WO2021167166A1 - Method for mass purification of highly pure cordycepin from cordyceps by using recycling liquid chromatography - Google Patents

Abstract

The present invention relates to a method for mass purification of highly pure cordycepin from Cordyceps militaris by using recycling liquid chromatography and provides a mass purification method for highly pure cordycepin, wherein naturally dried Cordyceps militaris is treated by hot water extraction, lyophilized, and developed using a 5-10% methanol solvent at a flow rate of 120 ml/min.

Description

Bulk purification method of high-purity cordycepin from Cordyceps Cordyceps using Recycled Liquid Chromatography

The present invention relates to a method for bulk purification of high-purity cordycepin isolated from Cordyceps Militaris containing cordycepin using recycling liquid chromatography (HPLC). More specifically, it relates to a method for rapidly purifying in large quantities high-purity cordycepin developed at a flow rate of 120 ml/min using a 5% methanol solvent after hot water extraction treatment of naturally dried Militaris Cordyceps Cordyceps and freeze-drying. .

This application claims priority based on Korean Patent Application No. 10-2020-0021490 filed on February 21, 2020, and all contents described in the specification and drawings of the application are incorporated herein by reference.

Cordyceps cordyceps is a kind of medicinal mushroom named because it stays in the body of insects in winter and appears like grass in summer, and there are currently hundreds of species around the world. There are three types of cordyceps, recognized as food and snow flower cordyceps (Paecilomyces japonica). In addition, cordyceps used for medicinal purposes includes Cordyceps ophioglossoides, Cordyceps sobolifera, Cordyceps beauveria or Cordyceps bassiana. have.

It belongs to the group with the highest protein content among the parasite crops, and contains a large amount of substances that enhance immunity. Among the immunoactive substances of Cordyceps Cordyceps, there is a substance called Cordycepin, and in Cordyceps, Militaris Cordyceps, the content of Cordycepin is much higher than that of other Cordyceps. Cordycepin is a nucleic acid substance that prevents normal cells from being converted into cancer cells by activating the reduced immune function while participating in the genetic information of cells. Cordycepin has been designated as a functional food that can help improve immune function by the Ministry of Food and Drug Safety (KFDA).

As such, Cordyceps Cordyceps is an important crop that is being continuously studied in the domestic medicine and food fields because of its high value as a crop. have.

In the prior invention related to the present invention, as a method for separating and purifying the cordycepin component, grapes and cordyceps cordyceps were mixed in a weight ratio of 1:1 to 20:1 and fermented and aged at a temperature of 5 to 30° C. for 20 to 120 days. Cordyceps militaris (Cordyceps militaris) containing a high content of Cordyceps militaris is disclosed in Korean Patent Registration No. 10-2058288, and Korean Patent Registration No. 10-1999273, obtained by separating the liquid phase. ) A method of producing Cordyceps by using a strain, and extracting and purifying high-purity cordycepin therefrom is disclosed.

The prior inventions disclose a general method for extracting cordycepin, and the process for purification with high purity consists of several steps. A simple and rapid method for extracting and purifying cordycepin has been developed.

An object of the present invention is to provide a method for simply and rapidly extracting and purifying high-purity cordycepin from a hot water extract of Cordyceps Cordyceps Militaris containing cordycepin using recycled liquid chromatography.

The present invention is Militaris Cordyceps pretreatment step; an extraction step of obtaining an extract of the Cordyceps; It provides a process for producing high-purity cordycepin, including a purification step of purifying the extract to obtain high-purity cordycepin.

As an embodiment of the present invention, a) manufacturing Militaris Cordyceps Cordyceps in a powder state using a grinder after natural drying; B) preparing a powder by mixing the Militaris Cordyceps Cordyceps powder and distilled water in a weight ratio of 1:100, extracting with hot water, and concentrating under reduced pressure; C) provides a method for purifying cordycepin from cordyceps by mixing the concentrated Cordyceps hot-water extract powder with a solvent and separating and extracting cordycepin in a recycling liquid chromatography.

