KR20070078167A - How to separate narirutin from citrus fruits - Google Patents

Abstract

The present invention relates to a method for separating narirutin from citrus fruits.

Specifically, the method for separating narirutin from citrus fruits according to the present invention comprises the steps of (a) preparing a dry powder of citrus fruits, (b) extracting the citrus dried powder with a polar solvent, and (c) step (b) It comprises a step of removing impurities from the extract obtained in.

Description

Method for Isolating Narirutin From Orange}

1 is a ¹ H NMR spectrum of a compound obtained in Examples of the present invention.

2 is a ¹³ C NMR spectrum of a compound obtained in the Examples of the present invention.

3 is a structural formula of a compound obtained in an embodiment of the present invention.

The present invention relates to a method for separating narirutine from citrus fruits.

Citrus fruits contain many physiologically active substances such as citric acid, vitamins, carotenoids, and flavonoids, which are known to be good for skin care and fatigue, help calcium absorption, and prevent arteriosclerosis and hypertension.

In particular, the tangerine peel has been used as a medicine for the dermis in traditional medicine, and has been used for influenza pandemic, gastrointestinal diseases, edema, and shellfish poisoning.

Flavonoids among the bioactive substances in citrus fruits are the most widely distributed polyphenolic compounds in plants, followed by chlorophyll and carotenoids, and are the most frequent pigments in plants, and vitamin P or vitamin C ₂ (vitamin C synergists). Also called).

Flavonoids have several physiological activities, including antioxidant activity, antimicrobial activity, antiviral activity, anti-inflammatory activity, anti-cancer activity, and also have the effect of preventing the atherosclerosis by inhibiting the oxidation of low density lipids in plasma.

There are thousands of flavonoids, and important flavonoids are flavonols, flavones, flavaones, flavanones, flavanones, flavanols, and ansocyanidins. ), Isoflavone, dihydroflavonl, chalcone and the like.

Narirutin belongs to flavanone compounds together with narigenin, hesperedin and the like.

It is well known that naciginine, hesperedin, and the like are contained in citrus fruits, but until now, narirutin is not known in citrus fruits.

The present inventors have completed the present invention by separating narirutin from citrus fruits.

Accordingly, the present invention provides a method for separating naruritin from citrus fruits.

Other objects or specific aspects of the present invention will be presented below.

We isolated narirutin from citrus fruits as follows.

First, the dried powder of tangerine was extracted with ethanol to obtain an extract from which the extraction solvent was removed, and the extract was dissolved in distilled water. Then, n-hexane was added, and the n-hexane layer and the aqueous layer were partitioned, and ethyl acetate was added to the aqueous layer, and the ethyl acetate layer and the aqueous layer were partitioned. The organic solvent was removed from the ethyl acetate layer fraction and dissolved in methanol to pass and elute the column filled with the polar filler and the column filled with the hydrophobic filler. The obtained fraction was dissolved in a suitable organic solvent and then developed using high performance liquid chromatography to separate a compound having a purity of 98%. The structural formula was then analyzed through NMR analysis to find narirutin (Naringenin-7-O-rutinoside; 5-hydroxy-2- (4-hydroxyphenyl) -7- [3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyl-oxan-2-yl) oxymethyl] oxan-2 -yl] oxy-chroman-4-one).

The present invention is provided based on these experimental results.

Therefore, the method for separating narirutin from citrus fruits according to the present invention comprises the steps of (a) preparing a dry powder of citrus fruits, (b) extracting the citrus dried powder with a polar solvent, and (c) step (b) It comprises a step of removing impurities from the extract obtained in.

Above and below, including the claims, the term “citrus fruits” is defined as fruit obtained from any kind of fruit tree belonging to taxonomic citrus fruits (ie, dicotyledonous clove and fruit citrus subfamily), Specifically, it is defined as a generic term for the fruit obtained from various fruits belonging to citrus, golden persimmon and tangerine, and fruit derived from these three genera.

Therefore, the "citrus fruits" is not only the citrus ( C. natsudaidai Hayata) used in the following examples but also citron (citron) cultivated in southern Europe, lemon (lemon) cultivated in the United States, California, etc. Paragraphs such as grapes and grapefruits, grape fruit, cultivated in Florida, USA, oranges grown in India, Italy, tangerines grown in India, China and Korea It is understood that all of the citron (재배 子) cultivated in, the tangerine cultivated in Jeju Island, etc., the golden persimmon cultivated in China, and other tangerines are included.

Nevertheless, the citrus fruit is preferably understood as a tangerine, in that it is preferably used in the following examples of the present invention.

On the other hand, as shown in the following examples of the present invention, the narirutin is separated much more when the immature fruit is used than when the mature fruit is used.

