KR100741350B1 - Extraction and purification method for effective component of plant - Google Patents

Abstract

The present invention dissolved the plant powder in ethanol as an extraction solvent, reflux cooling for 2-4 hours at boiling point, centrifuged for 7-15 minutes at 2,000-3,000 rpm and then extract the crude supernatant by filtering only the supernatant preparing a crude extract; Drying the crude extract to completely remove the extraction solvent and dissolving it in water; And eluting with a concentration gradient of 0% -100% ethanol in an ion exchange resin with respect to the crude extract dissolved in water to provide a step-by-step fraction extraction and purification.

According to the present invention, by simplifying the extraction and purification method of the existing plant extract in a single time, not only the use of the extraction solvent and column resin can be reduced, but also the organic solvent extraction process can be shortened, thereby improving the efficiency of time and cost. It can be increased.

Plant Extract, Purification Method, Chajeoncho, Plantazoside, Ethanol

Description

Extractive purification method of plant active ingredient {EXTRACTION AND PURIFICATION METHOD FOR EFFECTIVE COMPONENT OF PLANT}

1 is a view schematically showing a process for extracting and purifying the plantamazoside according to the prior art.

2 is a view schematically showing a process for extracting and purifying the plantamazoside according to the present invention.

Figure 3 is a graph showing the results of performing HPLC analysis on the chachochoe active ingredient extracted by the prior art.

Figure 4 is a graph showing the results of HPLC analysis for the chachochoe active ingredient extracted by the present invention.

FIG. 5 is a table showing ¹ H and ¹³ C NMR spectral data in CD ₃ OD for Plantamazoside.

Figure 6 is a view showing the structure of the plantazoside as an active ingredient of the chajeoncho.

7 is an illustration showing the structure of the purified extract chajeoncho active ingredient in the method of the invention, identified by the ¹ H- ¹ H COSY and HMBC experiments.

8 is a graph showing the results of experiments on the effect of inhibiting the gliding effect on the chajeoncho active ingredient extracted by the prior art.

Figure 9 is a graph showing the results of experiments on the effect of inhibiting the glycosis extract extracted by the present invention active ingredient.

10 is a graph showing the results of HPLC analysis after diluting the planamazoside by 25, 50, 75, 100, 200 uM / 200 μl concentration.

FIG. 11 is a graph showing a calibration curve prepared based on HPLC analysis after diluting the planamazoside at 25, 50, 75, 100, and 200 μM / 200 μl concentrations.

The present invention relates to a method for extracting and purifying a plant active ingredient, and more particularly, to a method for extracting and purifying a plant active ingredient efficiently in a short time.

Recently, as interest in health and beauty has increased, interest in active ingredients of plants has also increased. In particular, in Korea as well as in many countries, efforts are underway to search for substances with useful activities such as anticancer, anti-oxidant, or anti-glycation in plants.

To this end, it is a priority to identify the active ingredients inherent in plants, the operation and effects of such active ingredients. The next challenge is how to extract and purify the desired active ingredient from plants in large quantities. To this end, a number of extraction and purification methods have been proposed to date, and for example, Korean Patent Nos. 10-0302487, 10-0341797, and 10-0382170 disclose such methods.

As such, currently used extraction and purification methods are multi-step extraction using a variety of organic solvents, and then goes through several steps of column purification.

However, such an extraction and purification method is not only complicated, but also takes a long time (see FIG. 1). In addition, since a large amount and type of organic solvent toxic to the human body is used, there are also problems related to the safety of the extraction process and the safety of the active ingredient itself. Furthermore, since many kinds and amounts of column resins and solvents are required for multi-stage column purification, there is a problem in that a high cost is required.

Thus, there is an urgent need for a method for more effective extraction and purification of active ingredients in plants.

In order to solve the above problems, an object of the present invention is to provide a method for extracting and purifying the plant active ingredient that can reduce the amount of use and extraction purification time of the organic solvent, column by separating and purifying the active ingredient of the plant in a single time. have.

