WO2012011619A1

WO2012011619A1 - Method for separating ephedrine from ephedra sinica stapf at a high yield, and adjuvant for enhancing immunity containing ephedrine as an active ingredient

Info

Publication number: WO2012011619A1
Application number: PCT/KR2010/004764
Authority: WO
Inventors: 이현용; 김진철; 정향숙; 김승섭; 오성호; 정명훈; 최운용; 서용창; 황보영; 나천수
Original assignee: 강원대학교 산학협력단
Priority date: 2010-07-21
Filing date: 2010-07-21
Publication date: 2012-01-26

Abstract

The present invention relates to a method for isolating ephedrine from Ephedra sinica Stapf, which is a representative medicinal herb, at a high yield, and to an adjuvant for enhancing immunity containing the ephedrine as an active ingredient. The inventors of the present invention performed a process at an ultra-high pressure to efficiently obtain ephedrine from Ephedra sinica Stapf, and extracted ephedrine by using a solvent of sulfuric acid and ethyl alcohol mixed at a ratio determined through fractionation, without the conventional addition of diethyl ether.

Description

【Specification】

[Name of invention]

Ephedra from ephedra _. Method for Separation in High Yield and Immune Booster Using Ephedrine as Active Ingredient

Technical Field

The present invention relates to a method of isolating ephedrine (ephedrine) in a high yield from a representative stem medicinal herb ephedra 0¾¾ei / ra sinica Stapf) and an immunoadjuvant containing the ephedrine as an active ingredient.

In other words, the present invention relates to a method for isolating the base component ephedrine from ephedra, and to a technique utilizing the new body's immune activity of the isolated material. Crab is a stem plant herb that relieves the symptoms of colds, and it is possible to improve the yield of basal components through the introduction and fractionation of extreme extraction methods, and to promote immune cell growth, low cytotoxicity, and NK cell growth. In addition, the present invention relates to a technology that identifies and embraces the new immune activity of ephedra components of ephedra.

<3> In order to separate ephedrine from ephedra, there has been a complicated process of extracting sulfuric acid from the ephedra from diethyl ether in addition to adding sulfuric acid, adjusting pH, and removing salt. A simple and effective method for obtaining and separating is presented.

In order to effectively obtain ephedrine from ephedra, the inventors of the present invention performed an ultra-high pressure process, and extracted with sulfuric acid and ethyl alcohol as a solvent in a fraction determined during fractionation without adding diethyl etherol as before. That is, the process of obtaining ephedrine, a useful substance of ephedra, was simplified, and further, the yield improvement and new immune activity of ephedrine obtained through the isolation method were confirmed.

<5>

Background Art

<6> ephedra 0¾aferfra sinica STAPF) is a perennial herb and is distributed mainly in northern China and Bongol and has a length of 30 ~ 70cm. The main components of ephedra are ephedrine, pseudoephedrine, 1-N-methylephedrine, 1-norephedrine, dN- pseudomethyledphrine, d-demethyl- pseudoephedrine, ephedine, Nonacosanol, nonacosane, triacontaol, and the like.

<7> The medical effects of ephedra include flu symptoms such as coughing and high fever. It is characterized by having an anti-influenza virus effect. Ephedrine of ephedra is known to have antipyretic, anti-inflammatory and bile secretion, as well as the cardiovascular system, and has been used since ancient times for sweating, asthma and colds. However, its use has been noted for patients with hypertension because it has a blood pressure-boosting effect. As such, various pharmacological effects of ephedra are known, but until now, the specific effects of ephedra's direct immune activity enhancement are insignificant, and there is no industrial use as a functional material.

In addition, in the conventional extraction process, the sulfuric acid was extracted with a solvent for ephedra, and the salt was immediately removed. After re-extracted with diethyl ether to obtain ephedrine in a supercritical process. In order to separate ephedrine from ephedra by this method, there was a complicated process involving three consecutive extractions with diethyl ether in addition to sulfuric acid addition, pH adjustment, and salt removal. Not only was the process complicated, but the re-extraction of ephedra had to be re-extracted on several occasions, resulting in a high economic burden and consumption of extraction time to obtain the required solvents.

In this situation, the present inventors have effectively isolated ephedrine from ephedra and further revealed its immune cell growth promoting and NK cell growth promoting activity. This increases the added value as a functional material of the Chinese medicine ^' Ephedra ^' , and further prevents diseases caused by immunodeficiency, such as the swine flu, which is a problem with pandemic. Secondly, it relieves cough and high fever, which are representative symptoms of the flu. It is thought to open the possibility as an effective therapeutic agent to prevent the transmission of virus to diseases such as pneumonia and sepsis.

