KR102279861B1 - Method for extract of citrus preicarp and composition for preventing, improving or treating liver injury - Google Patents

Abstract

The present invention is an invention of a method for extracting and separating 5-O-dimethyl nobiletin from green citrus fruits through the following extraction method,
① pulverizing green citrus fruits or juice by-products through a grinder;
② extracting the pulverized product using ethyl acetate or methylene dichloride as a polar solvent;
③ removing impurities or coloring matter contained in the extract using activated carbon;
④ A vacuum drying step of removing the pesticide components remaining in the extract using NaOH, which is alkaline water, washing with water to remove alkali, and vacuum drying to obtain a powder;
⑤ After using the solvent, normal-hexane:ethyl acetate in a ratio of 2:1, 20 times the volume of the column was passed through a short silicagel column to remove nobiletin and synacetin, and to remove tangerine and 5-O-dimethyl nobiletin. separating the mixture;
⑥ Concentrating the mixture of tangeretin and 5-O-dimethyl nobiletin;
⑦ Dissolving the mixture in methylene dichloride and leaving it at -20°C for 6 hours to separate the solvent and the mixture (tangeretin is dissolved in the solvent) and 5-O-dimethyl nobiletin crystals;
⑧ After removing the residual solvent mixture in vacuum, obtaining the final crystal, 5-O-dimethyl nobiletin; consists of.

Description

Extraction/isolation of 5-O-dimethyl nobiletin from blue citrus fruits and a composition for protecting chronic alcoholic fatty liver damage {Method for extract of citrus preicarp and composition for preventing, improving or treating liver injury}

The present invention is a method for extracting 5-O-dimethyl nobiletin (5-O-Demethyl nobiletin) from blue citrus fruits using an extract of polymethyl flavonoid (abbreviated as 'PMF') using short flash column chromatography and The invention relates to a fatty liver damage protection composition using the same.

Drugs using synthetic chemicals have been constantly presented with problems such as side effects on the human body. Therefore, in recent years, in order to reduce the side effects caused by synthetic chemicals, various pharmaceutical compositions using natural products have been developed.

Natural materials have fewer side effects and are highly favored by consumers, so their development value as a raw material for pharmaceuticals is increasing. Nobletin, a cancer-suppressing component, is a type of polymethyl flavonoid, a special flavonoid, and is a component contained only in citrus fruits. It is known to be effective in preventing cancer, such as delaying invasion, preventing metastasis, and inhibiting the proliferation of tumor cells. Due to the pharmacological action as described above, the demand for nobiletin, tangeretin, and synacetin is increasing, and technologies for separating and purifying extracts containing high content of nobiletin, tangeretin and synacetin are being developed.

Looking at the prior art, nobiletin and tangeretin, which are extracts in the form of precipitates, are extracted by adding water-containing lower alcohol having an alcohol concentration of 50 to 80 wt% to the peel of citrus fruits in Korean Patent No. 1676751. In addition, Korean Patent No. 1588613 discloses (1) washing the pulp or peel of citrus fruits with a basic solution and recovering; (2) extracting the recovered product with an aqueous alcohol solution to obtain an extract; (3) concentrating the extract after filtration to obtain a concentrate; and (4) precipitating and drying the concentrate to obtain a final product. A method for producing a citrus extract containing Nobiletin or Tangeretin is disclosed, and Korean Patent No. 0770746 discloses citrus fruits. The pericarp was freeze-dried, extracted with 80% methanol, filtered and concentrated, and the concentrate was partitioned and extracted with hexane and water, and the aqueous layer was partitioned with ethyl acetate (EtOAc) and extracted with ethyl acetate. After concentrating the extract, it is fractionated by normal phase column chromatography, the fraction having the highest nobiletin content is selected using high performance liquid chromatography, and the selected fraction is purified using liquid chromatography for recycle preparative. A method is disclosed.

In addition, Korean Patent No. 1825150 and Korean Patent No. 0770746 disclose a method for producing a citrus dermis extract extracted through steps a to i, and a composition for preventing, improving or treating liver cancer containing the active ingredient. This is disclosed.