In addition, in step c), the hot water extract powder of Cordyceps cordyceps extract is prepared at a concentration of 20 mg/ml, the mobile phase of the recycling liquid chromatography uses 5-10% methanol, and the flow rate of the mobile phase in the recycling liquid chromatography is 100-130 ml/ It provides a method for purifying cordycepin from Cordyceps that develops in min.

The present invention provides a cordycepin purified from Cordyceps Cordyceps with a purity of 98% or more extracted by the above method.

According to the mass purification process of high-purity cordyceps isolated from Militaris Cordyceps of the present invention, when 20 mg/ml lyophilized powder was developed at a flow rate of 120 ml/min using recycled liquid chromatography, high-purity cordycepin in a short time of 32 min. The amount of solvent used can be reduced by separating the

The present invention claims priority based on Korean Patent Application No. 10-2020-0021490 filed on February 21, 2020, and all the drawing contents of the application are incorporated herein by reference.

1 shows a flow chart of the JAI Recycling Preparative HPLC (LC-250HS) system.

2 shows a chromatogram according to 10% methanol concentration and 100 ml/min flow rate as conditions for obtaining cordycepin from Militaris Cordyceps hot water extract powder.

3 shows a chromatogram according to a 10% methanol concentration and a flow rate of 120 ml/min.

4 shows a chromatogram according to a 5% methanol concentration and a flow rate of 100 ml/min.

5 shows a chromatogram according to a 5% methanol concentration and a flow rate of 120 ml/min.

6 shows a chromatogram obtained by fractionation of cordycepin under the conditions of a 5% methanol concentration and a flow rate of 120 ml/min.

7 shows an HPLC chromatogram of cordycepin obtained from the present invention.

8 shows an HPLC chromatogram of cordycepin obtained from the present invention.

Figure 9 shows a photograph of Cordyceps Cordyceps and cordycepin obtained by carrying out the present invention.

The measurement of cordycepin of the present invention means that it is measured by high performance liquid chromatography, and specifically, it may mean that it is measured using 5-10% methanol aqueous solution as a mobile phase. Cordyceps bacterium is not limited as long as it is a known Cordyceps bacterium belonging to Ascomycota, Pyrenomycetes, Clavicipitaceae, Codyceps, and the like, for example, Cordyceps militaris and the like. can Hereinafter, it is referred to as Militaris Cordyceps.

Cordyceps cordyceps used in the present invention can be purchased and used in general, and can also be used grown using insect pupae as a host.

The pupa refers to a normal pupa as a pupa of an insect, and any pupa capable of providing an environment in which Cordyceps fungi can grow as a host may be used. The insect may be various such as various butterflies and moths, and specific examples include silkworm moth.

The cultivation method of pupa Cordyceps Cordyceps according to the present invention may be performed by a manual cultivation method or may be performed through an automated machine. When an automated machine is used, it is possible to cultivate Cordyceps pupa with excellent reproducibility of the same quality, for example, having a content of cordycepin in the same range, and it can be advantageous for mass production. The cultivation method of pupa Cordyceps Cordyceps is as follows.

1. Cultivation method of pupa Cordyceps

1.1 Steps to prepare the pupa medium mixture

After putting 200 g of pupa and 200 g of water into a culture bottle at 25° C. and 65% relative humidity, and then sterilizing at 121° C. for 30 minutes, and cooling naturally to 25° C., a pupa medium was prepared. After mixing 5 g of yeast, 5 g of peptone, 15 g of sugar, and 1 kg of water at 25 ° C, sterilization at 121 ° C for 20 minutes and cooling naturally to 25 ° C. Cordyceps militaris ) After inoculation, it was cultured at 25° C. for 7 days to prepare a liquid seed.

