KR20190011691A - A method for acquiring quercetin from extract of Morus alba L by treating viscozyme L - Google Patents

Abstract

The present invention relates to a method of obtaining Morus alba leaf-derived quercetin comprising a step of treating Viscozyme L to Morus alba leaves and a method of producing a Morus alba leaf extract with increased content of quercetin. By using the method of obtaining Morus alba leaf-derived quercetin comprising the step of treating Viscozyme L to Morus alba leaves of the present invention, a large amount of quercetin can be obtained exhibiting anti-diabetic activity present in Morus alba leaves. Therefore, the method of the present invention can reduce a cost and effort in manufacturing an anti-diabetic drug, and thus can be usefully used for the manufacture of anti-diabetic medicines and manufacture of functional health foods capable of alleviating and preventing diabetes.

Description

[0001] The present invention relates to a method for obtaining quercetin in a mulberry leaf extract by treating a viscose extract,

The present invention relates to a method for producing Viscozyme L,Morus alba L) leaves, and a method for producing the mulberry leaf extract with increased quercetin content.

Chronic complicated diseases caused by population aging are increasing worldwide. Accordingly, there is an increasing interest and demand for drugs derived from natural products, which can be easily treated by multiple targets. Active materials derived from natural products and other natural products have less development time and cost than synthetic drugs and other synthetic products, and have good efficacy and fewer side effects, making active research and development investment. Domestic and overseas markets of biotech drugs are growing at a rate of 19.99% and 13.98%, respectively, in 2013 ~ 2018, and 10.59% at domestic and overseas markets for natural cosmetics, antibiotics and health functional foods. And 5.45%, respectively.

  The diverse active ability of mulberry, which is widely found not only in Korea but also in China, Japan, and the entire central region of the world, has been revealed. Among them, mulberry leaf has been mainly used in Oriental medicine. According to the Divine Husbandman's Medicinal Sculpture, Japan's Ogukyou, and Dongbuogaku, mulberry leaf has recorded its efficacy as medicinal plants such as angelic disease, edema, The efficacy of the mulberry leaf is reported to be manifested by various flavonoids present in the mulberry leaf. The mulberry leaf has various effects ranging from anticancer, antidiabetic, antioxidant, anti-obesity, whitening, anti-arteriosclerosis and anti-inflammation It is known. As a result, mulberry leaves, which have been used mainly in the form of tea, are now being commercialized as additives or products for various foods.

On the other hand, the rate of absorption of nutrients through food consumed by each person is different, and it is known that the absorption rate is different due to factors such as an individual's genetic or intestinal microorganisms.

Bioconversion technology is known to be involved in converting organisms such as plants or animal wastes into useful products or raw materials using biological methods or agents such as specific enzymes or microorganisms. The above-mentioned bioconversion technology is an effective method for improving physiological activity, and in particular, is used for enhancing physiological activity in various fields by using enzymes.

Therefore, there is a need for a bioconversion technology for extracting useful flavonoids present in mulberry leaves with high efficiency as described above.

Accordingly, the present inventors have studied a method for obtaining a large amount of an inactive substance present in a mulberry leaf as an active substance that is readily absorbable in vivo. When a bioconversion is carried out by treating viscozyme L enzyme, It was confirmed that the content of quercetin, which exhibits the antidiabetic activity present, is increased to obtain a large amount of quercetin, and the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for obtaining mulberry leaf-derived quercetin, which comprises treating Viscozyme L with Morus alba L leaves, and a method for producing mulberry leaf extract with increased quercetin content .

In order to accomplish the above object, the present invention provides a method for producing (a) Viscozyme L from Morus alba L) leaves; (b) extracting the substance to be treated in step (a) with a lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; And (c) separating the quercetin from the mulberry leaf extract; Wherein the mulberry leaf-derived quercetin is selected from the group consisting of mulberry leaf quercetin and mulberry leaf quercetin.

The present invention also relates to a process for producing (a) Viscozyme L from Morus alba L) leaves; And (b) extracting the substance to be treated in step (a) with a lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; , Wherein the quercetin content of the mulberry leaf extract is increased.

