KR102111969B1 - 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 for obtaining quercetin derived from mulberry leaves comprising the step of treating Viscozyme L on Morus alba L leaves and a method for producing a mulberry leaf extract with an increased content of quercetin. Using the method for obtaining quercetin derived from mulberry leaves comprising the step of treating Viscozyme L of the present invention on a mulberry leaf, it is possible to obtain a large amount of quercetin exhibiting anti-diabetic activity present in mulberry leaves. Therefore, the method of the present invention can reduce the manufacturing cost and effort of anti-diabetic agents, and can be usefully used for the manufacture of anti-diabetic drugs and health functional foods that can improve and prevent diabetes.

Description

{A method for acquiring quercetin from extract of Morus alba L by treating viscozyme L} by treating biskozyme L to obtain quercetin in mulberry leaf extract

The present invention Viscozyme (Viscozyme) L mulberry ( Morus alba L) A method for obtaining quercetin derived from a mulberry leaf comprising the step of treating the leaves and a method for preparing a mulberry leaf extract having an increased content of quercetin.

Chronic complex diseases due to the aging of the population are increasing worldwide. Accordingly, the interest and demand of natural-derived medicines that are easily targeted for multi-target treatment is increasing. Natural medicines and other active materials derived from natural products have less development period and cost compared to synthetic medicines and other synthetic products, and have good drug efficacy and fewer side effects, making active research and development investments. The growth rate of domestic and foreign markets for natural medicines among biopharmaceuticals in 2013-2018 has been increasing to 19.99% and 13.98%, respectively, and 10.59% for domestic and overseas active ingredients such as natural cosmetics, antibiotics, and health functional foods, respectively. And 5.45%.

  Various active abilities of mulberry trees, which are widely inhabited not only in Korea but also in China, Japan, and central regions of the world, have been revealed. Among them, mulberry leaves have been mainly used in oriental medicine, and according to Shinnongbonbyeonggyeong, Japan's Ochergyeong, and Donguibogam, mulberry leaves are recorded as medicinal plants such as beriberi, edema, urination, and tanghang. The efficacy of the mulberry leaf as described above is reported to be caused by various flavonoids present in the mulberry leaf, and the mulberry leaf has various effects ranging from anti-cancer, anti-diabetic, antioxidant, anti-obesity, whitening, anti-atherosclerotic, and anti-inflammatory. Is known. Due to this, mulberry leaves, which have been mainly taken in the form of tea, are currently commercialized as additives or products of various foods.

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

Bioconversion technology is known to involve the conversion of organic matter, such as plant or animal waste, into useful products or raw materials using biological methods or agents, such as certain enzymes or microorganisms. The bioconversion technology as described above is an effective method for improving physiological activity, and is particularly used to increase physiological activity in various fields using enzymes.

Therefore, as described above, the need for bioconversion technology of a mulberry leaf extract (Bioconversion technology) is emerging to absorb useful flavonoids present in the mulberry leaf with high efficiency.

Accordingly, the present inventors studied a method for obtaining a large amount of an inactive substance present in a mulberry leaf as an active substance that can be absorbed directly in vivo, and when biotransformation was performed by treating the enzyme Viscozyme L, the extract of the mulberry leaf It was confirmed that the content of quercetin, which represents the present anti-diabetic activity, can be obtained in large quantities, thereby completing the present invention.

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

In order to achieve the above object, the present invention (a) Viscozyme (Viscozyme) L mulberry ( Morus alba L) treating the leaves; (b) extracting the treatment material of step (a) with lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; And (c) separating quercetin from the mulberry leaf extract; It provides a method for obtaining a quercetin (quercetin) derived from mulberry leaves, comprising a.

In addition, the present invention (a) Viscozyme (Viscozyme) L Mulberry ( Morus alba L) treating the leaves; And (b) extracting the treatment material of step (a) with lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; It provides a method for producing a mulberry leaf extract containing an increased content of quercetin (quercetin).

Using the method for obtaining quercetin derived from mulberry leaves comprising the step of treating Viscozyme L of the present invention on a mulberry leaf, it is possible to obtain a large amount of quercetin exhibiting anti-diabetic activity present in mulberry leaves. Therefore, the method of the present invention can reduce the manufacturing cost and effort of anti-diabetic agents, and can be usefully used for the manufacture of anti-diabetic drugs and health functional foods that can improve and prevent diabetes.

