KR20060038027A - Effective extraction method for obtaining amygdalin from persicae semen and armenicae semen and the seperation method thereof using by hplc - Google Patents

Abstract

The present invention relates to an optimal extraction condition and separation method for inhibiting amygdalin degradation contained in hot water extracts of cabbage or algae. More specifically, the optimum amidaline extraction conditions to prevent inactivation and epimerization of emulsin, which degrades amidaline contained in the hydrothermal extracts of the causal or algae extract, and the mixture of amidaline and neoamigdaline with excellent resolution By providing a separation method using high-performance chromatography (HPLC) to separate by using, it is possible to develop a passerby or a seed of apricot ( Prunus armeniaca Linne Var. Ansu Maximowicz) as a safe and effective anticancer agent.

Pediatric, almond, hot water extraction, anticancer agent, amigdaline, emulsine, epimerization, reverse phase HPLC

Description

Effective extraction method for obtaining amygdalin from Persicae Semen and Armenicae Semen and the seperation method approximately using by HPLC}             

1 is a view illustrating the change in the extraction rate of amigdalin according to the extraction temperature, Figure 1a at 70 ℃, Figure 1b at 80 ℃, Figure 1c using the extraction temperature conditions of 90 ℃,

2 is a graph showing the amount of extraction of amigdaline in almond powder at intervals of 2 ° C. at an extraction temperature of 80 ° C. to 90 ° C.

3 is a chromatogram showing the degree of epimerization according to extraction time when the extraction temperature is 90 ℃, Figure 3a in 2 minutes, Figure 3b in 8 minutes, Figure 3c in 60 minutes Using the extraction time of

Figure 4 shows the separation of amigdaline and neoamigdaline by reverse phase HPLC separation, Figure 4a is looked at under buffer conditions containing 25% methanol, Figure 4b is looked at under buffer conditions containing 13% acetonitrile.

The present invention provides optimal extraction conditions for inhibiting amygdalin degradation in hot water or algae hydrothermal extract with anticancer effect, and reverse phase high performance chromatography (HPLC) separation method that separates the mixture of amigdaline and neoamigdaline with excellent resolution. It is about.

Cancer is the first priority to overcome in order to prolong the life span of humans in the 21st century. Currently, the global cancer incidence is increasing by more than 5% every year due to the increase of elderly population and deterioration of the environment. Ten thousand people are responsible for 12% of global mortality. The number of cancer patients in Korea is about 100,000 new cases every year, and about 50,000 people die every year. The increase rate of cancer patients is about 10% every year. In 1997, the number of cancer patients was 120,000, men were stomach cancer (21%) and liver cancer (12%). , Lung cancer (11%) in order, and cervical cancer (20%), stomach cancer (16%), breast cancer (13%) in order.

Most anticancer drugs intervene in various metabolic pathways of cancer cells and mainly inhibit the synthesis of nucleic acids or exhibit anticancer activity. However, these anticancer drugs not only act selectively on cancer cells but also damage normal cells, especially tissue cells with active cell division, resulting in various side effects such as bone marrow dysfunction, gastrointestinal disorders, and alopecia. Anticancer drugs currently used for cancer treatment are classified into six categories according to biochemical mechanisms of action.

The types of anticancer drugs are classified into chemotherapeutic and biotherapeutic agents. The chemotherapeutic agents are highly reactive, and alkylating agents that modify the structure of DNA and cause chain breakage when acting on cells, and metabolic processes necessary for the proliferation of cancer cells. It is a synthetic chemical substance such as metabolic antagonists (代 time 拮抗劑 olites) that have the effect of inhibiting it, antibiotics that show anticancer activity among antibiotics produced by bacteria, natural products, hormones, etc. It is an immunotherapeutic agent that removes cancer cells by increasing the immune response of cancer patients to cancer cells by using therapeutic vaccines, monoantibodies, and cytokines. It is a specific period of mitosis, which stops cell division in the metaphase. Vinca alkaloids, cancer patients caused by a lack of genes or mutations It is a gene therapy agent that treats cancer by administering a crab genetic material, modifies a wrong gene and generates therapeutic proteins in cells or tissues, and a nucleic acid fragment that can selectively bind to a carcinogen causing cancer. Antisense anticancer agents, angiogenesis inhibitors, and the like.

