of germs. If the fermentation period is less than 2 days, the fermentation period is not sufficient and the production of physiologically active substances may be poor. If the fermentation period is over 7 days, the physiologically active substance may be decomposed by over fermentation.

Iii) Aging

The fermented bellflower is aged at high temperature.

The ripening is aged at high temperature of 60 ~ 80 ℃ for 5 ~ 10 days.

If the aging temperature is less than 60 ° C or the aging period is less than 5 days, the aging is not smoothly performed. If the aging temperature is more than 80 ° C or the aging period is more than 10 days, the produced physiologically active substance may be decomposed and the content may be decreased .

The fermentation may be performed by, but not limited to, placing the fermented broth in a closed container.

The bellflower produced through the above steps of the present invention has proper lactic acid production to maintain its palatability, and not only strengthens physiologically active components such as protocatechyme, epicatechin and oleic acid, but also antioxidant activity, antidiabetic activity and anti-obesity activity (Test Examples 3 and 4).

According to yet another object of the present invention, there is provided a bellflower having the alpha-glucosidase inhibitory activity and the pancreatic lipase inhibiting activity, which are produced by the above-mentioned method and are enhanced and enhanced by protocatechuic acid, epicatechin and oleic acid .

According to another object of the present invention, there is provided a functional food having antioxidative activity, antidiabetic activity and anti-obesity activity, wherein said bellflower is an active ingredient.

In the present invention, the functional food may be prepared by drying and pulverizing the bellflower of the present invention to produce a powder, or a product in the form of a granule, a ring, a beverage, or a jelly.

The production method of the present invention produces bellflower with significantly increased contents of protocatechy acid, epicatechin and oleic acid as well as alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity compared with the conventional method.

The content of protocatechuic acid, epicatechin and oleic acid is remarkably improved, and also excellent alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity are exhibited. Thus, It can be used as a material for food and medicine.

The functional food according to the present invention has excellent antidiabetic and anti-obesity functions, and is useful for inhibiting fat production, controlling body weight, decreasing cholesterol, improving hyperlipidemia, alleviating atherosclerosis, alleviating diabetes, improving blood circulation, and improving immunity.

Fig. 1 is an example of a process diagram of the manufacturing method of the present invention and a photograph of a bellflower produced according to each step.
Fig. 2 shows the antioxidative activity of the rhizome of the present invention. FIG. 2A shows the DPPH radical scavenging activity, FIG. 2B shows the ABTS radical scavenging activity, and FIG. 2C shows the hydroxyl radical scavenging activity.
Fig. 3 shows digestive enzyme inhibitory activity of rhododendron according to the present invention. Fig. 3a shows the alpha-glucosidase inhibitory activity, and Fig. 3b shows the pancreatic-lipase inhibitory activity.

The present invention will be described in more detail by the following examples. These examples are for illustrating the present invention, and the scope of the present invention should not be limited by them.

Example

Manufacturing example . Bellflower manufacturing

Two to five years later, 1 kg of bellflower was washed thoroughly with tap water three times, and water was completely removed. Steamed potatoes were steamed at 100 ° C for one hour in an appropriate amount of water. Lactobacillus plantai P1201 and Lactobacillus brevis BMK184 were cultivated as seeds in a malt extract liquid medium, and mixed seedlings mixed at a ratio of 1: 1 before or after cultivation were inoculated into a vinegar broth at 5% (v / w) The fermented Dorado was fermented at 30 ° C for 7 days to prepare fermented Dorado, and the fermented Dorado was placed in a closed container and aged at 70 ° C for 7 days to complete the platycodon according to the present invention (Fig. 1).

The pH of the prepared bellflower was measured using a pH meter, and the total acidity was measured by neutralization titration and converted into lactic acid, and the results are shown in Table 1.

pH Total acidity (%, based on lactic acid) Example [ 3.71 0.64

The pH and total acidity of the bellflower prepared according to the present invention were 3.71 and 0.64%, respectively. Lactobacillus plantarum alone produces excess acid during fermentation, whereas lactobacillus brevis alone produces weak acidity. When excess acid is produced, the sour taste may be strengthened and palatability problems may occur. If the acid production is weak, chances of contamination with germs are increased.