1 is a result of normal phase thin layer chromatography on ethyl acetate fraction of mulberry leaf extract and irradiated with ultraviolet light (FIG. 1A), and then stained by spraying anise aldehyde sulfuric acid (FIG. 1B) and α-glucosidase inhibition It is a diagram showing the experimental results (Fig. 1C) confirming the activity.
FIG. 2 is a diagram schematically showing a method of performing an experiment to separate and purify the active flavonoid present in the ethyl acetate fraction.
3 is a result of performing thin layer chromatography on the acetate fraction of the mulberry leaf extract and MAV1, MAV2 and MAV3 separated from the fraction and irradiating with ultraviolet rays (FIG. 3A), and then staining by spraying anise aldehyde sulfuric acid (FIG. 3B) ) And High Pressure Liquid Chromatography (HPLC) to confirm the purified flavonoids (FIG. 3C).
4 is a view showing the results of the hydrogen nuclear magnetic resonance method for the separated MAV1, MAV2 and MAV3 compounds.
5 is a view confirming α-glucosidase inhibitory activity of a control ethyl acetate fraction not treated with biscozyme L, an ethyl acetate fraction treated with biscozyme L, and isolated MAV1, MAV2 and MAV3 compounds (* p <0.05).
FIG. 6 shows the substrate concentration (w / v) in FIG. 6A, the enzyme concentration in FIG. 6B, the reaction temperature in FIG. 6C, and FIG. 6C as a result of changing the reaction conditions in order to find the optimal bioconversion conditions through biscozyme. It is a diagram showing the change in the amount of quercetin obtained by changing the reaction pH in 6D and the reaction time in FIG. 6E.

The present invention (a) Viscozyme (Viscozyme) L Morus alba L) treating the leaves; (b) extracting the treatment material of step (a) with lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; And (c) separating quercetin from the mulberry leaf extract; It provides a method for obtaining a quercetin (quercetin) derived from mulberry leaves, comprising a.

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

The mulberry used in the present invention is a crop that is widely inhabited not only in Korea, but also in China, Japan, and the central region of the world. In particular, its leaves have anti-cancer, anti-diabetic, antioxidant, anti-obesity, whitening, anti-atherosclerotic, and anti-inflammatory properties. It is known to have a variety of active abilities, including.

The method of the present invention relates to a method capable of obtaining quercetin present in a mulberry leaf, which is one of flavonoids present in a mulberry leaf, through the enzyme by treating biscozyme L on the mulberry leaf. Bioconversion refers to the conversion of organic substances, 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 improve physiological activity, and in particular, it is used to increase physiological activity in various fields using enzymes. In the present invention, the biskozyme L was treated on the mulberry leaf to bioconvert the mulberry leaf by increasing the obtained content of quercetin having antidiabetic activity among the active flavonoids present in the mulberry leaf. The quercetin obtained in the present invention is one of flavonoids, widely distributed in vegetables and fruits as a glycoside, and obtained by hydrolyzing rutin (extract) with an acidic aqueous solution or enzyme, and resistant to heat. In particular, quercetin is a substance known to be usable as an antioxidant.

According to the method of the present invention, since it is possible to obtain quercetin in large quantities from the same amount of mulberry leaves, even if the same amount of mulberry leaf extract is used, it has been confirmed that the increase in anti-diabetic activity is superior to that of biscozyme L untreated mulberry leaf extract.

In the bioconversion method to step (a) of the present invention, the concentration of the mulberry leaf may be used at 15% (w / v) to 30% (w / v), but preferably 20% (w / v). Mulberry leaves can be used. In addition, the reaction time may be 20 to 40 hours, and preferably 24 hours. The concentration of biskozyme L used may be 3% (v / v) to 15% (v / v), but preferably 5% (v / v) to 10% (v / v) of biscozyme L is used. Can be used for conversion. In a preferred embodiment of the method of the present invention, in step (a), the reaction conditions are 20% (w / v) mulberry leaf concentration, 5% (v / v) enzyme concentration, the reaction time is 24 hours and 37 ° C. , pH 4 conditions to obtain a bio-converted mulberry leaf enzyme treatment.

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 is not limited thereto, and the present invention uses ethanol as a preferred example. In addition, in the above extraction method, any conventional methods known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction can be used.