In order to achieve the above object, the present invention is dissolved in the plant powder in ethanol as an extraction solvent, reflux cooling for 2-4 hours at boiling point, centrifuged for 7-15 minutes at 2,000-3,000rpm Separating and filtering only the supernatant to prepare a crude extract; Drying the crude extract to completely remove the extraction solvent and dissolving it in water; And eluting with a concentration gradient of 0% -100% ethanol in an ion exchange resin with respect to the crude extract dissolved in water to provide a step-by-step extract purification method of plant active ingredient.

Hereinafter, the configuration of the present invention in more detail.

The present invention dissolved the plant powder in ethanol as an extraction solvent, reflux cooling for 2-4 hours at boiling point, centrifuged for 7-15 minutes at 2,000-3,000 rpm and then extract the crude supernatant by filtering only the supernatant preparing a crude extract; Drying the crude extract to completely remove the extraction solvent and dissolving it in water; And eluting with a concentration gradient of 0% -100% ethanol in an ion exchange resin with respect to the crude extract dissolved in water to provide a step-by-step extract purification method of plant active ingredient.

In the above extraction and purification method, it will be said that the plant to be extracted is not particularly limited, and among these, a tea plant which contains abundantly an antioxidant component and an antiglycine component is preferable.

In the case of such a plant, it is preferable to use the powder by lyophilization to obtain a desired active ingredient in a higher yield.

In addition, the active ingredient of the plant is not particularly limited, but plantamajoside, which is known to contain abundantly in chajeoncho and is known to have antioxidant and anti-glycolytic effects, is preferable.

In the extraction and purification method according to the present invention, ethanol is used as the solvent for the extraction solvent and the purification. In this case, since ethanol corresponds to a solvent allowed by the KFDA, not only is it safe in the extraction and purification process, but also in the case of the plant active ingredient extracted and purified using this, it may be safely used for the use of food or medicine.

In the present invention, the step of preparing the crude extract may be performed only once, but if it is repeated twice, it is preferable because the yield can be further increased.

In the method according to the present invention, drying of the crude extract that proceeds after the preparation of the crude extract may be performed only by vacuum drying, but when the vacuum freeze drying is performed again after vacuum drying, the extraction solvent may be completely removed. More preferred.

In the extraction and purification method according to the present invention, the crude extract dissolved in water is eluted with a concentration gradient of 0% -100% ethanol in an ion exchange resin to obtain a stepwise fraction, whereby the purification is performed once. Is not particularly limited, and among them, it is preferable to use Diaion HP-20 resin.

Moreover, among the said ethanol concentration gradients, especially the fraction obtained by 25% or 50% of ethanol is the most efficient, More preferably, it is the fraction obtained by 50%.

As described above, the method according to the present invention can extract and purify the plant active ingredient only once. That is, according to the present invention, since the time required for one cycle of the extraction tablet is about 2 days, it is possible to shorten the extraction purification time by 1/7 or more than the prior art which takes 14 days or more.

In addition, in terms of the solvent to be used, the amount of the solvent required in one cycle in the case of the conventional technique is 50-100 liters, but according to the present invention, extraction and purification is possible in only about 10 liters.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments of the present invention. However, this embodiment is not intended to limit the scope of the present invention, but is presented by way of example only.

< Example >

Example  One : Chachocho  Extraction Purification of Active Ingredients

1) 100 g of the powder obtained by lyophilization of a plantago (Plantago asiatica plant) was dissolved in 2 L of ethanol, and extracted by reflux cooling at a boiling point for 3 hours.

2) The extract was centrifuged at 2,500 rpm at 4 ° C. for 10 minutes, and then only the supernatant was filtered again with filter paper.

3) only extracts were collected.

4) After repeating the steps 1) and 2) again for the remaining pellets (filter) after filtering, the obtained extract was combined with the extract of 3).

5) The extract obtained above was dried under reduced pressure under vacuum to completely remove the extraction solvent.

6) The extract was loaded on a column filled with Diaion HP-20 resin, and each fraction was collected by stepwise concentration gradient so that ethanol was 0, 25, 50, 75, 100%. At this time, 25% and 50% ethanol extracts were particularly active.

Comparative example  One : Chachocho  Extraction Purification of Active Ingredients

1) 100 g of a powder obtained by lyophilization of a plantago plant (Plantago asiatica plant) was dissolved in 2 L of methanol and reflux-cooled.

2) The extract was centrifuged at 2,500 rpm at 4 ° C. for 10 minutes, and only the supernatant was again filtered through a filter paper.