Anticancer drugs use the difference in sensitivity between drugs between normal cells and cancer cells, so they are relatively less toxic to normal cells and act more selectively against cancer cells, but they also cause side effects because normal cells are damaged to some extent. Since anticancer drugs have a characteristic of acting anywhere in fast-dividing cells, they act not only on rapidly dividing cancer cells, but also normal cells, bone marrow, gastrointestinal tract, and hair follicle cells, are also rapidly dividing. It is affected by anticancer drugs, and common side effects of these drugs include decreased blood cell counts, nausea, vomiting, diarrhea, loss of appetite, and hair loss.

Although the anticancer drugs used to treat cancer patients have developed considerably, there are still no perfect anticancer drugs without side effects, chemotherapy resistance, and recurrence.Therefore, there is no toxicity, so we want to develop natural herbal medicines to develop safe and effective anticancer drugs. will be.

The algae used in the present invention are seeds of Prunus armeniaca Linne Var. Ansu Maximowicz belonging to the genus Amygdalaceae or other related plants of the same genus and are widely used for asthma, dyspnea, edema, etc. It is used as a herbal medicine, and contains about 3% of amygdalin, 50% of fat oil (passenger oil), and various oily amino acids.Amigdalin, a vitamin B17, is used in the passage of amygdalase and pruna. Hydrolyzed by β-glycosidase, such as prunase, which in turn yields punasin and benzaldehyde, which is further degraded to produce benzaldehyde and hydrogen cyanide (HCN) ; Hyangjedae Dictionary, Younglim History, pp 625 ~ 626, 1998).

Also Christian (桃仁) is an herbal medicine that is widely used in peach trees belong to the rose family (Rosaceae) (Prunus persica Batsch) , one peach tree seed stand lubrication of (Prunus persica Batsch var. Davidiana Maximowicz ), herbal purposes in nine eohyeol, It contains about 3.6% of amidaline, 0.4% of essential oils, and 45% of fatty oils, and mainly contains olefin glycerin, a small amount of linol glycerin, palmitic acid, stearic acid, choline, and acetylcholine. And emulsin (Jung Bo-seop and Shin Min-gyo; Hyangjedae, Yeonglimsa, p632, 1998).

Recently, reports have shown that amigdaline, the principal ingredient of algae and causal, can specifically kill malignant cells in cancerous lesions without the physiological toxicity normally found in chemotherapeutic agents (Syrigos, KN, Rowlinson-Busza, G. and Epenetos, AA, International Journal of Cancer , 78 , pp712-719, 1998). In fact, studies using amidaline are being actively conducted. Amigdalin is a cyanide glycoside which is broken down into benzaldehyde, hydrogen cyanide (HCN) and glucose by manulonitrile by emulsin in the presence of water. In particular, it has been reported that amigdaline is hydrolyzed by emulsine when the extraction solvent is water, and in particular in the form of powder, it is mostly decomposed to detect only a few tenths of the original amount (Akahori A). , Kagawa K., Shoyakugaku Zasshi , 37, 241-247, 1983). In addition, amigdalin undergoes epimerization in boiling water and changes to neo-amigdalin (L-mandelonitrile-β-D-gentiobioside) without antitumor effect (Takayama Y., Kawai S., Chem. Pham. Bull. , 32, 778-781, 1984), should inhibit the amidarin epimerized to neoamigdaline in aqueous solution. On the other hand, a method of adding an acid has been used to increase the extraction rate in powder form and to prevent epimerization (Hwang EY, et al., Chem. Pharm. Bull. , 50, 1373-1375, 2002). However, when the acidic aqueous solution is used as the extraction solvent in the herbal complex preparation, there may be a safety problem, such that major components may be changed by acid hydrolysis.

Accordingly, the present inventors completed the present invention by establishing a reverse phase HPLC method capable of extracting and separating a high yield of amidaline in about 10 minutes using only pure water as an extraction solvent without pretreatment for treating enzymes or treatment of additives.

It is an object of the present invention to provide an optimum extraction condition and a separation method for inhibiting amygdalin degradation contained in hot water extracts of the causal or algae with anticancer effects.

In accordance with the above object, the present invention extracts amigdalin (amygdalin) from a hot water extract or doin (桃仁) with an anti-cancer effect with a hot water extracting solvent, and then amigdaline of Formula 1 and Formula 2 contained in the hot water extract The neoamigdaline mixture of provides a method for rapidly separating amigdaline using high performance liquid chromatography (HPLC).