In the present invention, the step (c) may include the step of obtaining an ethyl acetate fraction by adding water and ethyl acetate to the obtained mulberry leaf extract, wherein quercetin is an ethyl acetate fraction of the mulberry leaf extract. Can be obtained from In addition, the step (c) is the ethyl acetate fraction is then subjected to silica gel chromatography using chloroform and methanol solvent; And a fraction obtained through silica gel chromatography is performed with Sephatex LH-20 column chromatography using a methanol solvent. And the fraction obtained through Sephadex LH-20 column chromatography may be subjected to high-speed liquid chromatography (HLPC) again. In one embodiment of the present invention, the size of the silica gel column chromatography was Ø6 x 30cm, the size of the Sephadex LH-20 column chromatography was Ø3.5 x 90cm, and the high-pressure liquid chromatography had a flow rate of 2 It was carried out using a CAPCELL PAK C18 MG column having a size of Ø10 × 250 mm as a fixed phase in ml / min.

Quercetin obtained by the method of the present invention exhibits anti-diabetic activity, and the anti-diabetic activity specifically includes an activity that inhibits α-glucosidase.

In addition, the present invention (a) Viscozyme (Viscozyme) L Mulberry ( Morus alba L) treating the leaves; And (b) extracting the treatment material of step (a) with lower alcohol having 1 to 4 carbon atoms to obtain a mulberry leaf extract; It provides a method for producing a mulberry leaf extract containing an increased content of quercetin (quercetin).

A lower alcohol having 1 to 4 carbon atoms may be used to extract the mulberry leaf extract extracted by the method of the present invention, but is not limited thereto, and the present invention uses ethanol as a preferred example. In addition, the mulberry leaf extract extracted by the method of the present invention has an increased anti-diabetic activity, especially by increasing the obtained content of quercetin having anti-diabetic activity among the active flavonoids present in the mulberry leaf through bioconversion due to the treatment of biscozyme L. In particular, it is characterized by having excellent activity for inhibiting α-glucosidase.

Duplicate contents are omitted in consideration of the complexity of the present specification, and terms that are not otherwise defined herein have meanings commonly used in the technical field to which the present invention pertains.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example

Materials used in the experiment

Dried mulberry leaves were obtained in the Hadong Hat dimension and commercial enzyme Viscozyme L for bioconversion was purchased from Novozymes (Copenhagen, Denmark). Ethanol, ethyl acetate and n-butanol were purchased from SK Chemicals (Ulsan), HPLC grade ethanol was purchased from Burdick & Jackson (Muskygon, USA), and enzymes and substrates for biological analysis were Sigma-Aldrich (Yongin) , Korea). Silica gels 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 bioconverted mulberry leaf extract

Bioconversion of mulberry leaves was performed with a commercially available biscozyme L enzyme solution. The dried mulberry leaves were pulverized into a powder, and the 10% (w / v) powder was re-suspended in distilled water, and then 5% (v / v) of a viscozyme L enzyme solution was added. The conditions of the bioconversion reaction were set based on the conditions according to the product data sheet of the Biscozyme L enzyme, and the bioconversion reaction was performed for 48 hours with shaking at 150 rpm. Subsequently, ethanol extracts of mulberry leaves that were bio-converted by extracting three times with ethanol were obtained to perform sequential solvent extraction to divide fractions according to polarity of ethyl acetate, butanol, water, and thin layer chromatography (TLC) ).

TLC was performed by the method of normal phase and reverse phase. Table 1 shows the fixed and fluidized beds used in the chromatography.

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

A bioconverted mulberry leaf ethyl acetate or n-butanol extract was prepared at 10 mg / mL, and then 5 μL was spotted on the starting line of the TLC plate and separated with a developing solvent. Thereafter, the TLC plate was air dried and irradiated under ultraviolet rays (254 nm), and the experimental results of irradiating ultraviolet rays on the normal TLC plate are shown in FIG. 1A. Anise aldehyde sulfuric acid reagent was sprayed onto the TLC plate and dried for coloring of the separated compound, and the results of the experiment are shown in FIG. 1B.

Subsequently, an experiment was performed to confirm which fraction of the fractions separated had the highest α-glucosidase inhibitory activity. α-Glucosidase inhibitory activity is based on p-nitrophenyl glucopyranoside (pNPG) (Sigma-Aldrich) and α- derived from Saccharomyces cerevisiae (Sigma-Aldrich) It was measured by mixing 0.075 units of glucosidase with mulberry leaf extract bioconverted to various concentrations. PNPG was added to a concentration of 3 mM in 100 mM sodium phosphate buffer (pH 6.8) and incubated at 37 ° C for 30 minutes. α-glucosidase inhibitory activity was determined by measuring the amount of p-nitrophenol released from pNPG with a VERSA _max microplate reader (Molecular Devices, Toronto, Canada) at 405 nm. Percentage of activity measured using methanol as a negative control and glucosidase inhibitor-based oral hypoglycemic agents, 0.25, 1 and 4 mg / mL concentrations of acarbose (Sigma-Aldrich) as a positive control The results of performing the above experiment are shown in FIG. 1C. Lanes 1 and ME Vis are methanol extracts, lanes 2 and EL Vis are ethyl acetate fractions, lanes 3 and BL Vis are n-butanol fractions, and lanes 4 and WL Vis are experimental results of water fractions.