However, according to a recent study result, it was confirmed that 5-O-dimethyl nobiletin had a greater effect on alleviating alcoholic fatty liver damage than nobiletin, tangeretin, and synacetin, which are the PMF indicator components. Accordingly, the present inventors extracted poly methyl flavonoid extracts from blue citrus fruits with reference to prior art, and separated and purified these extracts using short flash column chromatography to remove nobiletin, tangeretin and synacetin, A new extraction method for extracting 5-O-dimethyl nobiletin was developed.

That is, the 5-O-dimethyl nobletin is filled with silica gel equivalent to 40 times the weight of the extract by filling the extract containing nobiletin, tangeretin and synacetin in a ratio of 2:1 with n-hexane:ethyl acetate. Nobiletin and synacetin were removed by passing a solvent corresponding to 20 times the volume of the filled silica gel as a mobile phase, and the recovered 5- O -dimethyl nobletin and tangeretine mixture was dissolved in CH ₂ Cl _{2 as a solvent.} After standing at -20°C for 6 hours, it was confirmed that 5-O-dimethyl nobletin was recrystallized to complete the present invention.

Domestic Registered Patent No. 1676751 Domestic Registered Patent No. 1588613 Domestic Registered Patent No. 0770746 Domestic Registered Patent No. 1825150 Domestic Registered Patent No. 0770746 Domestic Registered Patent No. 1449976 Domestic Patent Publication No. 2015-0020001

An object of the present invention is to develop a method for extracting 5-O-dimethylnoviletin, a kind of polymethyl flavonoid, from blue citrus fruits.

Another object of the present invention is to analyze each component of blue citrus fruits and provide a method for high-purity separation and purification of 5-O-dimethylnoviletin therefrom. In addition, an object of the present invention is to provide a fatty liver damage protection composition containing the 5-O-dimethyl nobiletin.

The problem to be solved by the present invention is to extract 5-O-dimethylnoviletin crystals from blue citrus fruits or juice by-products as a result of long-term research by the present inventors, for this purpose

① pulverizing green citrus fruits or juice by-products through a grinder;

② extracting the pulverized product using a polar solvent ethyl acetate (Ethtyl acetate) or methylene dichloride (CH ₂ Cl ₂ );

③ removing impurities or coloring matter contained in the extract using activated carbon;

④ A vacuum drying step of removing the pesticides remaining in the extract using NaOH, which is alkaline water, washing with water to remove alkali, and vacuum drying to obtain a powder;

⑤ to do;

⑥ Concentrating the mixture of tangeretin and 5-O-dimethyl nobiletin;

⑦ Dissolving the mixture in methylene dichloride and leaving it at -20°C for 6 hours to separate the solvent and the mixture (tangeretin is dissolved in the solvent) and 5-O-dimethyl nobiletin into crystals;

⑧ After removing the residual solvent mixture in vacuo, obtaining a final crystal of 5-O-dimethyl nobiletin; includes

High-purity 5-O-dimethyl nobiletin was extracted with a high content through the extract of blue citrus fruits according to the present invention.

The above 5-O-dimethyl nobiletin was confirmed to be quite effective in protecting chronic alcoholic fatty liver, as normal cells and nuclei were observed in the group treated with 5-O-dimethyl nobiletin by an organic synergistic action.

1 is a process diagram for extracting and separating 5-O-dimethylnoviletin from the peel of blue citrus fruits.

As for terms used in the invention, general terms that are currently widely used are selected as possible, and in specific cases, there may be terms arbitrarily selected by the applicant, but this takes into account the meanings described or used in the detailed description of the invention rather than the names of simple terms So the meaning should be understood. It has been reported that Nobiletin, Tangeretin, and Sinensetin, which are polymethyl flavonoids (PMF), are present in the peel of citrus fruits. Various methods have been developed for However, until now, it was not known that 5- O -dimethyl nobiletin was present in the peel of citrus fruits.

The present inventors confirmed the fact that green citrus fruits contain a large amount of 5-O-dimethyl nobiletin compared to general ripe citrus fruits through various repeated experiments.

In the present invention, 5-O-dimethyl nobiletin was extracted from green citrus fruits through the following extraction method.