The method of obtaining mulberry leaf-derived quercetin, which comprises treating the mulberry leaf with Viscozyme L of the present invention, can be used to obtain a large amount of quercetin exhibiting antidiabetic activity present in mulberry leaves. Therefore, the method of the present invention can reduce the manufacturing cost and effort of the anti-diabetic drug, and thus can be usefully used for the manufacture of anti-diabetic medicines and the manufacture of health functional foods capable of improving and preventing diabetes.

Figure 1 shows the result of thin layer chromatography of the normal phase on the ethyl acetate fraction of mulberry leaf extract and irradiation with ultraviolet light (Figure 1A), followed by spraying with anisaldehyde sulfuric acid (Figure 1B) and inhibition of? -Glucosidase (Fig. 1C). Fig.
Figure 2 is a schematic diagram of an experiment performed to isolate and purify active flavonoids present in the ethyl acetate fraction.
FIG. 3 shows the result of thin layer chromatography of the acetate fraction of mulberry leaf extract and MAV1, MAV2 and MAV3 isolated from the fractions and the result of irradiation with ultraviolet light (FIG. 3A), followed by spraying with anisaldehyde sulfuric acid ) And high-pressure liquid chromatography (HPLC) to confirm the purified flavonoid (FIG. 3C).
FIG. 4 is a graph showing the result of performing the hydrogen nuclear magnetic resonance (MR) method for the separated MAV1, MAV2 and MAV3 compounds.
FIG. 5 is a graph showing the inhibitory activities of α-glucosidase from the control ethyl acetate fraction, viscose L-treated ethyl acetate fraction and isolated MAV1, MAV2 and MAV3 compounds, which were not treated with viscose L. (* p <0.05).
FIG. 6 shows the results of varying the reaction conditions to find the optimum bioconversion condition through the viscosome, showing the substrate concentration (w / v) in FIG. 6A, the enzyme concentration in FIG. 6B, the reaction temperature in FIG. 6D shows the change in the reaction pH, and FIG. 6E shows the change in the quercetin gain obtained by varying the reaction time.

The present invention relates to a process for the preparation of (a) Viscozyme L from Morus alba L) leaves; (b) extracting the substance to be treated in step (a) with a lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; And (c) separating the quercetin from the mulberry leaf extract; Wherein the mulberry leaf-derived quercetin is selected from the group consisting of mulberry leaf quercetin and mulberry leaf quercetin.

Hereinafter, the present invention will be described in more detail.

The mulberry used in the present invention is widely used not only in Korea but also in China, Japan, and the middle parts of the world. Especially, the leaf has anticancer, antidiabetic, antioxidant, anti-obesity, whitening, anti-arteriosclerosis and anti- It is known to have a variety of active abilities, including.

The present invention relates to a method for producing quercetin, which is present in mulberry leaves, which is one of the flavonoids present in mulberry leaves through treatment with viscose L, on the mulberry leaves. Bioconversion is the conversion of organic matter, such as plant or animal wastes, into useful products or raw materials using biological methods or agents such as specific enzymes or microorganisms. Bioconversion technology is used to enhance physiological activity. Especially, it is used to enhance physiological activities in various fields by using enzymes. In the present invention, the mulberry leaves were biotransformed by increasing the content of quercetin having anti-diabetic activity among the active flavonoids present in mulberry leaves by treating viscozyme L on mulberry leaves. The quercetin obtained in the present invention is one of flavonoids, widely distributed in vegetables and fruits as glycosides, and obtained by hydrolysis of rutin (extract) with an acidic aqueous solution or enzyme, and is resistant to heat. In particular, quercetin is a substance known to be usable as an antioxidant.

According to the method of the present invention, quercetin can be obtained in large quantities from the same amount of mulberry leaves. Therefore, even when the same amount of mulberry leaf extract is used, an excellent antidiabetic activity is increased compared to the biscottin L mulberry leaf extract.