1A and 1B, it was confirmed that several separable components appeared in the ethyl acetate fraction. 1C, it was confirmed that ethyl acetate extract exhibited the highest α-glucosidase inhibitory activity at the same concentration compared to methanol, n-butanol, and water extract. Therefore, it was confirmed that the fraction extracted with ethyl acetate had the highest anti-diabetic activity. Therefore, in the following examples, an ethyl acetate fraction of a bioconverted mulberry leaf extract exhibiting high antidiabetic activity was used.

Example 2. Extraction separation and purification of active flavonoids from bioconverted mulberry leaf extract

An experiment was performed to separate and purify the active flavonoid present in the ethyl acetate fraction identified in Example 1, and the method of performing the experiment is schematically shown in FIG. 2. 5% (v / v) Viscozyme L was treated with 100 g of mulberry leaf powder in the same manner as in Example 1, and then extracted three times with 4 L of ethanol to obtain an ethanol extract. Thereafter, 1 part of ethyl acetate and water were mixed with ethanol extract at a ratio of 1 L to divide the fraction, and extraction was performed three times with ethyl acetate. After that, the silica gel column chromatography size was Ø6 x 30 cm, and flavonoid compounds were separated by silica gel column chromatography according to the polarity of the solvent using 100% chloroform to chloroform: methanol = 30: 1 mixed solvent.

Among the separated fractions, it was confirmed that anti-diabetic activity was observed in fractions 3 to 16 using chloroform: methanol mixed solvent as 30: 1. Accordingly, in order to separate the flavonoids present in the fraction according to the molecular weight, Sephadex LH-20 column chromatography was performed to separate the flavonoids with a size of Ø3.5 x 90 cm. The flavonoids isolated from fractions 48-60, 102-110, and 120-125 were named MAV1, MAV3, and MAV2, respectively.

Example 3. Identification of isolated and purified flavonoids and confirmation of enhancement of α-glucosidase inhibitory activity

3.1 Identification of flavonoids in fractions

The components were separated using high-pressure liquid chromatography to confirm which of the flavonoids isolated and purified in Example 2 exhibited anti-diabetic activity.

First, ethyl acetate extract of the bio-converted mulberry leaf and the MAV 1 to 3 were analyzed by thin layer chromatography (TLC), and the results of the analysis are shown as A and B in FIG. 3. Thereafter, high-pressure liquid chromatography (HPLC) was performed to identify purified flavonoids. Under the conditions of HPLC, a mobile phase with methanol and 0.1% acetic acid at a volume ratio of 60% was flowed for 4 minutes, and 60 to 100% methanol until 20 minutes. From 20 minutes to 30 minutes, a concentration gradient was given with 100% methanol, and the flow rate was 2 ml / min. In addition, a CAPCELL PAK C18 MG column (Shiseido Co., Japan) having a size of Ø10 × 250 mm was used as a stationary phase, and the result of the separation and identification was shown in FIG. 3C. EE Ctrl stands for Mulberry Leaf Extract, and EE Vis stands for Bioconverted Mulberry Leaf Extract into Viscozyme L. Afterwards, nuclear magnetic resonance spectroscopy (H-NMR spectroscopy) was performed to identify the identified flavonoid components, and the results are shown in FIG. 4.

3A and B, it was confirmed that the peak shape was changed when bioconverted using biscozyme L compared to EE Ctrl. In addition, as shown in FIG. 3C, it was confirmed once again that each MAV 1 to 3 isolated from the mulberry leaf extract was derived from a material that was bioconverted with a biscozyme enzyme at the same point.

As shown in Figure 4, it was confirmed that the flavonoids as a result of identifying MAV1 to 3 through hydrogen nuclear magnetic resonance method are caffeic acid, quercetin, and camperol (kaempferol), respectively.

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

To confirm whether the anti-diabetic activity of the MAV1 to 3 flavonoids isolated in Example 3.1 is caused by bioconversion by biscozyme enzyme, and to determine which component of the flavonoid is a substance exhibiting the increased anti-diabetic activity The experiment was conducted. The conditions of the experimental method of α-glucosidase inhibitory activity were measured in the same manner as in Example 1, and the activity of the flavonoids present in the isolated MAV1 to 3 was measured, and this is shown in FIG. 5.