<10>

[Detailed Description of the Invention]

[Technical problem]

It is an object of the present invention to provide a method for efficiently separating ephedrine from ephedra.

<12> It also reveals new features of ephedrine and takes advantage of it.

<13>

Although the components of the compounds suspected of having immune enhancing activity from ephedra have been reported to date, studies on their obtaining method have been insignificant. In the present invention, ephedrine is efficiently separated from ephedra, and then immune cells. Immune activities such as growth promotion and NK cell growth promotion were compared with conventional extracts. The inventors effectively separated ephedrine by extracting and fractionation of ephedra from ephedra as a medicinal herb that can be used for medicinal and edible foods, and identified physical elution through TLC and HPLC analysis. In addition, according to the process of the present invention, a variety of immunological experiments with ephedrine from ephedra by the practitioner derived basic data for the development of new immune-promoting food materials, which have not been reported before, thereby suggesting the possibility of practical use as a natural immune enhancer. Was intended.

<16>

Technical Solution

<Π> The high yield extraction method of the present invention for achieving the above object,

Ultra-high pressure extraction of ephedra at 300 Mpa;

Extracting the above extract from silver at 30-40 ^° C. for 10-14 hours using 0.5MH ₂ SO ₄ and 99% ethyl alcohol (1: 1, v / v) to obtain a soluble fraction;

<20> to obtain a powder by drying the fractions under reduced pressure;

<2i> Dissolve the above powder in methanol, 99% C0 ₂ , extraction temperature 30-40 ^° C, pressure 3,000

Performing supercritical extraction at a condition of ˜3,500 psi;

<22> 6M NaOH was added to the obtained product to adjust the pH to 11 ᅳ 13, and then NaCl was added to the salt.

It is characterized by consisting of; obtaining a pure ephedrine by removing (salt).

<23>

The present invention is also characterized in that it is an immunoadjuvant containing an ephedrine obtained by the separation method as an active ingredient.

<25>

In the present invention, an ultrahigh pressure extraction process is performed to obtain an effective bioactive substance from ephedra. In order to break the solid and hard cell wall of ephedra, 300Mpa ultra high pressure extraction method is used to maximize the dissolution of useful materials. Thereafter, in order to separate and fractionate the ephedrine component that enhances the ephedra's immune activity, 0.5 MH ₂ SO ₄ and 99% ethyl alcohol were extracted for 10-14 hours using a nonpolar solvent extraction method to obtain a soluble fraction. After drying the fractions under reduced pressure to obtain a powder, the powder was completely dissolved in methane and placed in a supercritical extraction device. Solvent 99% C0 ₂₎ Extraction temperature 30-40 ^° C, pressure

Set up 3,000 ~ 3,500 psi to perform supercritical extraction. Finally, 6M NaOH was added to the obtained product, adjusted to pH 11 13, and NaCl was added thereto. The salt is removed to obtain ephedrine.

Next, the immune immune activity of ephedrine is confirmed by measuring human immune B cell growth, evaluating human normal cytotoxicity, and measuring NK growth. In addition, IR, NMR structural analysis and MASS molecular weight analysis of the obtained separation material is finally confirmed that the material separated by ephedrine.

<28> The present invention is to increase the elution of the ephedra useful bioactive substance ephedrine through the extraction of ephedra, and the reaction of H ₂ S0 ₄ and ethyl alcohol directly with ephedra extract in the first fractionation conditions after the fractionation when the existing ephedrine extraction It is characterized by simplifying the hassle of three consecutive extractions of diethyl ether.

It was confirmed that the yield of ephedrine is improved through the isolation method of the present invention, and furthermore, it is also newly confirmed that the immune activity is improved according to the above method, thereby increasing its utilization. In particular, the inventors have for the first time found that the ephedrine has an immunological activity by increasing the yield by 15% through the step-separated fractionation of ephedrine from ephedra, the present invention provides a method for improving the yield of ephedrine material and its preparation It is characterized by new applications.