Subsequently, the liquid seed was mixed with a mixture of 80 g of yeast, 80 g of peptone, and 120 g of sugar at 25° C., and cultured for 7 days in an incubator at 23° C. and 65% relative humidity to prepare a seed mixture (inoculum). . The pupa medium mixture was prepared by introducing and inoculating the seed mixture medium into the culture bottle containing the pupa medium at 23°C.

1.2 Incubation step

At a relative humidity of 65%, the pupa medium mixture at 23° C. was rapidly lowered to a first temperature of 0° C. at a temperature reduction rate of 5° C./min, followed by a temperature reduction step (t1) of maintaining the first temperature of 0° C. for 2 days. . Subsequently, at a relative humidity of 65%, the pupa medium mixture at a first temperature of 0°C was gradually raised to a second temperature of 23°C at a temperature increase rate of 01°C/min, and then a temperature raising step of maintaining the second temperature of 23°C for 5 days (t2) ) was performed. Then, at a relative humidity of 65%, a maintenance step (t3) of maintaining the pupa medium mixture at a second temperature of 23° C. as it is at a second temperature of 23° C. for 35 days was performed and cultured for a total of 42 days in the culturing step.

1.3 Harvesting Steps

Pupa Cordyceps Cordyceps was harvested from the cultured pupa medium mixture.

1.4 Pretreatment Steps

The pupae were put in an amount of water that was sufficiently immersed in the pupae, and the pupae were soaked in water for 4 hours. The pupae in a swollen state with water were recovered, put in a freeze dryer, and dried at 5 torr for 24 hours while freezing to -60 °C at a temperature reduction rate of -1 °C/min to obtain a pupae that had undergone a pretreatment step.

2. Militaris Cordycepin tablet using Cordyceps Cordyceps

In the present invention, cordycepin purification using Militaris Cordyceps Cordyceps is composed of a pretreatment process, extraction and concentration, and a purification process.

2.1 Pretreatment Process of Militaris Cordyceps Cordyceps

Militaris Cordyceps was dried naturally and then made into a powder using a grinder.

2.2 Extraction and Concentration of Militaris Cordyceps Cordyceps

5L of distilled water was added to 50 g of Militaris Cordyceps Cordyceps powder, followed by hot water extraction at 100° C. for 3 hours. After repeating the above process 3 times, the extract was filtered and concentrated under reduced pressure to obtain 7.3067 g.

2.3 Purification of Cordycepin by HPLC

Cordycepin, an indicator component of Cordyceps Cordyceps, is a single compound commercially available as a derivative of adenosine as a kind of nucleoside, and can be measured using high performance liquid chromatography.

The produced cordyceps hot water extract powder was prepared at a concentration of 20 mg/ml and purified using high performance liquid chromatography equipment. As HPLC equipment, JAI Recycling Preparative HPLC (LC-250HS) and Column (JAIGEL-ODS-AP-50L, SP-120-20) were used. The differences were compared.

1 shows a flow chart of the JAI Recycling Preparative HPLC (LC-250HS) system. The HPLC device generally includes a degassing device for removing dissolved oxygen, nitrogen, and bubbles in the mobile phase, a pump that takes the mobile phase out of the mobile phase storage container and continuously pushes it to the sample injector, and a sample to be analyzed. A sample injector for loading the solvent into a flow of solvent, a column using a tubular container filled with filler, and a column temperature controller (TCC) to properly set and maintain the column temperature to improve resolution and ensure reproducibility of analysis results : Thermostatted Column Compartment), and when the sample separated from the column passes through the detector at regular intervals, the detector recognizes the presence and amount of the sample according to a certain rule and converts it into an electrical signal.

2.4 Purification of Cordycepin according to Mobile Phase Concentration

In the process of finding the optimal conditions for obtaining cordycepin, the same concentration and conditions were used. 2 is a chromatogram according to a flow rate of 100 ml/min using isocratic as a mobile phase mixed with 10% methanol and 001% formic acid in order to establish optimal conditions for obtaining cordycepin from Militaris Cordyceps hot water extract powder. , and FIG. 3 shows a chromatogram according to a flow rate of 120 ml/min using isocratic mixing with 10% methanol concentration and 001% formic acid.