Test Example  1. Analysis of oleic acid content in bellflower

For comparison, the broodstock of each step, i.e., the washed raw material broth (Comparative Example 1), the steaming broth (Comparative Example 2) roasted only to the steaming stage, and the fermentation The oleic acid content of each of the fermented dorados (Comparative Example 3) was analyzed.

The analytical sample was prepared by drying each of the bellflower at 55 ° C for about 3 days, preparing powders by pulverization, adding 3 ml of 0.5 N methanolic NaOH to 1 g of each powder, heating the mixture at 100 ° C for 10 minutes to hydrolyze the fatty acid and glycerol . Hydrolysis was followed by addition of 2 ml of boron trifluoride (BF ₃ ), stirring was continued for 30 minutes, and the methyl esterification of the fatty acid was proceeded. After the reaction was completed, 1 ml of isooctane was added and the mixture was vigorously shaken. Only the isooctane layer was recovered, dehydrated with anhydrous sodium sulfite, filtered through a 0.45 μm membrane filter (Dismic-25CS) and analyzed by gas chromatography. .

content (mg / 100 g) Comparative Example 1 Comparative Example 2 Comparative Example 3 Example C18: 1n9c Oleic acid 19.3 6.0 36.5 63.4

As shown in Table 2, as compared with Comparative Examples 1 to 3, it was confirmed that the content of oleic acid as the omega-9 unsaturated fatty acid was remarkably increased in the platycodon of the example according to the present invention.

Test Example  2. Analysis of physiologically active ingredient content of bellflower

Total flavonoids, browning, phenolic acid and flavonol were analyzed.

<Preparation of analytical sample>

10 ml of a 50% fermented alcohol was added to 10 g of each of the bellflower powder prepared as in Test Example 1, and the mixture was extracted at room temperature (20 ± 5 ° C) for 24 hours and centrifuged at a rate of 3,000 rpm to prepare an extract. Total extracts were analyzed by filtration, freeze - drying at 60 ℃, and samples prepared at 0, 0.25, 0.5 and 1.0 mg / ml concentration. And antioxidant activity and digestive enzyme inhibitory activity were analyzed.

<Total flavonoid content and browning analysis>

Total flavonoid contents were measured by Davis transformation. After adding 0.01 ml of 1 N NaOH to 0.1 ml of the sample prepared above, 1.0 ml of diethylene glycol was added, and the mixture was allowed to stand at 37 ° C for 1 hour, and the absorbance at 420 nm was measured. The total flavonoid content of rutin was 0.25, 0.5 and 1.0 mg / ml, and the content was determined from the standard curves. The results are shown in Table 3.

10 mg of distilled water was added to 1 g of each of the blooming powders, followed by extraction at 25 ° C. for 1 hour. The supernatant was measured at 420 nm using a spectrophotometer (Spectronic 2D). The results are shown in Table 3.

Total flavonoid (mg / g) The browning material (OD 420 nm) Comparative Example 1 0.11 1.595 Comparative Example 2 0.16 1.396 Comparative Example 3 0.37 1.267 Example 0.59 3.006

As shown in Table 3, as compared with Comparative Examples 1 to 3, the total flavonoid content of the platycodon of the examples according to the present invention was increased by about 1.6 to 5 times and the browning material was also increased by about twice.

<Content of phenolic acid and flavonol compound>

Analysis of phenolic acid and flavonol content was carried out using HPLC (high performance liquid chromatography) for each of the samples prepared above.

The analytical column used was XBridge ^™ C18 (4.6 × 250 mm, 5 μm, Waters Corp., Milford, Mass., USA) column and eluted with 0.5% glacial acetic acid (mobile phase solvent A) and 100% (280 nm) and flavonol (270 nm) were detected with a UV detector at a rate of 1 ml per minute for 60 minutes at 30 ° C in a linear gradient of 0-100% 4 and Table 5, respectively.

Analysis item * (μg / ml) Comparative Example 1 Comparative Example 2 Comparative Example 3 Example Gallic acid 37.14 54.00 66.33 88.99 Protocatechuic acid 109.08 197.11 222.34 309.81 Chlorgenic acid 59.59 58.90 74.74 87.95 t-Cinnamic acid 39.86 - 45.49 51.00

As shown in Table 4, the content of the phenolic acid compound of the platycodon of the examples according to the present invention was improved, and the content of protocatechuic acid was increased by about 1.5 to 2 times .