<30>

Ultra-high pressure treatment is a processing technology that is recently attracting attention in the field of extracting natural products to improve the preservation, physical properties and functionality of natural water. Ultra high pressure is the principle of instantaneously and evenly transmitting the pressure of water or oil to the pressure medium by using the pressure of 100 ~ 1000 Mpa. In food processing, heat treatment and pressure treatment are mainly used. While heat treatment has many chemical changes, pressure treatment has an advantage of not causing chemical change. Therefore, the ultra-high pressure process is a non-heating processing method, so it is evaluated as a processing technology that can maintain freshness without denaturing the main ingredients in the food, and can overcome the deterioration of texture and flavor of the food by the conventional heating treatment. Herbal medicines such as ephedra need to introduce ultra-high pressure that is different from the existing processing technology in order to obtain a bioactive substance due to its strong cell wall. In particular, the high energy and pressure of the ultra-high pressure acting on the herbal medicine enables us to obtain useful physiologically active substances that cannot be obtained by the conventional extraction process, thereby re-evaluating the natural products contained in the herbal medicine. By using the ultra-high pressure technology that can be most efficiently used for the extraction of chemical components of herbal medicines, the problems of high energy, low efficiency, heat denaturation and loss of useful components, and solubility-oriented extraction methods have been improved. From the medicinal plants, it is possible to extract the desired single component with high purity and low impurities in a short time. In the present invention, after extracting ephedra using ultra-high pressure energy, the useful materials were separated and purified through sequential fractionation. Through the introduction of extreme processes such as high pressure, the elution of the effective base components of ephedra was maximized, the yield was increased, and the purification purity of ephedrine in the extract could be increased by performing supercritical extraction after performing the stepwise fractionation. The optimum conditions for yield improvement in the separation of ephedrine from ephedra were presented, and the extraction amount was experimentally separated and compared using TLC / HPLC analysis. In addition, it was confirmed that the ephedrine through the IR, NMR structural analysis and MASS molecular weight analysis of the finally obtained separation material.

<33>

Sulfuric acid and ethyl alcohol used in the fractionation step are characterized by synthesis of diethyl ether when heat is applied. Since sulfuric acid acts as a catalyst for ethyl alcohol, ¾ alcohol is easily synthesized into ether 5. When diethyl ether is obtained through chemical reaction between sulfuric acid and ethyl alcohol in the fraction, the molecular formula is C ₂ ¾0C ₂ H ₅ , which has a molecular structure with two ethyl groups centered on an oxygen atom. Irritating and sweet ethyl ethers are widely used as solvents for bromine, iodine, most fats and resins, gasoline, pure rubber, and vegetable alkaloids. In the existing process, the ephedra was extracted with sulfuric acid as a solvent, and then the salt was removed. After re-extracting with diethyl ether, ephedrine was obtained in a supercritical process. Not only was the process complicated, but the re-extraction of ephedra would have to be repeated several times, resulting in a high economic burden and extraction time to obtain the required solvents. However, if the present invention obtains ephedrine, it is possible to synthesize diethyl ether by the direct reaction of solvent sulfuric acid and ethyl alcohol, thereby reducing the economic burden and reducing the time consumption, and in contrast to the complicated stepped process, The loss can be enjoyed, so the yield can be effectively increased.

<35>

Anti-allergic agent (anti-allergic extract preparation method, Republic of Korea Patent Registration 10-0527912), anti-atopic agent (a composition for the prevention or treatment of atopic dermatitis, and preparation method thereof, Korea registration) Patent 10-0483539) and the like, but the separation of these immunoactive substances from ephedra is the first attempt in the field of yield enhancement and pure substance separation when applied to commercialization of the functional food and pharmaceutical materials of ephedra in the future It is expected to create new high added value and give high economic feasibility and preoccupy one field of biological business. Advantageous Effects

While the conventional method of separating ephedrine from ephedra from the ephedra has a yield of 0.002, the yield of ephedrine obtained through this isolation method has been increased to 0.0085¾ (85.46 ± 0.42; ag / g). Until now, the useful components derived from natural products have been complicated to isolate the isolated material, and the low immune activity compared to the complex process of obtaining a useful material has been a problem. The present invention provides a systemic and effective method for the separation of ephedrine having body immune activity from ephedra, has been used in cardiovascular as well as antipyretic, anti-inflammatory, bile secretion, sweating, asthma, cold It is thought that the bioavailability of ephedra may improve the yield and body immunity effectively. The present invention facilitates the dissolution of useful physiologically active substances to enhance the immune function, lowers the toxicity of natural water-derived cells, and facilitates the separation of pure substances, thereby becoming a cornerstone in the development of functional foods and pharmaceuticals for improving the immune function in the body. Could be.

