KR20100001907A - Method for preparation of quercetin -3-glucoside from rutin using rhamnosidase originated from aspergillus niger - Google Patents

Abstract

PURPOSE: A method for preparing quercetin glucoside from rutin using rhamnosidase(EC 3.2.1.40) is provided to improve productivity of quercetin-3-glucoside having apoptosis ability to cancer cells. CONSTITUTION: A quercetin-3-glucoside having anticancer activity is converted from Aspergillus niger(KCTC 6906)-derived rhamnosidase. A method for isolating the rhamnosidase comprises: a step of shaking Aspergillus niger(KCTC 6906) in PD(potato dextrose) and ME(malt extract) at 30°C for five days; a step of filtering the culture medium using Whatman Glass Microfibre filter; a step of filtering again with bottle top filter at 0.22μm; a step of dissolving the filtered liquid in 85% ammonium sulfate, centrifuging to remove supernatant, and dissolving precipitate in buffer; and a step of isolating through DEAE-cellulose ion chromatography sephacryl S-200 gel chromatography.

Description

Method for preparation of quercetin -3-glucoside from rutin using rhamnosidase originated from Aspergillus niger}

The present invention is produced by bioconversion of rutin, one of the phytochemicals present in plants, such as buckwheat, with a rhamnosidase (EC 3.2.1.40) enzyme from Aspergillus niger. It relates to quercetin glycosides (isoquercitrin, quercetin-3-glucoside), quercetin-3-glucoside shows apoptosis effect on cancer cell lines at a much lower concentration than the various phytochemicals known to date.

Plant-derived phytochemicals, especially flavonoids, are known to perform various biological functions on the human body, including high antioxidant capacity such as free radical scavenging ability, inhibition of free radical formation, and prevention of oxidation of intracellular lipids and proteins. have. Among the more than 4,000 of these flavonoids, quercetin, which is found in onions and apples, is not only for its antioxidant capacity, but also for its cytotoxic and apoptosis effects and its mechanisms against cancer cells in vitro, animal, human, and epidemiological studies. Studies on the anticancer effect through the has been performed.

 Quercetin belongs to the flavonol family of flavonoids, and is one of the aglycones representing them together with kaempferol, myricetin and the like. However, in the plant, quercetin is present in the form of glycoside combined with several single or multiple sugars such as glucose, rhamnose, galactose, and arabinose rather than nonglycoside. Rutin, which is abundantly contained in tea and buckwheat, is also one of the glycosides in which quercetin is combined with rutinose consisting of glucose and rhamnose. (Figure 1)

In terms of anti-cancer activity, the research results thus far have been focused on the activity of the non-glycoside quercetin, which does not show the ability to kill cancer cells in the glycoside routine. In terms of anticancer activity studies, quercetin has been evaluated for its anticancer activity through studies on cell proliferation inhibition and apoptosis induction, and tumor suppression studies in a real tumor mouse model. The effective concentration of quercetin (evaluated by IC50 concentration) showing anticancer activity is shown to be approximately 50 μM or more depending on the experimental method used, the type of cell line used in the evaluation, and the experimental model. However, the quercetin-3-glucoside obtained by the method newly proposed by the present technology development leads to the invention that the antitumor activity is superior to other materials.

The biofunctional utility of phytochemicals derived from plants, in particular flavonoids, is influenced by the content and type of flavonoids present in food, in vivo absorption and exposure concentrations. At this time, the functional effective concentration seen in the existing in vitro experiments is considerably high in terms of the prevention of cancer cell production, and there are many differences from the actual exposure concentration. On the other hand, if the flavonoids present in the form of glycosides in combination with sugar is transformed into a more easily absorbed and effective form, its functionality can be further enhanced.

The present invention is to provide a method for increasing the productivity and use of the quercetin-3-glucoside, a glycoside that exhibits the ability to kill cancer cells at a lower concentration than other flavonoids show the ability to kill cancer cells.

In order to achieve the object of the present invention,

Rhamnosidase enzyme from Aspergillus niger strain capable of degrading the rhamnose bond of lutinose was used for the conversion of quercetin-3-glucoside of rutin. As a result, the enzyme isolated from the A. niger strain converted rutin to quercetin-3-glucoside. (Figure 2)

Using several dozen probiotic and edible food microbial strains, strains capable of hydrolyzing glycosides of glycosides can be evaluated in advance, resulting in Aspergillus being able to degrade the rhamnose bonds of lutinose. niger strains were selected.