As shown in FIG. 5, in MAV 2 and 3, high α-glucosidase inhibitory activity was confirmed. In particular, when bioconversion was performed by treatment with biskozyme, a significant increase in α-glucosidase inhibitory activity was observed in the ethyl acetate fraction, and it was confirmed that the increase in activity was due to quercetin of MAV2. In addition, showing the enhanced anti-diabetic activity as described above is due to the increase in the content of quercetin, which is MAV2, at the same concentration, inhibiting α-glucosidase of 80% or more by the high content of quercetin when the concentration of the extract is 15 μg / mL or more. It was confirmed that the anti-diabetic activity of glucosidase inhibitor-based oral hypoglycemic agents was higher than that of acarbose used in clinical practice. In addition, when the concentration of the extract was 50 μg / mL, it was confirmed that the inhibitory activity of α-glucosidase was increased by more than 90%, and the anti-diabetic activity was significantly increased due to the increase in the content of quercetin, an active substance due to bioconversion. there was.

Example 4. Identification of bioconversion conditions for optimally obtaining active quercetin from mulberry leaf extract

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

As shown in FIG. 6A, when the substrate concentration was increased to 5, 10, 15 and 20% (w / v), the concentration-dependent increase was confirmed by confirming that the amount of quercetin quantified increased. Particularly, when the concentration of the substrate was 15% or more, it was confirmed that the yield was increased by a factor of 2 compared to when the substrate concentration was 5%. However, when the concentration of the substrate reached 20% or more, it was confirmed that the saturation was reached and no further increase was observed, and the optimum substrate concentration was 20% (w / v).

As shown in Figure 6B, it was confirmed that the amount of quercetin obtained increases as the concentration of the enzyme also increases as the substrate. However, when the concentration was 3% or more, it was confirmed that the yield was increased about twice as much as when the concentration was 0.3%, and a significant increase in the amount of quercetin obtained was confirmed. However, when the concentration of the enzyme was 5% or more, it was confirmed that it reached the saturation state and was no longer increased. In the method of optimizing the quercetin content, it was confirmed that the optimal enzyme concentration was 5% (v / v).

6C, the reaction temperature was 15 ° C, 37 ° C, and 55 ° C. As a result, the highest temperature could be obtained at 37 ° C and 1/2 of the amount of quercetin obtained at 37 ° C at 15 ° C and 55 ° C. It was confirmed that it can be obtained at a level, it was confirmed that the optimum reaction temperature is 37 ° C.

As shown in FIG. 6D, the best activity when performing bioconversion in the pH range of 4 to 6 among various pH ranges (2, 4, 6, 8, 10, 12) ranging from acidic to neutral and basic with respect to pH It confirmed that this appeared.

In addition, as shown in FIG. 6E, as a result of performing the experiment with reaction times of 0, 3, 6, 12, 24, and 48 hours, the yield of quercetin increases by about 1.5 times as compared with the case of 6 hours after 24 hours. It was confirmed that the reaction time reached a saturation state after 24 hours had elapsed, and it was confirmed that 24 hours was a condition for obtaining an optimal amount of quercetin. Taken together, the amount of quercetin (5,282μg / 1g mulberry leaf) obtained at a basic bioconversion condition of 10% (w / v) substrate, 5% (v / v) enzyme, 37 ° C, pH 4, 48 hours The optimal reaction conditions identified in this example are 20% (w / v) substrate, 5% (v / v) enzyme, 37 ° C, pH 4, and 24 hours of quercetin that can be obtained when bioconversion is performed. It was confirmed that the amount was increased by about 32% with 6,994 μg / 1g mulberry leaf, which was a reaction condition to obtain optimal quercetin.

Claims (8)

delete delete delete delete delete delete (a) treating Viscozyme L on Morus alba L leaves under conditions of 37 ° C., pH 4 for 24 hours,
The mulberry leaf concentration is 20% (w / v) and the Viscozyme L concentration is treated with 5% (v / v) to bioconvert; And
(b) extracting the treatment material obtained in step (a) with ethanol to obtain a mulberry leaf extract;
As a method of producing a mulberry leaf extract containing an increase in the content of quercetin, including,
The total content of quercetin contained in the mulberry leaf extract is 6.994 mg / g mulberry leaf;
The mulberry leaf extract is characterized in that the anti-diabetic activity is increased, the method of manufacturing a mulberry leaf extract with increased content of quercetin (quercetin). delete

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