Therefore, the method of separating narirutin from citrus fruits according to the present invention is preferably to use immature fruit rather than mature fruit.

Here, "mature fruit" refers to the fruit in the state that the flesh and skin are not yet differentiated and joined to each other.

On the other hand, the term "polar solvent" refers to a solvent composed of polar molecules, such as water, distilled water, alcohol (preferably lower alcohols having 5 or less carbon atoms), ethyl acetate, acetone, chloroform, methylene chloride or their Mixed solvents are mentioned.

In addition, "impurity" as used above means all polar or nonpolar compounds other than narirutin.

In a preferred aspect, the step of removing impurities in the method of separating narirutin from citrus fruits according to the present invention is to add a non-polar solvent to the extract obtained in step (b) to remove the fraction of the non-polar solvent layer and the fraction of the polar solvent layer And obtaining.

The extract obtained in the step (b) is a liquid extract in the state that the extraction solvent of step (b) is not removed, or when the extraction solvent is removed in step (b), the solid extract is dissolved in a polar solvent. Refers to an extract of the form. Here, the polar solvent may be understood as defined and exemplified above.

In addition, the nonpolar solvent is a solvent composed of nonpolar components, for example, n-alkanes having 6 or more carbon atoms, in particular n-hexane used in the following examples.

The reason why the nonpolar solvent is added in the above is to remove the nonpolar impurities from the impurities by adding the nonpolar solvent.

In a more preferred aspect, the step of removing impurities in the method for separating narirutin from citrus fruit according to the present invention after the step of obtaining a fraction of the polar solvent layer by adding a polar solvent having a different polarity to the fraction of the polar solvent layer The method may further include obtaining two polar solvent layer fractions.

For example, if the fraction of the polar solvent layer is water or distilled water as a solvent, the polar solvent having a different polarity may be methanol, ethanol, ethyl acetate, acetone, chloroform, methylene chloride or a mixed solvent thereof. In the following examples, when the fraction of the polar solvent layer is distilled water as a solvent, ethyl acetate was used as the polar solvent having a different polarity.

The reason for adding polar solvents having different polarities is to remove impurities having polarity.

In a more preferred aspect, the step of removing impurities of the method for separating narirutin from citrus fruits according to the present invention, after obtaining a fraction of the polar solvent layer or after obtaining the two polar solvent layer fractions, The polar solvent layer may further comprise the step of passing through the column filled with the polar filler and the column filled with the non-polar filler, eluting.

In the above, in the case of passing through the step of obtaining the two polar solvent layer fractions, and then passing through the step of eluting, any polar solvent layer fraction of the two polar solvent layer fractions may be passed through the elution step. However, in consideration of the polarity of narirutin, it is preferable to use a polar solvent layer fraction having a polarity similar to that of narirutin. This is because the polarity should be similar so that the content of narirutin contained in the polar solvent layer fraction will be higher. For example, if the two polar solvent layer fractions are the aqueous layer fraction or the ethanol layer fraction, respectively, it is preferable to perform the passage and elution step using the ethanol layer fraction, and the two polar solvent layer fractions are the ethanol layer fraction and the ethyl, respectively. If it is an acetate layer fraction, it is preferable to perform the said pass-through elution step using an ethyl acetate fraction.

On the other hand, when performing the pass-through elution step using the polar solvent layer fraction, the polar solvent layer fraction may be used as it is, but the solid extract obtained by removing the polar solvent is dissolved in another suitable polar solvent Pass-through and elution steps can also be performed. Suitable polar solvents here will be water, distilled water, alcohols with up to 5 carbon atoms, and the like.

In addition, when performing the pass-through elution step using the polar solvent layer fraction, a polar filler or a non-polar filler may be used in the art known in the art, the elution solution also depending on the polar filler or non-polar filler used and Any elution solution known in the art may be used in consideration of the polarity of narirutin.

Also in a more preferred aspect, the step of removing impurities in the method of separating narirutin from citrus fruit according to the present invention, after obtaining the fraction of the polar solvent layer or after obtaining the two polar solvent layer fraction, or After the passage and the elution step, the step of passing the polar solvent layer fraction or the eluate through liquid chromatography may be performed one or more times.

Here, preferably, the step of passing the liquid chromatography is performed by sequentially passing Prep-LC and HPLC. It is in order to maximize the purity of the final narirutin obtained.

In a most preferred aspect, the method for separating narirutin from citrus fruits according to the present invention is a case where all of the processes of the following examples are performed.