In step (a) of the present invention, the concentration of mulberry leaves may be 15% (w / v) to 30% (w / v), preferably 20% (w / v) Mulberry leaves can be used. The reaction time may be 20 to 40 hours, preferably 24 hours. The viscose L concentration used may be 3% (v / v) to 15% (v / v), but preferably 5% (v / v) to 10% Can be used for conversion. As a most preferred example of the method of the present invention, the reaction conditions are set at 37 ° C for 24 hours with 20% (w / v) mulberry leaf concentration and 5% (v / v) , and pH 4 to obtain a biologically converted mulberry leaf enzyme-treated product.

In the method of the present invention, a lower alcohol having 1 to 4 carbon atoms may be used as a solvent for extracting the mulberry leaf extract, but the present invention is not limited thereto. For example, ethanol is used as a preferred example of the present invention. In the above extraction method, any conventional method known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used.

In the present invention, the step (c) may include a step of adding ethyl acetate to water and ethyl acetate to obtain the ethyl acetate fraction. In the mulberry leaf extract, ethyl acetate fraction &Lt; / RTI &gt; The step (c) may further include the steps of: performing silica gel chromatography using chloroform and a methanol solvent; And the fraction obtained through silica gel chromatography was subjected to Sephatex LH-20 column chromatography using a methanol solvent; And separating the fractions obtained through Sephadex LH-20 column chromatography, again performing high performance liquid chromatography (HLPC). In a most preferred embodiment of the present invention, the size of the silica gel column chromatography is ø6 x 30 cm, the size of the Sephadex LH-20 column chromatography is ø3.5 x 90 cm, and the high pressure liquid chromatography has a flow rate of 2 ml / min, and the fixed phase was a CAPCELL PAK C18 MG column having a size of 10 mm x 250 mm.

The quercetin obtained by the method of the present invention exhibits an anti-diabetic activity, and the anti-diabetic activity particularly includes an activity to inhibit? -Glucosidase.

The present invention also relates to a process for producing (a) Viscozyme L from Morus alba L) leaves; And (b) extracting the substance to be treated in step (a) with a lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; , Wherein the quercetin content of the mulberry leaf extract is increased.

In order to extract the mulberry leaf extract extracted by the method of the present invention, a lower alcohol having 1 to 4 carbon atoms may be used, but the present invention is not limited thereto. For example, ethanol is used as a preferred example of the present invention. The mulberry leaf extract extracted by the method of the present invention also exhibits an antidiabetic activity due to an increase in the content of quercetin having an anti-diabetic activity among the active flavonoids present in the mulberry leaves through biotransformation due to treatment with viscose L, Particularly, an activity of inhibiting? -Glucosidase.

The redundant contents are taken into consideration in the complexity of the present specification, and terms not otherwise defined herein have the meanings commonly used in the art to which the present invention belongs.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example

Material used in the experiment

Dried mulberry leaves were obtained in the Hadang Hat dimension and the commercial enzyme Viscozyme L for bioconversion was purchased from Novozymes (Copenhagen, Denmark). Ethanol, ethyl acetate and n-butanol were purchased from SK Chemical (Ulsan). HPLC grade ethanol was purchased from Burdick & Jackson (Merzkowon, USA) and enzymes and substrates for biochemical analysis were purchased from Sigma-Aldrich , Korea). Silica gel 60F ₂₅₄ and 60RP-18F _254S for thin layer chromatography were purchased from Merck (Darmstadt, Germany).

Example 1 Confirmation of Enhancement of α-Glucosidase Inhibitory Activity in Biotransformed Mulberry Leaf Extract

Bioconversion of mulberry leaves was carried out with commercially available viscose L - enzyme solution. The dried mulberry leaves were pulverized into powder, and the 10% (w / v) powder was resuspended in distilled water, and then a 5% (v / v) viscose L enzyme solution was added. The conditions of the bioconversion reaction were set on the basis of the conditions according to the product data sheet of Viscozyme L enzyme, and the bioconversion reaction was carried out for 48 hours with shaking at 150 rpm. Subsequently, ethanol extracts of mulberry leaves were obtained by three times extraction with ethanol (4 L), sequential solvent extraction was performed, and the fractions according to the respective polarity of ethyl acetate, butanol, and water were separated and analyzed by thin layer chromatography ).