The poultry is preferably a size that passes through No. 20 body, the material of the size below is not preferable in terms of extraction efficiency.

The hydrothermal extraction is characterized in that the extract of the algae or algae marinated in water at a temperature of 90 to 95 ℃ extracted for 2 to 8 minutes, preferably for 3 to 6 minutes.

Separation using HPLC is characterized in that the separation using the 10 to 15% acetonitrile as a mobile phase, using a 9 to 11 mM sodium phosphate buffer, depending on the conditions of maintaining a column temperature of 5 to 10 ℃ It is done.

Hereinafter, the present invention will be described in detail.

Crude or passerby of the present invention is prepared, and the initial temperature of 90 to 95 ° C. is adjusted using water of about 5 to 20 times, preferably about 10 to 15 times, the weight (g) of each dried attendant and passerby. Extraction and concentration of the water extract by extraction methods such as reflux extraction and reduced pressure for about 2 to 8 minutes at the extraction temperature can be obtained.

Specifically, for example, as a result of examining the change in the extraction rate of amigdalin according to the extraction temperature during extraction of hot water of algae and algae, the emulsin is not inactivated at the temperature of the extraction aqueous solution at 60 ° C., so most of the amigdaline is decomposed, which cannot be measured. In addition, the inactivation of the emulsine is started from 70 ℃ to slightly extract the amigdaline, the emulsifier is inactivated by increasing the temperature to inhibit the hydrolysis of amigdaline, so the extraction rate of the amigdaline is increased to show the highest extraction rate above 90 ℃.

On the other hand, in the extraction time, the extraction rate is low at 60 ℃ and the change according to the extraction time cannot be measured, but the extraction rate increases with the extraction time above 70 ℃, but the extraction time does not change when the extraction time is over 6 minutes. It is not possible to extract at an extraction temperature of 90 to 95 ℃ for 2 to 8 minutes shows the best extraction rate. In addition, in the case of pure water extraction, amigdaline is converted to neoamigdaline at a high rate at 70 ° C. or higher, and when extracted for 2 to 8 minutes at an extraction temperature of 90 to 95 ° C, the conversion of amigdaline to neoamigdaline is suppressed. It is possible to extract amigdaline in high yield.

High performance liquid chromatography (HPLC) for separation of amigdaline and neoamigdaline contained in the water extract extracted by the above method uses 10 to 15% acetonitrile as the mobile phase, and 9 to 11 mM sodium Using phosphate buffer, it is preferable to isolate | separate according to the conditions which hold the column temperature of 5-10 degreeC. This allows neoamigdaline to be separated from amigdaline within a short time within 10 minutes to obtain a high yield of amigdaline.

The present invention is described in more detail based on the following Reference Examples and Examples, but the present invention is not limited thereto.

Reference Example 1. Preparation of Experimental Materials

Amigdalin was used as the standard solution, analytical amigdalin (Tokyo Kasei Chemical Co., Tokyo Japan) was used, methanol was purchased from Merck (Darmstadt, German), and tertiary distilled water was used for automatic aquarius AW-. It was obtained by the 1001 (Top Trading co., Seoul, Korea) apparatus. Acetonitrile was used for HPLC and other reagents and solvents were used for guranteed grade or analytical grade. The passerby purchased the one that meets the specifications of the Korean Pharmacopoeia (VII) in Gyeongdong market. 8-amino-2-naphthalenesulfonic acid (8,2-ANS) was used as an internal standard.

Reference Example 2 High Performance Liquid Chromatography (HPLC) Apparatus

High performance liquid chromatography (HPLC) was measured at 214 nm using a Nanospace SI-2 / 3001 pump (Shiseido, Tokyo, Japan) equipped with a Nanospace SI-2 / 3002 UV detector and the column was luna ( Luna) 3u C18 (2) 100A (4.6 mm x 150 mm, 3 μm, phenomenex, Torrance, CA, USA) was used. The column oven was kept at 8 ° C. using nanospace SI-2 / 3004, autosampler used nanospace SI-2 / 3023, and the flow rate was 600 μl / min. The mobile phase was a 10 mM sodium phosphate buffer containing 25% methanol and acetonitrile, and a 600 MHz NMR Spectrometer (Bruker Advance-600, Germany) was used to obtain proton NMR.