Specifically, (i) extracting the citrus dry powder using ethanol and removing the extraction residue and the extraction solvent to obtain a solid extract, (ii) dissolving the solid extract in distilled water, (iii) in distilled water Adding n-hexane to the dissolved extract to obtain an n-hexane layer fraction and an aqueous layer fraction, (iv) adding ethyl acetate to the aqueous layer fraction to obtain an aqueous layer and an ethyl acetate layer fraction, and (v) an ethyl acetate layer fraction. Ethyl acetate was removed and dissolved in methanol again and passed through a column filled with polar silica gel to elute with a mixed solution of chloroform and methanol. (Vi) The eluate was passed through a column filled with hydrophobic octadecylsilane and eluted with methanol. (Iv) passing the eluate through Prep-LC to dissolve the compound obtained in step (viii) in step (vii) in methanol and then through HPLC. Refers to a case include the step.

In particular, all of the above steps may be performed under the specific conditions disclosed in the following examples.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

< Example > From citrus fruits Narirutin  Separation and Identification

< Example  1> Citrus From immature  Separation of Pure Compound

<1-1> Preparation of Dry Powder

Citrus unripe fruit is a pesticide-free mandarin plant grown in Seogwipo-si, Jeju Island, and used in mid-July. First it was washed with water, crushed with a wet mill, lyophilized and powdered.

<1-2> Ethanol Extract of Citrus Immature

500 g of the citrus immature and dried powder were put into a 10 L volume round bottom flask, and 5 L of 70% ethanol was added. The process of reflux extraction was performed twice at a room temperature (25 ± 5 ° C.) for about 48 hours (2 days). After repeated 3 to 3 times to obtain an extract of about 10L to remove the extraction residue using a filter paper (0.25mm size). The filtrate was completely removed using a rotary pressure reducer, and then frozen in a cryogenic freezer at -80 ° C. for about a day with little water remaining, and then lyophilized to obtain an extract of about 50 g.

<1-3> Distribution of Organic Solvents from Citrus Immature

The extract obtained in <1-2> was dissolved in 500 mL of distilled water, and then, 1 L of n-hexane was mixed in a 2 L separatory funnel, and fractionated twice into a hexane layer and an aqueous layer. 1 L of ethyl acetate was added to the aqueous layer, and the mixture was partitioned twice into an ethyl acetate layer and an aqueous layer.

The ethyl acetate layer fractions obtained above were dried under reduced pressure to remove ethyl acetate and lyophilized to obtain 15.6 g of ethyl acetate fraction.

<1-4> column chromatography purification

The ethyl acetate fraction of <1-3> was dissolved in 10 mL of methanol, adsorbed onto the upper portion of the first column (4 × 25 cm) filled with polar silica gel (Silica gel 60), and then chloroform and methanol were 3: 1 ratio. 4.1 g of the active fraction eluted with the mixed solution was adsorbed onto a second column (2 × 25 cm) filled with hydrophobic octadecyl silane (ODS), and eluted with 40% methanol to obtain 492 mg of the active fraction.

<1-5> Separation using Prep-LC

The 492 mg obtained in <1-4> was dissolved in 10 mL of methanol for HPLC, and then passed through a 0.45 μm sample filter. The result was to isolate using stabilized high performance liquid chromatography. The high-performance liquid chromatography uses Waters Co model DeltaPrep equipped with US Waters Co. SunfirePrep (5μm, 19 × 150mm), and 10 acetonitrile and distilled water. The mobile phase solvent mixed and degassed at the rate of 90 was separated by flowing at a flow rate of 10 mL / min. Waters dual λ Absorbance detector (Detector) was used to isolate the compounds using the ultraviolet absorption pattern of the material and the absorbance of the wavelength (254nm) was able to obtain 312mg of the compound in 10 minutes.

<1-6> Separation using HPLC

The 312 mg obtained in <1-5> was dissolved in 4 mL of methanol for HPLC, and then passed through a 0.2 μm sample filter to be separated using stabilized high performance liquid chromatography. High-speed liquid chromatography uses Waters Co Model 2695 alliance equipped with US Waters Co Atlantis columns (Atlantis, 5μm, 4.6 × 250mm) and uses acetonitrile and distilled water 20 The flow was analyzed at a flow rate of 1 mL / min of the mixed and degassed mobile phase solvent at a ratio of 80 to 80. The Waters 2996 Photodidde array (PDA) detector (Detecter) was used to look at the UV absorption pattern of the material. The maximum absorption wavelength was 283 nm and the retention time was 12 minutes to obtain a pure compound to obtain 80 mg (purity 98%; Waters Photodioder array detector (Model No .: 2695 allience)).

< Example  2> Citrus With maturity  Separation of Pure Compound

Narirutin was isolated from the tangerine harvested in November by the same process using the same conditions and solvent as in <Example 1>. The final amount of pure compound was 6.43 mg (purity 98%).