① pulverizing green citrus fruits or juice by-products through a grinder;

② extracting the pulverized product using ethyl acetate or methylene dichloride as a polar solvent;

③ removing impurities or coloring matter contained in the extract using activated carbon;

④ A vacuum drying step of removing the pesticides remaining in the extract using NaOH, which is alkaline water, washing with water to remove alkali, and vacuum drying to obtain a powder;

⑤ After using the solvent, normal-hexane:ethyl acetate in a ratio of 2: 1, pass 20 times the column volume through a short silicagel column to remove nobiletin and synacetin, and to remove tangerine and 5-O-dimethyl nobiletin. separating the mixture;

⑥ Concentrating the mixture of tangeretin and 5-O-dimethyl nobiletin;

⑦ Dissolving the mixture in methylene dichloride and leaving it at -20°C for 6 hours to separate the solvent and the mixture (tangeretin is dissolved in the solvent) and 5-O-dimethyl nobiletin crystals;

⑧ After removing the residual solvent mixture in vacuum, obtaining a final crystal, 5-O-dimethyl nobiletin; consists of.

Specifically, in step ⑤ of the present invention, synacetin of formula (1) and nobiletin of formula (2) are removed.

Nobiletin : 1H NMR (400MHz, CDCl ₃ ): δ7.60(dd, J=8.4Hz, J’=2.4Hz, 1H), δ7.42(d, J=2Hz, 1H), δ7.00(d, J=8.8Hz, 1H), δ6.67(s, 1H), δ 4.11(-CH ₃ , 3H), δ4.03(-CH ₃ , 3H), δ3.98 CH ₃ , 3H), δ3.97 (-CH ₃ , 3H), δ3.96(-CH ₃ , 6H) ; 13C NMR (100MHz, CDCl ₃ ): δ177.1, δ161.10, δ152.0, δ151.5.6, δ149.3, δ149.5, δ148.4, δ147.7, δ144.1, δ138.0, δ123 .9, δ119.7, δ114.8, δ111.3, δ108.6, δ106.8, δ62.3, δ61.9, δ61.7, δ56.1, δ55.9

Sinensetin : 1H NMR (400MHz, CDCl ₃ ): δ7.50(dd, J=8.8Hz, J'=2.4Hz, 1H), δ7.03(d, J=9.2Hz, 2H), δ6.67(s) , 1H), δ 4.11 (s , -OCH 3, 3H), δ4.03 (s, - OCH 3, 3H), δ3.95 (s, -OCH 3, 3H), δ3.89 (s, -OCH ₃ , 3H) ; 13C NMR (100 MHz, CDCl ₃ ): δ177.3, δ161.2, δ 157.8, δ154.8, δ154.6, δ152.7, δ151.9, δ149.4, δ140.5, δ124.2, δ119. 7, δ112.9, δ111.2, δ108.7, δ107.5, δ96.4, δ62.3, δ62.7, δ61.7, δ56.4, δ56.2, δ56.2

In step ⑦, tangeretin of formula (3) is dissolved in a solvent, and crystals of 5-O-dimethyl nobiletin of formula (4), which is an extract, are precipitated.

Tangeretin : 1H NMR (400 MHz, CDCl ₃ ): δ7.89(d, J=8.84Hz, 2H), δ7.03(d, J=9.2Hz, 2H), δ6.67(s, 1H), δ 4.11 _{(s, -OCH 3, 3H)} , δ4.03 (s, - OCH 3, 3H), δ3.95 (s, -OCH 3, 3H), δ3.89 (s, -OCH 3, 3H); 13C NMR (100 MHz, CDCl ₃ ): δ177.4, δ162.4, δ 161.4, δ151.5, δ148.4, δ147.8, δ144.2, δ138.1, δ 127.8, δ 123.8, δ114.8, δ114.6, δ106.6, δ62.3, δ62.1, δ61.8, δ61.7, δ55.5