TLC was performed by a normal phase and a reverse phase method. The fixed and fluid phases used in the chromatography are shown in Table 1.

Stationary phase Fluid phase Normal phase Silica gel 60 F ₂₅₄ Chloroform / methanol / water
(4: 2: 1, v / v / v, lower phase) Inverse phase Silica gel 60 RP-18 F ₂₅₄ s 70% methanol

The biotransformed mulberry leaf ethyl acetate or n-butanol extract was prepared at 10 mg / mL and then 5 μL was spotted onto the beginning of the TLC plate and separated by developing solvent. Then, the TLC plate was air-dried under ultraviolet light (254 nm), and the result of the experiment in which the TLC plate of the normal phase was irradiated with ultraviolet rays is shown in FIG. 1A. TLC plates were sprayed with anisaldehyde sulfuric acid reagent and heat dried for coloration of the separated compounds, and the results of the experimental runs are shown in Figure 1B.

An experiment was then conducted to determine whether the fraction of the separated fractions had the highest inhibitory activity against? -Glucosidase. α-Glucosidase inhibitory activity was determined by using α-glucosidase derived from Saccharomyces cerevisiae (Sigma-Aldrich) using p-nitrophenyl glucopyranoside (pNPG) (Sigma- 0.075 units of glucosidase were mixed with various concentrations of the mulberry leaf extract. PNPG was added to 100 mM sodium phosphate buffer (pH 6.8) at a concentration of 3 mM and incubated at 37 占 폚 for 30 minutes. alpha -glucosidase inhibitory activity was determined by measuring the amount of p-nitrophenol released from pNPG to a VERSA _max microplate reader (Molecular Devices, Toronto, Canada) at 405 nm. Methanol was used as a negative control and acarbose (Sigma-Aldrich) at a concentration of 0.25, 1 and 4 mg / mL, which is an oral hypoglycemic agent of the glucosidase inhibitor series, was used as a positive control, And the results of performing the above experiment are shown in FIG. 1C. Lane 1 and ME Vis denote the methanol extract, lane 2 and EL Vis denote the ethyl acetate fraction, lane 3 and BL Vis denote the n-butanol fraction, and lane 4 and WL Vis denote the experimental results of the water fraction.

As can be seen in Figures 1A and 1B, several separable components appeared in the ethyl acetate fraction. As shown in FIG. 1C, it was confirmed that the ethyl acetate extract showed the highest inhibitory activity of? -Glucosidase at the same concentration as that of methanol, n-butanol and water extract. Therefore, it was confirmed that the fraction extracted with ethyl acetate had the highest antidiabetic activity. Therefore, the ethyl acetate fraction of the biotransformed mulberry leaf extract exhibiting high antidiabetic activity was used in the following examples.

Example 2. Extraction and Purification of Active Flavonoids from Biotransformed Mulberry Leaf Extracts

Experiments were performed to isolate and purify the active flavonoids present in the ethyl acetate fraction as identified in Example 1, and a method of performing the experiment was shown in FIG. In the same manner as in Example 1, 100 g of mulberry leaf powder was treated with 5% (v / v) Viscozyme L and extracted with 4 L of ethanol three times to obtain an ethanol extract. Then, 1 L of ethyl acetate and water were mixed at a ratio of 1: 1 to the ethanol extract, and the fraction was separated and extracted three times with ethyl acetate. Then, the flavonoid compound was separated by silica gel column chromatography according to the polarity of the solvent using a silica gel column chromatography size of ø6 × 30 cm and a solvent mixture of 100% chloroform and 30: 1 chloroform: methanol.

