KR102164943B1 - Methods for simultaneous analysis of five types of physiological active substances contained in Noni berries - Google Patents

Abstract

The present invention relates to a simultaneous analyzing method of five types of physiological active materials contained in a Noni fruit, the method comprising: a step of preparing a sample solution for analysis by using an extracting solvent from the Noni fruit or food containing the Noni fruit; a step of injecting the prepared sample solution into a high-performance liquid chromatography column having a non-polar column as a stationary phase; and a step of flowing a mobile phase mixed solvent to the high-performance liquid chromatography column into which the sample solution is injected, and analyzing contents of the five types of the physiological active materials contained in the Noni fruit through absorbance measurement of high-performance liquid chromatography. The present invention rapidly and accurately detects the five types of the physiological active materials contained in the Noni fruit at the same time; and analyzes each content. Therefore, the present invention is usefully used to analyze the physiological active materials contained in the Noni fruit or the food containing the Noni fruit.

Description

Methods for simultaneous analysis of five types of physiological active substances contained in Noni berries}

The present invention relates to a method for simultaneous analysis of five physiologically active substances contained in Noni fruit, and more specifically, listed as a raw material that can be used as food. In order to standardize quality for the development of health functional foods from noni fruit or foods containing noni fruit, 5 types of physiologically active substances contained in noni fruit can be detected simultaneously and accurately and contained in noni fruit that can analyze its content It relates to a method for simultaneous analysis of 5 types of bioactive substances.

Noni (Morinda citrifolia L) is an evergreen shrub belonging to the genus Morinda citrifolia, and contains abundant vitamins A and C. As physiologically active substances, deacetylasperulosidic acid and asperuloside acid (Asperulosidic acid), Rutin, Scopoletin, Rubiadin, etc. are contained, so it has antioxidant, anti-inflammatory, anti-tumor, anti-viral, and cytotoxic effects. In particular, South Pacific Polynesian Tahitian natives) have long used noni for edible and therapeutic purposes, and it is a tropical plant that is drawing attention as a health food as it is still used as a raw material for juice and raw food (powder).

Meanwhile, as a general method for analyzing components contained in the formulation, an analysis method by chromatography is used. Chromatography refers to various solids or liquids as a stationary phase and a gas or liquid as a mobile phase, allowing the mobile phase to pass through the stationary phase, while introducing a sample into the mobile phase, It refers to a method of separating samples for each component by using the difference in adsorption properties or partition coefficients of the samples between stationary beds.

Chromatography methods have been developed in many different types, and are widely used including separation of natural products, purification of pharmaceuticals, and purification of other chemical substances. Depending on the type of mobile phase, gas chromatography in which gas is used as a mobile phase and liquid chromatography in which a liquid is used as a mobile phase can be broadly classified, and are commercially developed and commercially available. In addition, ion exchange chromatography, which separates and analyzes by using bonds between ions instead of using the difference in partition coefficient, has been developed and used. Although various types of chromatography have been developed and commercialized, the chromatography is fundamentally based on separation using the physical properties of the sample to be analyzed by chromatography, that is, the partition coefficient between the stationary phase and the mobile phase, molecular weight, and ionicity. Therefore, the optimal separation and analysis conditions for a desired sample cannot be uniformly defined or determined. In addition, various analysis factors such as the selection of the stationary phase and the mobile phase, the flow rate of the mobile phase, and the selection of a detector that detects a sample that is separated and eluted according to the target sample must be determined through interaction. Since it is an important thing that can influence the results, there is a problem in that an optimal analysis method by chromatography must be selected for each material.

Therefore, in order to standardize the quality for the development of health functional foods through the fruits of Noni registered as raw materials that can be used as food, we optimize the conditions for simultaneously analyzing 5 types of physiologically active substances contained in Noni fruits, and analyze them quickly and accurately There is a need for a way to do it.

KR 10-2082106 B1 (2020.02.21.)

The present invention was devised to solve the problems of the prior art, and the problem to be solved in the present invention is to standardize the quality for the development of health functional foods from the fruits of Noni registered as raw materials that can be used as food, Its purpose is to optimize conditions for simultaneous analysis of five physiologically active substances contained in fruit, and to provide a method for analyzing quickly and accurately.

The simultaneous analysis method of five physiologically active substances contained in noni fruit according to the present invention for achieving the above object is the step of preparing a sample solution for analysis using an extraction solvent from noni fruit or food containing noni fruit And, injecting the prepared sample solution into a high performance liquid chromatography column having a non-polar column as a stationary phase, and flowing a mobile phase mixed solvent into the high performance liquid chromatography column into which the sample solution is injected, and absorbance of the high performance liquid chromatography It characterized in that it comprises the step of analyzing the content of five physiologically active substances contained in the noni fruit through measurement.