5-O-Demethylnobiletin : 1H NMR (400MHz, CDCl ₃ ): δ12.53(s, -OH), δ7.57(dd, J=8Hz, J'=2.2Hz, 1H), δ7.40(d, J=2.4Hz, 1H), δ6.98(d, J=8.8Hz, 1H), δ6.59(s, 1H), 4.10(-CH ₃ , 3H), δ3.97(-CH ₃ , 3H) , δ3.96 CH ₃ , 3H), δ3.94(-CH ₃ , 3H), δ3.93(-CH ₃ , 3H); 13C NMR (100MHz, CDCl ₃ ): δ183.1, δ164.0, δ153.1, δ152.6, δ149.6, δ149.5, δ145.9, δ136.7, δ133.0, δ123.8, δ120 .2, δ111.4, δ108.9, δ107.1, δ104.1, δ62.2, δ61.8, δ61.2, δ56.2, δ56.1

In the above, the green citrus fruits are Citrus depressa, C. tachibana, Koji (C. leiocarpa), Kiri tangerine (C. tardiva), Jimigang (C. succosa), and Kishiu (C. kinokuni). , Kobeni mikang (C.erythrosa), Chichuukaimandaring (C.deliciosa), King (C.nobilis), Main building (C.retuculata), Tangierin (C.tangerina), Hana (C.hanaya), You can choose any one of Kourai Tachibana (C.nippokoreana), Ginger Tangerine (C.sunki hort. ex Tanaka), Hallabong (C.hybrid Shiranuhi), Radish Bong, Onju Tangerine (citrus unshiu), and Citrus aurantium. have.

[Effect of 5-O-dimethyl nobiletin on alcoholic liver damaged tissue]

The results of H&E staining and immunostaining to confirm the liver histopathological change and fatty liver relief effect through the administration of alcohol and PMF indicator components are shown in FIG. 2 . In the normal control group, which consumed only distilled water without any alcohol intake, there was no alcohol-induced liver damage and only normal hepatocytes could be observed. It was not observed, lipid accumulation was also not observed, and the shape of the nucleus was relatively clearly observed. Unlike the hepatocytes of the normal control group, damage to the liver tissue was clearly observed in the control group treated with alcohol during the experiment period. Due to fat deposition in hepatocytes, balloon-shaped deformation of cells, mallysosomes, and destroyed nuclei were observed, and these types of cells and destroyed nuclei are indicators for judging fatty liver. As normal cells and nuclei were observed in the MIX (D), tangeretin (F), and 5-O-dimethyl nobiletin (G) treated groups, it was observed that the treatment was significantly effective in the protection of chronic alcoholic fatty liver. On the other hand, nobiletin treatment group (E) showed no alleviating effect (see Figure 1).

[Figure 1] Hepatic histopathological analysis of PMF in blue citrus extract

In particular, in order to determine the effect of the PMF indicator component on alcoholic fatty liver, immunostaining was performed using the pro-inflammatory enzyme TNF-α. As a result, in the normal control group and the group taking 5-O-dimethyl nobiletin (G), No TNF-α positive reaction was observed. However, a strong positive reaction was observed centered on blood vessels in the group that consumed alcohol and the group that consumed nobiletin (D), and a relatively weak positive reaction was also observed in the group that consumed MIX (D) and tangeretin (F). could be observed (see Figure 2).

[Figure 2] Hepatic immunohistopathological analysis using TNF-α distribution for green citrus PMF

[Effect of 5-O-dimethyl nobiletin on the biochemical composition of blood]

As the liver is damaged by alcohol, there are significant hematologic changes, so the blood test results confirming the changes in the biochemical components in the blood, etc. in the animal model ingesting the PMF indicator component and ingesting alcohol are as follows. Changes in levels of ALT, AST, and triglycerides (TG), which are representative proteins related to liver damage, were measured in the blood serum of mice administered with alcohol and PMF indicator components.

Blood ALT and AST are generally known as enzymes released into the blood when liver tissue is damaged and are widely used as indicators of liver damage, and were significantly higher in the alcohol (NC) treatment group than in the normal control group (CON). Blood ALT and AST activity were shown. As shown in Figure 3, in the mixture (hereinafter referred to as MIX) and the nobiletin (NB) treatment group containing all of the PMF indicator components, the blood ALT and AST activity were significantly lower than in the alcohol treatment group, The tangeretin and 5-O-dimethyl nobiletin-treated groups showed significantly similar ALT and AST activities than the normal control group. In conclusion, all of the green citrus indicator components were effective in reducing ALT and AST, and in particular, the tangeretine and 5-O-dimethyl nobiletin-treated groups showed very effective efficacy close to that of the normal group.