When 5 ml of a sample with a concentration of 20 mg/ml of hot water extract powder was injected and the mobile phase 10% Methanol (01% Formic acid) was processed as Isocratic, separation was seen at a flow rate of 100 ml/min, but another compound in the front part of the peak identified as cordycepin Because the peaks of the overlapped (indicated by the arrow), the purity of the cordycepin dropped to 80% (see FIG. 2), and all came out at the beginning at a flow rate of 120ml/min (see FIG. 3)

4 shows a chromatogram according to a flow rate of 100 ml/min by mixing 5% methanol concentration and 001% formic acid, and FIG. 5 is a chromatogram according to a flow rate of 120 ml/min by mixing 5% methanol concentration and 001% formic acid. is shown.

When a sample with a concentration of 20 mg/ml of hot water extract powder was processed with the mobile phase 5% Methanol (01% Formic acid) isocratic, when the flow rate was 100 ml/min, the purity of cordycepin was high at 90%, but The peaks of other compounds overlapped in front of the identified peaks (refer to FIG. 4 indicated by an arrow), and at a flow rate of 120ml/min, high separation and 98% purity of cordycepin were confirmed (see FIG. 5).

Cordycepin purity according to difference in mobile phase concentration and flow rate

optimum condition	mobile phase concentration	flow rate	Cordycepin Purity
condition 1	10% methanol	100ml/min	80%
condition 2	10% methanol	120ml/min	-
condition 3	5% methanol	100ml/min	90%
condition 4	5% methanol	120ml/min	98%

From the above results, the conditions were established with a 5% methanol mobile phase at a sample concentration of 20 mg/ml, and a flow rate of 120 ml/min, and 10 ml was injected to obtain high-purity cordycepin from 32 min to 98%. 6 shows a chromatogram obtained by fractionation of cordycepin under the established conditions. 7 and 8 show HPLC chromatograms of cordycepin obtained from the present invention. Looking at the HPLC chromatogram of regions 1 and 2 of the cordycepin separated in FIG. 6 , it can be seen that high purity cordycepin of 98% or more is confirmed, respectively.

Figure 9 shows a photograph of Cordyceps Cordyceps and cordycepin obtained by carrying out the present invention. After drying the raw material of Militaris Cordyceps naturally, it was made into a powder state using a grinder, distilled water was added to the Militaris Cordyceps cordyceps powder, extracted with hot water at 100 ° C. for 3 hours, the extract was filtered, and the resulting powder was concentrated under reduced pressure using HPLC. You can check the result of purification with cordycepin.

The present invention is thought to have high industrial applicability because it is possible to easily obtain cordycepin with excellent pharmacological activity by developing a technology that can be mass-produced inexpensively with a simple process of purifying high-purity cordycepin isolated from Militaris Cordyceps. .

Claims (3)

1. A) manufacturing Militaris Cordyceps Cordyceps in a powder state using a grinder after natural drying; B) preparing a powder by mixing the Militaris Cordyceps Cordyceps powder and distilled water in a weight ratio of 1:100, extracting with hot water, and concentrating under reduced pressure; C) Purification method of cordycepin from Cordyceps Cordyceps, characterized in that the reduced pressure-concentrated Cordyceps hot-water extract powder is mixed with a solvent, and cordycepin is separated and extracted in a recycling liquid chromatography
2. The method of claim 1, wherein step c) prepares the concentrated Cordyceps hot water extract powder under reduced pressure at a concentration of 20 mg/ml, the mobile phase of the recycling liquid chromatography uses 5-10% methanol, and the flow rate of the mobile phase is 100-120 ml Purification method of cordycepin from Cordyceps Cordyceps, characterized in that it is developed at /min to isolate and extract cordycepin
3. Cordycepin purified from Cordyceps Cordyceps with a purity of 98% or more, extracted by the purification method of claim 1

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