In addition, the five physiologically active substances contained in the fruit of Noni are Deacetylasperulosidic acid, Asperulosidic acid, Rutin, Scopoletin, and rubiadine. It is characterized by being (Rubiadin).

In addition, the column of the high performance liquid chromatography is characterized in that the inner diameter is 4 ~ 5mm, the length is 200 ~ 300mm, the diameter of the particles charged in the column is 4 ~ 7㎛.

In addition, the absorbance measurement is characterized by using a wavelength of 254nm.

In addition, the mixed solvent is used by mixing 0.1% formic acid aqueous solution and acetonitrile by volume in a ratio of 0.01 to 98.00: 2.00 to 99.99, but depending on the transfer time, when the transfer time is less than 35 minutes, 98% by volume of 0.1% formic acid aqueous solution, Mix 2% by volume of acetonitrile and mix 0.01% by volume of 0.1% formic acid aqueous solution and 99.99% by volume of acetonitrile when the transfer time is 35 minutes or more and less than 37 minutes, and 0.1% when the transfer time is more than 37 minutes and less than 52 minutes It is characterized by mixing so that 98% by volume of an aqueous formic acid solution and 2% by volume of acetonitrile.

According to the present invention, not only can the 5 kinds of physiologically active substances contained in the noni fruit be detected simultaneously and accurately, but also the content of each can be analyzed, so that the physiology contained in the fruit or food containing the noni fruit There is an effect that can be usefully used to analyze the active substance.

In addition, quality standardization for the development of health functional foods through the fruit of Noni registered as a raw material that can be used as a food, and increase economic and temporal efficiency in research and development of products containing noni fruit ingredients or quality control of manufacturing. It can have an effect.

1 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in Noni fruit under final analysis conditions according to the present invention.
Figure 2 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in Noni fruit using YMC-Pack ODS-AM (YMC, 4.6 × 250 mm, 5 μm) as a high-performance liquid chromatography column.
3 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in Noni fruit using μBondapak C18 Prep (Waters, 7.8 × 300 mm, 10 μm) as a high-performance liquid chromatography column.
4 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in Noni fruit using XBridge C18 (Waters, 3 × 250 mm, 3 μm) as a high-performance liquid chromatography column.
5 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in Noni fruit using methanol as an organic solvent for a mobile phase solvent.
6 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in Noni fruit using acetonitrile as an organic solvent for a mobile phase solvent.
7 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in noni fruit by setting the absorbance to 254 nm.
8 shows a chromatogram of simultaneous analysis of five physiologically active substances contained in noni fruit by setting the absorbance to 225 nm.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In the present invention, (A) preparing a sample solution for analysis using an extraction solvent from a noni fruit or food containing noni fruit, and (B) a high-performance liquid chromatography column having a non-polar column as a stationary phase. Injecting the sample solution prepared in step) and (C) pouring a mobile phase mixed solvent into the column of high-performance liquid chromatography injected with the sample solution in step (B), and measuring the absorbance of the high-performance liquid chromatography It relates to a method for simultaneous analysis of five kinds of bioactive substances contained in Noni fruit, comprising the step of analyzing the content of five kinds of bioactive substances contained in

The five physiologically active substances contained in Noni fruit analyzed according to the simultaneous analysis method of the present invention are Deacetylasperulosidic acid, Asperulosidic acid, Rutin, and Scopol. Retin (Scopoletin), rubiadin (Rubiadin).

Step (A) is a process of preparing a sample solution for analysis using an extraction solvent from noni fruit or food containing noni fruit.

In more detail, the extraction solvent is mixed in a ratio of 1: 5-10 by weight to noni fruit or food containing noni fruit, and extracted to prepare a sample solution for analysis.

In the present invention, one or two or more of water, methanol, ethanol, and ethyl acetate may be mixed and used as an extraction solvent.

Step (B) is a process of injecting the sample solution prepared in step (A) into a column of high performance liquid chromatography having a non-polar column as a stationary phase.

In the column used in the present invention, the length and inner diameter of the column are reduced to minimize the analysis time, and the size of the charged particles in the column is reduced to ensure the degree of separation, and the mobile phase solution or sample solution passing through the column is caught in the column. It should not contain insoluble particulates or microorganisms that can increase pressure or affect the analysis.