[Figure 3] Hemopathological analysis of blue citrus PMF

[Effect of 5-O-dimethyl nobiletin on CYP2E1 expression]

The cause of oxidative stress is an increase in free radicals caused by alcohol, which plays a key role in alcoholic liver damage. Accordingly, in vivo, high levels of CYP2E1 expression are shown to alleviate oxidative stress, which is a major indicator to measure the degree of liver damage. Therefore, in this experiment, western blot was performed to quantify the expression level of CYP2E1 in the liver. In the normal treatment group administered only with distilled water, the protein level of CYP2E1, the protein to be measured, was almost not expressed, and in the alcohol treatment group, the expression level of CYP2E1 was approximately twice that of the normal treatment group, which was statistically significant. was found to be highly expressed. Also, as shown in Figure 4, the expression level of CYP2E1 decreased in the group administered with tangeretin and 5-O-dimethyl nobiletin among the PMF indicator components, similar to that of the normal group.

[Figure 4] Analysis of CYP2E1 protein expression in blue citrus PMF

[Effect of 5-O-dimethyl nobiletin on TNF-α expression]

An experiment was performed to quantify the NF-α expression level, and the normal treatment group administered only with distilled water showed almost no expression of the level of TNF-α to be measured, and the alcohol treatment group showed about 2% compared to the normal treatment group. It could be seen that the expression level of TNF-α was close to twice that of TNF-α, and the expression level was statistically significantly high. In addition, it could be seen that the expression level of TNF-α in the group administered with the PMF indicator component decreased similarly to the normal group, and in particular, the expression level of tangeretin and 5-O-dimethyl nobiletin treated group was similar to that of the normal group. As a result, the entire index component was very effective in inhibiting TNF-α expression, and as shown in Figure 5, tangeretin and 5-O-dimethyl nobiletin showed the best results.

[Figure 5] TNF-α expression analysis for green citrus PMF

Claims (6)

A first step of pulverizing green citrus fruits or juice by-products through a grinder;
a second step of extracting the pulverized product using ethyl acetate or methylene dichloride as a polar solvent;
a third step of removing impurities or coloring matter contained in the extract using activated carbon;
a fourth step of vacuum-drying to obtain a powder by removing the pesticides remaining in the extract using NaOH, which is alkaline water, washing with water to remove alkali, and vacuum drying;
After using the solvent, normal-hexane:ethyl acetate in a ratio of 2:1, 20 times the volume of the column was passed through a short silicagel column to remove synacetin of Formula (1) and nobiletin of Formula 2, and formula (3) a fifth step of separating a mixture of tangeretine and formula (4) 5-O-dimethyl nobiletin;
a sixth step of concentrating a mixture of tangeretin and 5-O-dimethyl nobiletin;
a seventh step of dissolving the mixture in methylene dichloride and leaving it to stand to separate crystals of 5-O-dimethyl nobiletin;
Extraction separation method of 5-O-dimethyl nobiletin of formula (4) from blue citrus fruits, characterized in that consisting of.

According to claim 1,
Green citrus fruits include Citrus depressa, C.tachibana, Koji (C.leiocarpa), Giri mandarin (C.tardiva), Jimigang (C.succosa), Kishiu (C.kinokuni), and koji. C.erythrosa, C.deliciosa, King (C.nobilis), Main Building (C.retuculata), Tangerina (C.tangerina), Yumo Hana (C.hanaya), Kourai Tachibana (C.nippokoreana), ginseng (C.sunki hort. ex Tanaka) and hallabong (C.hybrid Shiranuhi), radish, onju tangerine (citrus unshiu), light tangerine (Citrus aurantium) 5- Extraction separation method of O-dimethyl nobiletin. According to claim 1,
A method for extracting and separating 5-O-dimethyl nobiletin from blue citrus fruits, characterized in that the residual solvent is removed in vacuum in step 7 and 5-O-dimethyl nobiletin crystals are precipitated. delete delete delete

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