Analysis item (μg / ml) Comparative Example 1 Comparative Example 2 Comparative Example 3 Example Catechin 30.40 54.19 61.81 57.88 Epicatechin 19.53 59.47 34.89 79.59 Routine 24.38 39.30 87.11 70.34 Catechin gallate 33.41 20.87 19.25 34.11 Quercetin 60.40 38.07 39.25 59.24 Naringin 16.08 19.64 25.35 27.39 Naringenin 21.48 5.99 25.55 26.53

As shown in Table 5, the content of flavonol compounds in the bellflower of the examples according to the present invention was improved, and the content of epicatechin was increased about 2 to 4 times, in particular, as compared with Comparative Examples 1 to 3.

Test Example  3. Antioxidant activity analysis of bellflower

The antioxidative activity of the platycodon according to the present invention was measured by measuring DPPH, ABTS and OH radical scavenging activity.

To DPPH radical scavenging activity, 0.8 ml of DPPH solution (1.5 x 10 ^-4 M) was added to 0.2 ml of each of the samples prepared above (three kinds of 0.25 mg / ml, 0.5 mg / ml and 1 mg / ml concentration) After incubation for 30 minutes, the absorbance was measured at 525 nm. The negative control of DPPH radical scavenging activity proceeded in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) by the following equation, and the result is shown in FIG.

Radical scavenging activity (%) = [1- (negative control absorbance / experiment absorbance)] × 100

The ABTS radical scavenging activity was obtained by mixing 5 ml of 7 mM ABTS reagent and 5 ml of 140 mM K ₂ S ₂ O ₈ (FW 270.3, Sigma 9392) in a dark place for 16 hours to produce a cation radical. And the absorbance of the control was adjusted to 0.7 ± 0.02. 0.1 ml of each sample (three kinds of concentrations of 0.25 mg / ml, 0.5 mg / ml and 1 mg / ml) and 0.9 ml of ABTS solution were mixed and reacted for 3 minutes and absorbance was measured at 732 nm. The ABTS radical inhibitory activity was also measured in the same manner using distilled water instead of the sample as the negative control, and the difference in absorbance was calculated as a percentage (%) according to the above equation. The results are shown in FIG. 2B.

Hydroxyl (OH) radical scavenging activity was measured 10 mM FeSO _{4. 7H 2 0-EDTA 0.2ml, 10} mM 2- deoxyribose _{0.2 ml, 10 mM H 2 O} 2 0.2 ml, samples (0.25 mg / ml, 0.5 mg / ml, 1 mg / ml concentration of each of three kinds) 1.4 ml 1 ml of thiobarbituric acid / distilled water and 2.8% trichloroaceric acid / distilled water were added to the mixture, and the mixture was incubated at 100 ° C for 1 hour. Lt; 0 &gt; C for 20 minutes, cooled, and then measured for absorbance at 520 nm. The hydroxyl radical scavenging activity was calculated by the above equation using the above equation, and the results are shown in FIG. 2C.

As shown in FIG. 2, Dorago according to the present invention significantly enhanced the scavenging activities of the DPPH radical, the ABTS radical and the hydroxyl radical in a concentration-dependent manner, compared with Comparative Examples 1 to 3, , The DPPH radical scavenging activity was 84.72%, the ABTS radical scavenging activity was 82.97%, and the hydroxyl radical scavenging activity was 56.05%.

Test Example  4. Digestive enzyme inhibition activity of bellflower

Alpha-glucosidase inhibitory activity, which is an index of anti-diabetic (diabetic) effect, was assayed on the rhizomes according to the present invention and the activity of inhibiting pancreatic lipase, which is an anti-obesity index, was examined.

&Lt; Alpha-Glucosidase Inhibitory Activity >

50 μl of 0.5 U / ml α-glucosidase enzyme solution was added to 50 μl of samples (three kinds of 0.25 mg / ml, 0.5 mg / ml and 1 mg / ml concentration, respectively) mM phosphate buffer solution (pH 6.8) were mixed and preliminarily cultured at 37 ° C for 10 minutes. 100 μl of sodium phosphate buffer (pH 6.8) was added and reacted at 37 ° C for 10 minutes. The reaction was stopped by adding 0.75 ml of 100 mM Na ₂ CO ₃ to the reaction mixture, and the absorbance at 420 nm was measured. Negative control was carried out in the same manner using distilled water instead of the sample, and the difference in absorbance was expressed as a percentage (% ). The results are shown in Fig. 3A.