Antifibrinolytic activity was observed in fractions 3 to 16 using a 30: 1 mixture of chloroform and methanol in the separated fractions. Thus, in order to separate the flavonoid present in the fraction according to the molecular weight, the size of the phlorobonoid was determined by Sephadex LH-20 column chromatography to be ø3.5 × 90 cm. The flavonoids isolated from fractions 48 to 60, 102 to 110, and 120 to 125 were named MAV1, MAV3, and MAV2, respectively.

Example 3. Identification of separated and purified flavonoids and confirmation of the activity of inhibiting? -Glucosidase inhibitory activity

3.1 Identification of flavonoids in fractions

The components were separated using high pressure liquid chromatography to determine the antidiabetic activity of any of the flavonoids isolated and purified in Example 2 above.

First, the ethyl acetate extract of the biotransformed mulberry leaves and the MAVs 1 to 3 were analyzed by thin layer chromatography (TLC). The results of the analysis are shown in FIGS. 3A and 3B. Then, the purified flavonoids were subjected to high pressure liquid chromatography (HPLC) to determine the flavonoids. The HPLC was carried out for 4 minutes with methanol and 0.1% acetic acid in a volume ratio of 60% for 60 minutes. , And a gradient of 20% to 30% with 100% methanol was carried out at a flow rate of 2 ml / min. A CAPCELL PAK C18 MG column (Shiseido Co., Japan) having a size of 10 mm x 250 mm was used as a stationary phase, and the results of the above separation and identification are shown in Fig. 3C. EE Ctrl refers to extracts of mulberry leaves, and EE Vis refers to extracts obtained by biotransformation of mulberry leaf extract into Viscozyme L. H-NMR spectroscopy was performed to identify the components of the identified flavonoids, and the results are shown in FIG.

As shown in FIGS. 3A and 3B, it was confirmed that the shape of the peak was changed when bioconversion was performed using Bioscience L compared to EE Ctrl. In addition, as shown in FIG. 3C, the MAVs 1 to 3 isolated from the mulberry leaf extract were once confirmed that the peaks were identified at the same spot and were derived from the bioconverted material of biscottin.

As shown in FIG. 4, it was confirmed that the flavonoids resulting from the determination of MAV 1 to 3 through hydrogen nuclear magnetic resonance were caffeic acid, quercetin, and kaempferol, respectively.

3.2 Identification of increased α-glucosidase inhibitory activity by bioconversion in isolated flavonoids

To confirm whether the antidiabetic activity exhibited by the flavonoids of MAV1 to MAV3 isolated in Example 3.1 was caused by bioconversion by the biscozyme enzyme and which of the flavonoids exhibited the elevated antidiabetic activity Experiments were performed. The activity of the α-glucosidase inhibitory activity was measured in the same manner as in Example 1, and the activity of the flavonoid present in the MAVs 1 to 3 isolated was measured and shown in FIG.

As shown in FIG. 5, it was confirmed that MAV 2 and 3 showed a high inhibitory activity of α-glucosidase. In particular, in the case of treatment with viscose biosynthesis, it was confirmed that the α-glucosidase inhibitory activity was significantly increased in the ethyl acetate fraction, and the increase in the activity was confirmed by the quercetin of MAV2. In addition, the above-mentioned enhanced antidiabetic activity is due to the increase of the quercetin content of MAV2 at the same concentration. When the concentration of the extract is higher than 15 μg / mL, the α-glucosidase inhibition by 80% Activity, which is a glucosidase inhibitor-based oral hypoglycemic agent, was found to exhibit a higher degree of anti-diabetic activity than acarbose used in clinical practice. In addition, when the concentration of the extract was 50 μg / mL, the inhibitory activity of α-glucosidase was increased to 90% or more, and the antidiabetic activity was remarkably increased due to the increase of the content of quercetin, there was.