3) only extracts were collected.

4) After filtering the remaining pellets, the steps 1) and 2) were repeated, and the obtained extract was combined with the extract of 3).

5) The obtained extract was dried under reduced pressure under vacuum to remove the extraction solvent, and the concentrate was dissolved in 100 ml of water.

6) The concentrate dissolved in water was placed in a 2L separatory funnel and 1.5L of hexane was shaken for 30 minutes.

7) If the layer separation occurred reliably, the water layer was separated, and the process was repeated again 6) with the water layer until the color of the hexane layer became colorless. At this time, the organic solvent extracts for each step were collected.

8) The resulting water layer was extracted with chloroform, ethyl acetate and butanol in a multistage organic solvent as in 6) and 7).

9) Among them, the highly active ethyl acetate extract was put on a column filled with silica gel, and the fractions were collected by stepwise gradient of methanol from 0 to 100%.

10) Again, the most active 10% methanol fraction was loaded on a column filled with sephadex LH-20 resin and gradientd from 0 to 100% methanol to collect each fraction to obtain a highly active fraction.

Experimental Example  One : HPLC  And NMR analysis

HPLC was performed on the active ingredients extracted and purified in Example 1 and Comparative Example 1. As a result, as shown in Figures 3 and 4, the active ingredients extracted in Example 1 and Comparative Example 1 showed the same retention time, it was confirmed that they are the same material.

In addition, NMR structural analysis of these materials revealed that these materials were identified as planamazosides, as can be seen in Figures 5-7 below.

Experimental Example  2 : Gliding ( GLYCATION Inhibitory Effect Confirmation Experiment

The glyphage inhibitory effect experiment was performed on the active ingredients extracted in Example 1 and Comparative Example 1. At this time, as a control, it was reported that 100 gl did not occur at all, and the inhibitory activity according to the concentration of the extract was observed.

In addition, as a comparative control, 25 mM aminoguanidine known as a glycation inhibitory substance was used.

The results are shown in FIGS. 8 and 9, respectively.

From the results of the experiment, it was confirmed that the active ingredients extracted in Example 1 and Comparative Example 1 were almost similar to the inhibitory effect of glycation, and both were excellent in the inhibitory effect of glycation.

Experimental Example  3: Of Planta Mazoside  Calibration analysis

The analytical analysis of the plantarmazoside extracted and purified in Example 1 was carried out. That is, after diluting the planamazoside by the concentration of 25, 50, 75, 100, 200 uM / 200 μl, the results of HPLC analysis and a calibration curve based on the same are shown in FIGS. 10 and 11, respectively. Indicated.

From the above experimental results, the plantarmazoside content in the extract obtained in Example 1 could be estimated to be 510g / 1kg D.M.

As described above, according to the present invention, instead of using a toxic organic solvent, by using ethanol allowed by the KFDA as an extraction and purification solvent, it is possible to safely use the extracted and purified active ingredient as a food additive or a functional substance. do.

In addition, according to the present invention, by simplifying the extraction and purification method of the existing plant extract in a single step, not only can reduce the use of the extraction solvent and column resin, but also shorten the organic solvent extraction process to reduce the time and cost efficiency It can be increased.

Thus, according to the present invention, it is possible to industrially extract a large amount of plant active ingredient exhibiting efficacy as an antioxidant or an anti-glycological agent as described above.

Claims (5)

After discharging the chajeoncho powder in ethanol as an extraction solvent, reflux cooling for 2-4 hours at the boiling point, centrifuged for 7-15 minutes at 2,000-3,000 rpm and then filtered only the supernatant crude extract (crude extract) Preparing); Drying the crude extract to completely remove the extraction solvent and dissolving it in water; And Extraction and purification method of the plantamajoside (plantamajoside) comprising the step of obtaining a step fraction by eluting the crude extract dissolved in water in the concentration gradient of ethanol 0% -100% in ion exchange resin. The method of claim 1, Drying of the crude extract is vacuum extraction drying method of the plantar mazoside is achieved by vacuum drying or vacuum freeze drying after vacuum drying. delete delete The method of claim 1, The step fraction is extracted and purified method of plantarmazoside is obtained from ethanol 25% or 50%.

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