Example 1 Preparation of Standard Solution and HPLC Analysis

The amigdaline standard solution of Reference Example 1 was dissolved in water as it is commercially available, and neoamigdaline was used according to Fischer's method (Fisher E., Ber. Deut. Chem. Ges. , 28, p1508, 1895). 10 mg of amigdaline and 10 ml of 0.005 M ammonia aqueous solution were added and left at room temperature for 2 hours to obtain a mixture of amigdaline and neoamigdaline (10 mg), using preparative HPLC (Youngrin, Korea) to separate neoamigdaline. Neoamigdaline was aliquoted and concentrated and used as neoamigdaline standard solution.

At this time, the conditions of HPLC were set at a flow rate of 5 ml / min at room temperature, 6% acetonitrile was used as the mobile phase, and the column was octadecylsilane (Nucleosil 100-5 C18 (250 mm x 10 mm ID)). .

As a result of analyzing the neoamigdaline isolated as described above by mass spectrometry (FAB-HR mass) and nuclear magnetic resonance (NMR), the molecular weight of amigdaline is 458.1672 [M + 1], and neoamigdaline is 458.1674 [M + 1]. It was confirmed that the same, the chemical shift on the NMR (chemical shift) was 5.89 ppm in the case of amigdaline, 6.07 ppm in the neo-amigdalin bar, they can be confirmed that the mutual optical isomer (epimer) relationship.

Example 2 Preparation and HPLC Analysis of Sample Liquids with Constant Extraction Temperature

2-1. Changes of Amigdaline Extraction Rate with Extraction Temperature

The powder passed through No. 20 was ground as a test material. 250 ml of distilled water was added to a round flask on a water bath, and the starting temperature was kept at 50 ° C., 60 ° C., 70 ° C., 80 ° C. and 90 ° C., respectively. After the addition to the round flask, about 1 ml of the sample was taken at regular time intervals (2 minutes apart) and placed in a microtube. Each was filtered through a 0.2 μm filter membrane to prepare a sample for HPLC, and then measured according to the HPLC conditions of Reference Example 2, and the change of the amigdaline extraction rate according to the extraction temperature was examined (see FIG. 1). On the other hand, in order to find the temperature range showing the highest extraction rate, more specifically, the amount of extraction of Amig with the algae powder was measured at intervals of 2 ° C. at an extraction temperature of 80 ° C. to 90 ° C. (see FIG. 2).

As a result, since the aqueous solution was unable to inactivate the emulsin at 60 ℃, most of the amigdaline was decomposed and could not be measured, and the inactivation of the emulsine started at 70 ℃, and the amidaline was slightly extracted. Increasing the extraction rate of amigdaline increased. Specifically, the extraction amount of amigdaline was small at 80 ° C., but increased rapidly at 84 ° C. and showed the highest extraction rate at 90 ° C., and the same at higher temperatures. Therefore, it was confirmed that the extraction at 90 ℃ or more for complete extraction without the effect of emulsine.

2-2. Changes of Amigdaline Extraction Rate with Extraction Time

Samples prepared in Example 2-1 were taken about 1 ml at regular time intervals (2 min intervals) and placed in a microtube. Each sample was filtered with a 0.2 μm filter membrane to prepare an HPLC sample solution, and measured according to the HPLC conditions of Reference Example 2, and the change of the amidaline extraction rate according to the extraction temperature and the extraction time was examined (see Table 1). ).

Temperature / hour 2 minutes 4 minutes 6 minutes 8 minutes 10 minutes 20 minutes 30 minutes 60 minutes 60 ℃ N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 70 ℃ 0.38 ± 0.13 0.41 ± 0.14 0.40 ± 0.13 0.44 ± 0.11 0.49 ± 0.12 0.85 ± 0.03 0.96 ± 0.15 1.03 ± 0.14 80 ℃ 14.50 ± 0.95 15.84 ± 1.01 16.12 ± 0.82 16.13 ± 1.02 16.12 ± 1.02 16.30 ± 1.12 16.56 ± 1.34 16.73 ± 1.50 90 ℃ 50.50 ± 0.55 51.10 ± 0.64 50.65 ± 0.68 50.13 ± 0.64 49.75 ± 0.67 50.07 ± 0.96 50.17 ± 0.68 50.30 ± 0.81