Example 4. Identification of Bioconversion Conditions for Optimum Activity Quercetin in Mulberry Leaf Extracts

Experiments were conducted by setting standards for the amount of substrate, the concentration of enzyme, temperature, pH, and reaction time in order to find reaction conditions for obtaining a high yield of quercetin, which is a flavonoid in mulberry leaf biotransformation produced by viscose . Experiments were carried out with 10% (w / v) substrate, 5% (v / v) enzyme, 37 ° C, pH 4 and reaction time of 48 hours for basic bioconversion conditions. As a result, the substrate concentration (w / v) was shown in FIG. 6A, the enzyme concentration was shown in FIG. 6B, the reaction temperature was shown in FIG. 6C, the reaction pH was shown in FIG. 6D, and the reaction time was shown in FIG. 6E.

As shown in FIG. 6A, when the substrate concentration was increased to 5, 10, 15 and 20% (w / v), the amount of quercetin to be quantified was confirmed to increase and a concentration-dependent increase was confirmed. Especially, when the substrate concentration was more than 15%, it was confirmed that the yield was doubled more than that when the substrate concentration was 5%. However, when the concentration of the substrate was 20% or more, it reached saturation and it was confirmed that it did not increase any more, and it was confirmed that the optimum substrate concentration was 20% (w / v).

As shown in FIG. 6B, it was confirmed that the amount of quercetin obtained increases as the concentration of the enzyme increases as the substrate. However, a remarkable increase in the amount of quercetin, which can be obtained by confirming that the concentration was more than 2%, when the concentration was more than 3%, was confirmed. However, it was confirmed that when the enzyme concentration was 5% or more, the enzyme reached saturation and was no longer increased. It was confirmed that the optimum enzyme concentration was 5% (v / v) in the method of optimizing the quercetin content.

As can be seen in FIG. 6C, the reaction temperature was found to be 15 ° C., 37 ° C. and 55 ° C., and it was found to be the highest at 37 ° C. The half-life of quercetin at 37 ° C. at 15 ° C. and 55 ° C. , And it was confirmed that the optimum reaction temperature was 37 ° C.

As shown in FIG. 6D, when bioconversion is performed in a range of pH 4 to 6 among various pH ranges (2, 4, 6, 8, 10, 12) ranging from acidic to neutral and basic in relation to pH, .

As shown in FIG. 6E, when the reaction time was set to 0, 3, 6, 12, 24, and 48 hours, the yield of quercetin increased about 1.5 times as compared with that of 6 hours after 24 hours , And it was confirmed that the reaction reached saturation after the reaction time of 24 hours had elapsed, and it was confirmed that the condition for obtaining the optimum amount of quercetin for 24 hours was obtained. Compared with the amount of quercetin (5,282 μg / 1 g mulberry leaf) obtained at 10% (w / v) substrate, 5% (v / v) enzyme, 37 ° C, pH 4 and 48 hours, The amount of quercetin that can be obtained when bioconversion is carried out with 20% (w / v) substrate, 5% (v / v) enzyme, 37 ° C, pH 4, And 6,994 μg / g of mulberry leaves increased by about 32%, confirming that the optimum conditions for obtaining quercetin were obtained.

Claims (8)

(a) treating Viscozyme L with Morus alba L leaf;
(b) extracting the substance to be treated in step (a) with a lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; And
(c) separating quercetin from the mulberry leaf extract; Wherein the mulberry leaf-derived quercetin is selected from the group consisting of: The method according to claim 1, wherein the mulberry leaf concentration is 15% (w / v) to 30% (w / v) in step (a). The method according to claim 1, wherein the step (a) is carried out at a reaction time of 20 to 40 hours, wherein quercetin derived from mulberry leaves is obtained. The method according to claim 1, wherein the concentration of viscose L in the step (a) is 3% (v / v) to 15% (v / v). The method according to claim 1, wherein the quercetin separation in step (c) is performed in an ethyl acetate fraction of a mulberry leaf extract. The method according to claim 1, wherein the obtained quercetin has an antidiabetic activity, wherein quercetin derived from mulberry leaves is obtained. (a) treating Viscozyme L with Morus alba L leaf; And
(b) extracting the substance to be treated in step (a) with a lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; Wherein the quercetin content of the mulberry leaf extract is increased. The method according to claim 7, wherein the mulberry leaf extract has increased antidiabetic activity, wherein the content of quercetin is increased.

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