Alpha-glucosidase inhibitory activity (%) =

 [1- (negative control absorbance / experimental absorbance)] × 100

As shown in FIG. 3A, the platelet of the present invention according to the present invention showed an alpha-glucosidase inhibitory activity in a concentration-dependent manner, compared to Comparative Examples 1 to 3. In particular, about 1 mg / It can be confirmed that it is enhanced 1.5 to 5 times (FIG. 3A).

<Pancreatic-lipase inhibitory activity>

50 μl of 1.0 U / ml pancreatic lipase enzyme solution, 50 μl of 200 mM sodium phosphate buffer solution (pH 6.8) 50 μl of each sample (each of 0.25 mg / ml, 0.5 mg / ml and 1 mg / ml concentration of pancreatic-lipase inhibitory activity) Were mixed and pre-incubated at 37 占 폚 for 10 minutes. Then, 100 占 퐇 of a sodium phosphate buffer solution (pH 6.8) was added and reacted at 37 占 폚 for 10 minutes. The reaction was stopped by adding 0.75 ml of 100 mM Na ₂ CO ₃ to the reaction solution, and the absorbance at 420 nm was measured. Negative control was carried out in the same manner using distilled water, and the difference in absorbance was calculated by the following formula (% And the results are shown in FIG. 3B.

Pancreatic-lipase inhibitory activity (%) =

[1 - (negative control absorbance / experimental absorbance)] × 100

As shown in FIG. 3B, the platelets of Comparative Examples 1 and 2 did not show any inhibitory activity against pancreatic-lipase, and the platycodon according to the present invention according to the present invention exhibited a concentration- -Lipase inhibitory activity, and in particular, it was confirmed that the concentration was increased more than two times based on the concentration of 1 mg / ml (FIG. 3b).

From the above functional test results, it can be seen that the bellflower prepared according to the production method of the present invention not only enhances physiologically active components such as protocatechuic acid, epicatechin and oleic acid, but also exhibits antioxidant activity, alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity And thus it is useful as a material for functional foods having excellent antioxidant, antidiabetic and anti-obesity activities.

Agricultural Biotechnology Research Institute KACC91848P 20130719 Agricultural Biotechnology Research Institute KACC92156P 20161212

Claims (7)

A method for the production of bellflower having enhanced alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity, which are enhanced and enhanced by protocatechyme, epicatechin and oleic acid,
I) ripening the bellflower at 100 DEG C for 30 to 120 minutes;
Ii) fermenting the cooked bellflower with a mixed seed of Lactobacillus plantai P1201 strain and Lactobacillus brevis BMK184 strain; And
Iii) aging the fermented bellflower.
The method according to claim 1, wherein the fermentation in step ii) is carried out by inoculating the cultured broth of the mixed broth with a concentration of 2 to 10% (v / w) in the broth, and fermenting the broth at 25 to 40 ° C for 2 to 7 days. A process for the production of bellflower having enhanced alpha - glucosidase inhibitory activity and pancreatic lipase inhibitory activity enhanced and enhanced by acetic acid, epicatechin and oleic acid.
The method according to claim 1, wherein the aging is carried out at a temperature of 60 to 80 ° C for 5 to 10 days, wherein protocatechuic acid, epicatechin and oleic acid are strengthened and enhanced, and alpha-glucosidase inhibitory activity and pancreatic lipase- Gt;
A broth prepared by the manufacturing method according to claim 1 and having alpha-glucosidase inhibiting activity and pancreatic lipase inhibiting activity enhanced and enhanced by protocatechy- cate, epicatechin and oleic acid.
An antioxidative active functional food comprising the bellflower of claim 4 as an active ingredient.
An antidiabetic and anti-obesity functional food comprising the platycodon according to claim 4 as an active ingredient.
The functional food according to claim 5 or 6, wherein the functional food is any one selected from the group consisting of powder, granule, ring, beverage, and jelly.

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