Therefore, it is preferable to use a non-polar column filled with octadecylsilane (C18), dodecylsilane (C12) or octasilane (C8) having non-polarity as a fixed phase, and more preferably, components having similar chemical properties are overlapped with each other to reduce the degree of separation. In order to prevent this, it is preferable to use a non-polar column filled with octadecylsilane (C18) having strong non-polar properties.

The non-polar column filled with octadecylsilane (C18) is filled with a material having a non-polar 18 molecular chain functional group of carbon in the column, so that the polar component is rapidly eluted because it does not bind with the non-polar column functional group. In the case of the component, it binds to a non-polar functional group and elutes relatively slowly.

The size of the column can be within the range used normally. However, since the length, inner diameter and particle size of the column are related to the analysis time and degree of separation, it is preferable to use it in an appropriate category in consideration of speed and selectivity.

In the present invention, in consideration of analysis efficiency, the inner diameter of the column is 4 to 5 mm, the length is 200 to 300 mm, and the diameter of the particles charged in the column can be used within the range of 4 to 7 μm, most preferably The column has an inner diameter of 4.6 mm, a length of 250 mm, and a particle size of 5 μm. It is preferable to use YMC-Pack ODS-AM (YMC, 4.6 x 250 mm, 5㎛) as the product name.

A column that satisfies the above-described standard has excellent rapidity and selectivity, so that the efficiency of simultaneous analysis of noni fruit can be improved.

The above configuration will be described in more detail through the selection of the optimal column in Experiment 1 below.

In step (C), the mixed solvent of the mobile phase was flowed into the column of high-performance liquid chromatography in which the sample solution was injected in step (B), and the 5 kinds of physiologically active substances contained in the noni fruit were measured by measuring the absorbance of the high-performance liquid chromatography. This is the process of analyzing the content.

The mixed solvent used in the present invention is a mobile phase used in liquid chromatography, and various types of organic solvents such as ethanol, methanol or acetonitrile, which are commonly used, can be used. However, when only the organic solvents are used, some components having similar polarities may not be separated.

In the present invention, in order to solve this problem, a mixed solvent in which an aqueous solution is mixed with an organic solvent in a certain ratio as a mobile phase may be used, and acetonitrile having a relatively low polarity may be used as the organic solvent.

However, in the case of using an acetonitrile mixed solvent, the peak may be non-polar or peak separation may be observed when water is used as an aqueous solvent, and this problem can be solved by using formic acid.

The above configuration will be described in more detail through the selection of the optimal organic solvent in Experiment 1 below.

In the present invention, a mixed solvent is used including a 0.1% aqueous formic acid solution and acetonitrile, and in this case, the mixing ratio is preferably mixed with a 0.1% aqueous formic acid solution and acetonitrile in a ratio of 0.01 to 98.00 by volume: 2.00 to 99.99. Do.

The mixed solvent used as the mobile phase can be separated by varying the mixed volume of 0.1% formic acid aqueous solution and acetonitrile depending on time in order to selectively separate the physiologically active substances of Noni at the same time without overlapping.

More specifically, when the transfer time is less than 35 minutes, 98% by volume of 0.1% formic acid aqueous solution and 2% by volume of acetonitrile are mixed, and when the transfer time is more than 35 minutes and less than 37 minutes, 0.01% by volume of 0.1% aqueous formic acid solution % And 99.99% by volume of acetonitrile are mixed, and when the transfer time is more than 37 minutes and less than 52 minutes, use by mixing so that a 0.1% aqueous formic acid solution is 98% by volume and acetonitrile is 2% by volume.

If the above mixing conditions are exceeded, 5 kinds of physiologically active substances in the fruit of Noni (Deacetylasperulosidic acid, Asperulosidic acid), Rutin, Scopoletin, Rubiadin) may not be detected at the same time.

The absorbance in the present invention is preferably analyzed at 254 nm, which is 5 types of physiologically active substances in the fruit of Noni (Deacetylasperulosidic acid, Asperulosidic acid), and Rutin. , Scopoletin, rubiadin) has a maximum absorbance at 225 to 254 nm, and the absorbance is 225 nm and 254 nm with reference to FIGS. 7 to 8 showing the selection of the optimal wavelength and the result of the following experiment 1 This is because the analysis can be made more clearly as the baseline is stabilized at 254 nm.

In addition, when simultaneously analyzing 5 types of physiologically active substances contained in Noni fruit in step (C), the mobile phase flow rate of high performance liquid chromatography is preferably set to 1 mL/min in order to maximize analysis efficiency and minimize analysis time.

According to the present invention, not only can the 5 types of physiologically active substances contained in the noni fruit be detected simultaneously and accurately, but also the content of each can be analyzed, so that the noni fruit or the food containing the noni fruit There is an effect that can be usefully used to analyze bioactive substances.