In this study, quercetin-3-glucoside (Q3G), which is produced when the routine is converted to glycosylase from Aspergillus niger, is lower than the glycoside quercetin. Cancer cell death was also observed at the concentration of 20 μM.

Rhamnosids were isolated and purified from the culture solution of Aspergillus niger KCTC 6906 belonging to Filamentous Fungi.

Cell culture was carried out in PD (potato dextrose), ME (malt extract) medium generally used for culturing fungal strains. Shaking culture was carried out under aerobic conditions and the incubation temperature was 30 ℃. The incubation period was the highest in the production of rhamnosides when shaken for 5 days.

The enzyme is obtained by culturing the strain under the culture conditions as described above. Since the active substance is present in the culture solution, after culturing, the culture solution was filtered with a Whatman Glass Microfibre filter, and then filtered again with a 0.22 μm bottle top filter containing enzyme to completely remove spores. In order to obtain only the protein containing the enzyme, the filtered culture solution was dissolved in 85% ammonium sulfate, the precipitated protein was centrifuged to remove the supernatant, and then dissolved in 20 ml of buffer.

The enzyme solution thus obtained was separated by a gradient of 0-0.5M NaCl using DEAE-Cellulose ion chromatography (pH4.0, piperazine buffer). A fraction representing the enzyme activity was obtained near the NaCl concentration of 0.17M, and once again separated and purified under the same conditions to increase the resolution. Thereafter, the active fractions were separated by molecular weight through Sephacryl S-200 Gel chromatography to obtain about 85 kDa of rhamnosidase enzyme.

At 25 μM, the concentration of quercetin that showed cancer cell death, when the 20 or more flavonoids were treated preferentially, the MTT assay method used to measure the survival of the cells was screened for the most active substance. (Figure 3) On the other hand, in order to evaluate which glycostructures have a significant effect on the antitumor activity of quercetin in various structurally related glycosides, quercetin (QUE) (aglycone) and rutin (RUT) ( glycone; QUE-rutinose), quercitrin (QUI) (glycone; QUE-rhamnose), isoquercitrin (IQI) (glycone; QUE-glucose), hyperin (HYP) (glycone; QUE-galactose), and reinutrin (RIN) (glycone Cell death of glycosides such as QUE-xylose) was also evaluated. As a result, among the quercetin glycosides, only quercetin-3-glucoside was shown to have higher anticancer activity than quercetin.

The types of cancer cell lines used in the experiment to evaluate the specificity and sensitivity change according to the difference of cell lines were diversified into three types of colorectal cancer cell lines, breast cancer, prostate cancer, lung cancer, and melanoma cells. The impact was also evaluated.

In all cancer cell lines, the IC50 concentration of quercetin-3-glucoside was at or above 25 μM, including colorectal cancer cell lines HT-29, HCT-116 and breast cancer cell lines MCF-7 and prostate cancer cell lines PC. -3 showed high sensitivity and death induction pattern.

The HT-29 and MCF-7 cell lines were used to evaluate apoptosis patterns according to the concentrations of quercetin, a glycoside and a non-glycoside such as rutin and quercetin-3-glucoside (Q3G), as shown in FIG. 4. Among them, when the HT-29 cancer cell line was cultured for 3 days at a concentration of 25 μM, the morphological changes appearing with the induction of apoptosis are as shown in FIG. 5.

1 shows the structure of rutin, quercetin-3-glucoside, quercetin

  2 is a TLC analysis of Q3G conversion when the glycoside routine is incubated with Aspergillus niger-derived enzymes over time.

  3 is a cell death capacity evaluation of various flavonoids for various cancer cell lines

  4 is a cell death capacity evaluation according to the concentration of glycosides and non-glycosides for HT-29, MCF-7 cell line

  Figure 5 shows the morphological changes of the HT-29 colorectal cancer cell line when cultured for 3 days at 25 μM level.

Claims (3)

Quercetin-3-glucoside with anticancer activity. A method for preparing quercetin-3-glucoside from rutin using rhamnosidase from Aspergillus niger . 3. The method of claim 2, wherein Aspergillus niger is Aspergillus niger KCTC 6906. 4. A method of preparing quercetin-3-glucoside according to claim 2, wherein Aspergillus niger is Aspergillus niger .

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