As a result, the extraction rate was too low to measure (N.D.) at 60 ℃, the extraction rate gradually increased with the extraction time at 70 ℃, but only a fraction of the extraction rate was 1.03mg / g at 60 minutes. At 80 ° C., the extraction rate was almost constant after 6 minutes, and the extraction rate of 6 minutes was 16.13 mg / g. At 90 ° C., the extraction rate reached a maximum of 51.10 mg / g at 4 minutes, showing an almost constant extraction rate over the entire time. The extraction effect was the same as that of 90 ° C. even at temperatures of 90-95 ° C., but decreased at higher temperatures. That is, the extraction temperature is the most important in the water extraction of almond powder, and most of amidaline could be extracted without loss when maintained above 90 ℃.

Example 3 Preparation and HPLC Analysis of Specimen Using Extraction Temperature Different from Start Temperature and Final Temperature

The powder passed through No. 20 was ground as a test material. 250 ml of distilled water was added to a round flask on a water bath, and then the start temperature of the extraction solvent was maintained at 60 ° C., 70 ° C. and 80 ° C., respectively. After the addition, each temperature was raised directly to 90 ° C. Thereafter, about 1 ml of the sample was taken at regular time intervals (two minutes apart) and placed in a microtube. Each sample was filtered with a 0.2 μm filter membrane to prepare a sample for HPLC, and then measured according to the HPLC conditions of Reference Example 2, and the change of the amidaline extraction rate according to the extraction temperature and the extraction time was examined (see Table 2). ). At this time, the extraction time was compared based on 6 minutes.

Temperature / hour 2 minutes 4 minutes 6 minutes 8 minutes 10 minutes 20 minutes 30 minutes 60 minutes 60 ℃ 10.72 ± 0.65 10.87 ± 0.85 10.79 ± 0.94 10.74 ± 1.14 10.96 ± 1.19 11.35 ± 0.72 11.15 ± 0.62 11.39 ± 0.75 70 ℃ 19.09 ± 1.26 19.52 ± 2.27 19.00 ± 1.69 19.78 ± 1.82 19.13 ± 2.18 19.37 ± 1.46 19.13 ± 1.57 19.06 ± 0.59 80 ℃ 24.49 ± 0.77 25.09 ± 0.87 24.80 ± 0.82 25.12 ± 1.48 24.77 ± 1.17 25.14 ± 0.46 24.83 ± 0.67 25.39 ± 0.13 90 ℃ 50.50 ± 0.55 51.10 ± 0.64 50.65 ± 0.68 50.13 ± 0.64 49.75 ± 0.67 50.07 ± 0.96 50.17 ± 0.68 50.30 ± 0.81

As a result, it took only 1 minute and 40 seconds to raise the temperature when the initial temperature was 60 ° C, but the extraction rate of amigdaline was 10.79 mg / g, and the extraction rate was low. This indicates that the hydrolysis rate of amigdaline by the emulsion was faster than the rate of inactivation of the emulsion at below 60 ° C. On the other hand, when the initial temperature was 70 ℃, it took about 1 minute and 10 seconds to raise the temperature to 90 ℃, the extraction rate of amigdaline increased to 19.00 mg / g, and at 80 ℃ it took 35 seconds to raise the temperature. , The extraction rate of amigdaline was 24.80 mg / g. This shows that about 50% of amidaline was decomposed by the hydrolysis action of the emulsion before the emulsion was inactivated. However, when the initial temperature was 90 ° C., most of the amidaline present in the pedestrians was detected without degradation, and the extraction rate of the amigdaline was 50.65 mg / g, which was the best. This shows that the emulsion is completely inactivated before it is hydrolyzed.

As described above, since the hydrolysis of the emulsion was completed very quickly within 2 minutes, it could be confirmed that the hydrolysis of the emulsion could not be completely suppressed no matter how quickly the temperature was raised from the initial temperature of the reaction to 90 ° C. In addition, in the extraction of amigdaline, the initial temperature setting of extraction is important, and it was confirmed that the temperature should be set above 90 ° C. in order to completely inactivate the emulsion.