In addition, quality standardization for the development of health functional foods through the fruit of Noni registered as a raw material that can be used as a food, and increase economic and temporal efficiency in research and development of products containing noni fruit ingredients or quality control of manufacturing. It can have an effect.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrative purposes only, and are not intended to limit the scope of the present invention. That is, simple modifications or changes of the present invention can be easily made by a person skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Example 1: Preparation of sample solution (dried noni fruit)

Example 1-1: After crushing dried noni fruit having a moisture content of less than 5% to a size of 300 to 500 mesh, 50 ml of 50% ethanol was mixed with 10 g of pulverized dried noni fruit and extracted for 30 minutes using an ultrasonic extraction method To prepare Sample Solution 1-1.

Example 1-2: After pulverizing the dried noni fruit having a moisture content of less than 5% to a size of 300 to 500 mesh, 50 ml of methanol was mixed with 10 g of the pulverized dried noni fruit and extracted for 30 minutes using an ultrasonic extraction method. Prepare solution 1-2.

Example 1-3: After pulverizing the dried noni fruit having a moisture content of less than 5% to a size of 300 to 500 mesh, 25 ml of 50% ethanol and 25 ml of methanol were mixed with 10 g of the pulverized dried noni fruit, and using an ultrasonic extraction method. Extract for 30 minutes to prepare Sample Solutions 1-3.

Example 1-4: After pulverizing dried noni fruit having a moisture content of less than 5% to a size of 300 to 500 mesh, 50 ml of ethyl acetate was mixed with 10 g of pulverized dried noni fruit, and extracted for 30 minutes using ultrasonic extraction. Prepare sample solution 1-4.

Example 2: Preparation of sample solution (Noni juice (100%))

Sample Solution 2 was prepared by mixing 25 ml of water and 25 ml of methanol in 10 g of noni juice with 100% noni fruit content.

Example 3: Preparation of sample solution (fermented noni beverage (100%))

Sample Solution 3 was prepared by mixing 25 ml of water and 25 ml of methanol in 10 g of fermented noni juice with 100% noni fruit content.

Example 4: Preparation of sample solution (composite noni stick)

Sample Solution 4 is prepared by mixing 25 ml of water and 25 ml of methanol in 10 g of complex noni stick.

Experiment 1: Optimal conditions for simultaneous analysis

1-1. Optimal column selection

To select the optimal column for high performance liquid chromatography, three products (YMC-Pack ODS-AM (YMC, 4.6 × 250 mm, 5㎛), μBondapak C18 Prep (Waters, 7.8 ×300 mm, 10㎛), XBridge As a result of analysis using a column of C18 (Waters, 3 × 250 mm, 3 μm)), sample analysis due to longer retention time when using a YMC-Pack ODS-AM (YMC, 4.6 × 250 mm, 5 μm) column It was more advantageous for the separation of each material. The results are shown in FIGS. 2 to 4.

In the present invention, in consideration of the separation and rapidity of the analysis result, the inner diameter of the high performance liquid chromatography column is 4 to 5 mm, the length is 200 to 300 mm, and the diameter of the particles charged in the column is set to 4 to 7 μm.

Condition by column
Column YMC-Pack ODS-AM
(YMC, 4.6×250 mm, 5㎛) μBondapak C18 Prep
(Waters,7.8×300mm,10㎛) XBridge C18
(Waters, 3×250mm, 3㎛) Detector 254 nm 254 nm 254 nm Temp. 25 ℃ 25 ℃ 25 ℃ Injection vol. 10 uL 10 uL 10 uL Flow rate 1 mL/min 1 mL/min 1 mL/min
Mobile phase A: 0.1% Formic acid
in Water
B: Acetonitrile A: 0.1% Formic acid
in Water
B: Acetonitrile A: 0.1% Formic acid
in Water
B: Acetonitrile

1-2. Selection of optimal organic solvent

In order to select the optimal organic solvent as the mobile phase solvent for high performance liquid chromatography, the results of analyzing using methanol and acetonitrile respectively showed that acetonitrile has a higher nonpolarity than methanol, so it has good elution power. I made my retention time shorter, and through this, I could see that the analysis time was shortened. The results are shown in FIGS. 5 to 6.

Therefore, in the present invention, the mobile phase organic solvent was used as acetonitrile.