< Example  3> Structure Identification of Compound

The <Examples 1 and 2> and by each compound completely separated from the drying dissolved in CD ₃ OD (1mL) was poured into 5mm NMR tube using a FT-NMR to jiol model aircraft (JMX-700) ¹ H- NMR and ¹³ C-NMR were analyzed.

The results are shown in <Figure 1> and <Figure 2>, respectively, and the chemical structural formulas are shown in <Figure 3>.

This result indicates that the compound isolated in <Examples 1 and 2> is narirutin.

As mentioned above, according to the present invention there is provided a method for separating narirutin from citrus fruits.

In particular, it can be seen that the yield from the immature fruit is higher than that from the mature fruit.

Claims (15)

A method for separating narirutin from citrus fruits comprising the following steps (a) to (c). (a) preparing a dry powder of citrus fruits; (b) extracting the citrus dried powder with a polar solvent; And (c) removing impurities from the extract obtained in step (b) The method of claim 1, The tangerine is a method of separating narirutin from citrus fruit, characterized in that the mandarin. The method according to claim 1 or 2, The tangerine is a method of separating narirutin from citrus, characterized in that immature fruit. The method of claim 1, The polar solvent is water, distilled water, a lower alcohol having a carbon number of 5 or less, ethyl acetate, acetone, chloroform, methylene chloride and a solvent selected from the group consisting of a mixed solvent thereof. The method of claim 1, Removing the impurities of step (c) comprises adding a non-polar solvent to the extract obtained in step (b) to remove the fraction of the non-polar solvent layer and to obtain a fraction of the polar solvent layer How to isolate narirutin. The method of claim 5, The extract obtained in step (b) is a liquid extract in a state in which the extraction solvent of step (b) is not removed or a solid extract obtained by removing the extraction solvent in step (b) is dissolved in a polar solvent. Method of separating narirutin from citrus fruits, characterized in that the extract. The method of claim 5, The nonpolar solvent is a method for separating narirutin from citrus fruit, characterized in that the carbon number of 6 or more n-alkanes. The method of claim 5, Removing the impurities of step (c) further includes obtaining two polar solvent layer fractions by adding polar solvents having different polarities to the fraction of the polar solvent layer after obtaining the fraction of the polar solvent layer. Method for separating narirutin from citrus fruits, characterized in that. The method of claim 5, Removing the impurities of step (c) may include passing the eluted fraction of the polar solvent layer through the column filled with the polar filler and the column filled with the nonpolar filler after the step of obtaining the fraction of the polar solvent layer. A method for separating narirutin from citrus fruits, further comprising. The method of claim 8, Fraction of the polar solvent layer is distilled water as a solvent, the polar solvent is different polarity added is a solvent selected from the group consisting of methanol, ethanol, ethyl acetate, acetone, chloroform, methylene chloride and a mixed solvent thereof How to separate narirutin from citrus fruits. The method of claim 8, Wherein the polar solvent layer fraction is distilled water as a solvent, and the polar solvent of different polarity is added ethyl acetate, the method of separating narirutin from citrus. The method of claim 8, In the removing of the impurities in the step (c), after obtaining the two polar solvent layer fractions, one of the polar solvent layer fractions is sequentially filled with a polar filler column and a non-polar filler column. A method of separating narirutin from citrus fruits, further comprising passing and eluting. The method of claim 12, The fraction to be passed and eluted out of the two polar solvent layer fraction is a polar solvent layer fraction having a polarity similar to the polarity of naruritin separation method of the nacirutin from citrus. The method according to any one of claims 5, 8, 9 and 12. Removing the impurities in step (c) may include performing the step of passing the polar solvent layer fraction or the eluate through liquid chromatography after the step of obtaining the polar solvent layer fraction or after the passage and elution step. Method for separating narirutin from citrus fruits, characterized in that. A method for separating narirutin from citrus fruits comprising the following steps (i) to (vii). (i) extracting the tangerine dry powder using ethanol and removing the extraction residue and the extraction solvent to obtain a solid extract; (ii) dissolving the solid extract in distilled water, (iii) adding n-hexane to the extract dissolved in distilled water to obtain an n-hexane layer fraction and an aqueous layer fraction, (iv) adding ethyl acetate to the aqueous layer fraction to obtain an aqueous layer and an ethyl acetate layer fraction, (v) removing ethyl acetate from the ethyl acetate layer fraction and dissolving it in methanol again, passing through a column filled with polar silica gel and eluting with a mixed solution of chloroform and methanol, (vi) eluting the eluate with methanol through a column filled with hydrophobic octadecylsilane, (vii) passing the eluate through Prep-LC and (viii) dissolving the compound obtained in step (vii) in methanol and then passing through HPLC.

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