Example 4 Search for Extraction Conditions for Epimerization Inhibition

In the case of water extracts using only pure water as the extraction solvent without pretreatment, the conversion of amigdaline to neoamigdaline is effected only by heat, and it is influenced by the extraction time, and it is affected by the extraction temperature and the extraction time. In order to determine the degree of conversion, the conversion ratio of neoamigdaline to neoamigdalin (neoamigdalin / amigdalin, average% ± SD%) was examined (see Table 3).

Temperature / hour 2 minutes 4 minutes 6 minutes 8 minutes 10 minutes 20 minutes 30 minutes 60 minutes 60 ℃ N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 70 ℃ N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 80 ℃ 0.73 ± 0.10 1.46 ± 0.16 2.05 ± 0.10 2.78 ± 0.16 3.30 ± 0.38 6.81 ± 0.42 9.23 ± 0.60 17.22 ± 0.20 90 ℃ 1.22 ± 0.38 2.46 ± 0.16 3.75 ± 0.36 4.97 ± 0.26 6.64 ± 0.71 11.83 ± 0.22 16.58 ± 0.57 27.40 ± 0.43

As a result, in the case of 60 degreeC and 70 degreeC, conversion rate was too low and it was impossible to measure (N.D .; not detected). In the case of 80 ° C, the conversion rate to neoamigdaline was the lowest as 3.30% at 10 minutes and 6.81% at 20 minutes. However, despite this advantage, the extraction rate is not sufficient at 80 ℃. In the case of 90 ° C, the conversion rate was 1.22% at 2 minutes of extraction time, but almost no conversion was achieved, but gradually increased to 4% at 4.97% at 8 minutes of extraction time. When the extraction time was within 8 minutes, it was practically sufficient to be used. 3 is a chromatogram showing the degree of epimerization according to extraction time when the extraction temperature is 90 ℃.

Example 5 Separation of Amigdaline and Neoamiglin

In experiments for measuring amigdaline in crude extracts, the HPLC mobile phase used 10 mM sodium phosphate buffer (pH 2.6) containing 25% methanol (see FIG. 4A). In the separation and quantitation experiments of amigdaline and neoamigdalin, the mobile phase used 10 mM sodium phosphate buffer (pH 2.6) containing 13% acetonitrile (see Figure 4b). On the other hand, the column oven temperature was adjusted to 8 ℃, lowering the temperature of the resolution of amigdaline epimer was improved. The complete resolution (Complete baseline resolution, Rs) was achieved under the above conditions, and the separation time was shortened to within 10 minutes, which was good for practical use. Quantitative analysis is possible using internal standards. The caliber curve shows a linear range of UV ranges depending on the concentrations of amigdaline and neoamigdalin (0.05 to 0.5 mM) ( r2 values were 0.9998 for D-amygdalin and 0.9994 for neoamygdalin) and the search was within 5M ( S / N). = 3) (see Figure 4).

From the above results, we found an effective condition for inhibiting the conversion of amigdaline into neoamigdaline contained in the indoin or algae, and also confirmed the method of controlling the surface area of the cut surface of the indoin or algae without being affected by the emulsion.

As described above, the present invention provides an HPLC method for optimizing the extraction conditions and quantitative analysis of the amidaline epimers for the inhibition and epimerization of the emulsine to inactivate the amigdaline contained in the water or algae hydrothermal extract As a result, it is possible to quantitatively analyze amidaline with complete resolution (baseline resolution, Rs) in less than 10 minutes in comparison with the conventional HPLC method. have.

Claims (5)

After extracting amygdalin (amygdalin) with a hot water extracting solvent from the Doin (행 仁) or hangin (가지는 仁) with the anti-cancer effect, the mixture of amigdaline of formula 1 and neoamigdalin of formula 2 contained in the hydrothermal extract is a high performance liquid A method for separating amidaline using chromatography (HPLC). (Formula 1)

(Formula 2)

The method of claim 1, wherein the hydrothermal extraction is characterized in that the extraction of almonds or almonds in hot water of 90 to 95 ℃ temperature for 2 to 8 minutes. The method of claim 2, wherein the hydrothermal extraction is for 3 to 6 minutes. The method according to claim 1, wherein the powder has passed through No. 20 sieve. The method of claim 1, wherein the separation using HPLC is performed by using 10 to 15% acetonitrile as a mobile phase and using a 9 to 11 mM sodium phosphate buffer to maintain a column temperature of 5 to 10 ° C. Separating according to the method.

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