Conditions according to organic solvent
Column YMC-Pack ODS-AM
(YMC, 4.6 ×250 mm, 5㎛) YMC-Pack ODS-AM
(YMC, 4.6 ×250 mm, 5㎛) Detector 254 nm 254 nm Temp. 25 ℃ 25 ℃ Injection vol. 10 uL 10 uL Flow rate 1 mL/min 1 mL/min
Mobile phase A: 0.1% Formic acid
in Water
B:Methanol A: 0.1% Formic acid
in Water
B: Acetonitrile

1-3. Optimal wavelength selection

In the case of wavelength, 5 kinds of physiologically active compounds contained in Noni fruit had maximum absorbance at 225~254nm. Therefore, in order to select the optimal wavelength during the analysis of high performance liquid chromatography, the results of analyzing the wavelengths at 254 nm and 225 nm respectively, it was found that the baseline was stable at 254 nm. The results are shown in FIGS. 7 to 8.

Therefore, in the present invention, 254 nm was used as the optimal wavelength.

Conditions according to wavelength
Column YMC-Pack ODS-AM (YMC, 4.6 ×250 mm, 5㎛) YMC-Pack ODS-AM (YMC, 4.6 ×250 mm, 5㎛) Detector 254 nm 225 nm Temp. 25 ℃ 25 ℃ Injection vol. 10 uL 10 uL Flow rate 1 mL/min 1 mL/min
Mobile phase A: 0.1% Formic acid
in Water
B: Acetonitrile A: 0.1% Formic acid
in Water
B: Acetonitrile

1-4. Final analysis result

Table 4 shows the results of analyzing the contents of five physiologically active substances contained in the noni fruits of Examples 1 to 4 based on the optimal analysis conditions selected through 1-1 to 1-3 of Experiment 1.

sample

Bioactive substance name
Analysis result (mg/g)
Example 1-1
50% ethanol Example 1-2
Methanol Example 1-3
Methanol, ethanol Example 1-4
Ethyl acetate Example 1 Deacetylasperulosidic acid 18.58 6.69 2.16 <0.01 Asperulosidic acid 1.50 1.31 0.79 - Rutin 0.01 <0.01 <0.01 - Scopoletin <0.01 <0.01 <0.01 <0.01 Rubiadin <0.01 <0.01 <0.01 <0.01 Example 2 Deacetylasperulosidic acid 2.67 Asperulosidic acid 0.67 Rutin 0.01 Scopoletin <0.01 Rubiadin <0.01 Example 3 Deacetylasperulosidic acid 1.97 Asperulosidic acid 0.32 Rutin 0.01 Scopoletin 0.01 Rubiadin <0.01 Example 4 Deacetylasperulosidic acid 4.29 Asperulosidic acid 0.96 Rutin 0.0 Scopoletin 0.01 Rubiadin <0.01

Claims (5)

(A) preparing a sample solution for analysis by mixing the extraction solvent in a ratio of 1:5 to 10 by weight to noni fruit or food containing noni fruit;
(B) injecting the sample solution prepared in step (A) into a column of high performance liquid chromatography having a non-polar column as a stationary phase; And
(C) Flowing the mobile phase mixed solvent into the column of high-performance liquid chromatography injected with the sample solution in step (B), and measuring the absorbance of the high-performance liquid chromatography to determine the content of five physiologically active substances contained in the fruit Analyzing;
Including,
The five physiologically active substances contained in the Noni fruit,
Deacetylasperulosidic acid, Asperulosidic acid, Rutin, Scopoletin, Rubiadin,
The extraction solvent of step (A),
It is a mixture of one or more of water, methanol, ethanol, ethyl acetate,
The column of the high performance liquid chromatography has an inner diameter of 4.6 mm and a length of 250 mm, and the diameter of the particles charged in the column is 5 μm,
In the absorbance measurement,
It uses a wavelength of 254 nm,
The mixed solvent of step (C) is used by mixing 0.1% aqueous formic acid solution and acetonitrile in a ratio of 0.01 to 98.00 by volume: 2.00 to 99.99,
Depending on the transfer time, when the transfer time is less than 35 minutes, 98% by volume of 0.1% formic acid aqueous solution and 2% by volume of acetonitrile are mixed,
When the transfer time is 35 minutes or more and less than 37 minutes, 0.01% by volume of 0.1% formic acid aqueous solution and 99.99% by volume of acetonitrile are mixed,
When the transfer time is more than 37 minutes and less than 52 minutes, the mixture is mixed so that 98% by volume of 0.1% formic acid aqueous solution and 2% by volume of acetonitrile,
Simultaneous analysis of five physiologically active substances contained in Noni fruit in step (C), wherein the mobile phase flow rate of high-performance liquid chromatography is 1 mL/min. Analysis method.







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