<39>

[Brief Description of Drawings]

1 shows a method of obtaining ephedrine purely separated from ephedra, comparing the difference between the process (A) of the present invention and the existing process (B).

<41>

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in detail with examples by process.

<43>

1. First Step: Obtaining Extreme Extract from Ephedra

In order to obtain the desired active ingredient, 50 g of ephedra pulverized to a suitable size was extracted with an ultrahigh pressure extraction apparatus (Ilshin autoclave, Korea) using an extraction solvent as distilled water. The ultrahigh pressure condition for improving the extraction yield was set to 300Mpa and the extraction time was set to 15 minutes. Then, separation fractions were performed as described in the second step.

<46>

2. Second step: Step fractionation step

100 mg of ephedra extract obtained from the above process was completely dissolved (1: 1, v / v) in 70 ^ of 0.5M sulfuric acid (¾S0 ₄ ) and 70 ^ of 99% ethyl alcohol, and then Extracted at ^° C. Subsequently, the extract was filtered under reduced pressure and concentrated (Rotary Vacuum Evaporator NN series, EYELA, Germany) to obtain a powder through lyophilization. Since powder phase After dissolving Taetae extract in 10 m of methane, solvent 99% C0 _2> Extraction temperature 35 ^° C, pressure

Supercritical extraction was performed at 3,000 3,500 psi. Finally, 6M NaOH was added to the extract to adjust the pH to 11-13, and again 16 g of NaCl was added to remove salt to obtain ephedrine (see FIG. 1). Meanwhile, 50 g of ephedra was extracted as a control group (see FIG. 1).

<49>

Example 1. Measurement of yield of useful substance of extract

According to the experimental results of the inventors, the yield of ephedrine obtained by separating and fractionating the ephedra extract of 300 Mpa ultra-high pressure 15 minutes which is considered as the optimum extraction condition is shown in Table 2. When the ephedrine was obtained according to the process proposed in the present invention, the total extraction time was reduced by about three times compared to the existing separation process time of 12 hours, thereby economically reducing the time required for extraction. In addition, it is possible to improve the yield of separation of ephedrine by 25% compared to the 0.0022% yield of the existing separation process (B of Table 1), so the process of obtaining useful substances by this isolation method is an efficient process of obtaining ephedrine. .

Table 1. Comparison of ephedrine yield of extracts. Conditions Total extraction time (πώι) Number (%, us / s) vacant point of the present invention 240 0.0085 (85.46 ± 0.42)

Existing Separation Process 720 0.0022 (22.12 土 0.54)

<53>

<54>

Example 2 Isolation Aliquots of Useful Bioactive Ingredients

All fractions, reverse phase silica TLC plate (RP 18 W, 20 x 20 cm, Merck, USA) for stationary phase, butanol (Sigma, USA) for mobile phase: 6% acetic acid (Merck, USA): H ₂ 0 (Merck, USA) (4: 1: 1, v / v / v). After saturating the organic solvent to form a chromatograph, 10% sulfuric acid prepared in a brown glass sprayer was evenly sprayed on a dried TLC plate and baked in an oven at 85 ° C. It was.

Pseudoephedrine, ephedrine and norpseudoephedr ine were used as standard for comparative qualitative analysis of this study. As a result of comparing the spots, it was found that the largest amount of ephedrine was eluted from the fraction according to the present invention (Fig. 1).

„Standard 1 2-1 2-2

<59>

<60>. 1. TLC analysis (1: ephedra fraction obtained by the existing process, 2-1: ephedra fraction obtained by the process except ultra-high pressure treatment in the process of the present invention, 2-2: ephedra fraction obtained by the process of the present invention, * : Ephedrine spot of ephedra fraction obtained by the process of the present invention)

<61>

Example 3 Analysis of Separation Yield of Ephedrine by Prep-HPLC

For analysis, pseudoephedrine, ephedrine, and methylephedrine were mixed (l: l: l = v / v / v) as a comparative standard (Fig. 2). As a result of analyzing the ephedra fraction according to the embodiment of the present invention by Prep-HPLC, as shown in (B) the ephedra extreme extract fraction showed an increase in content of 100 mV or more compared to the ephedra fraction obtained by the conventional process as about 300 mV . Ephedrine, a physiologically active substance that is effective in treating colds and asthma through HPLC analysis of ephedra, is higher than the existing 0.0022% (22.12 ± 0.54 / g / g) through the fractionation process. The largest increase was found to be 0.0085% (85.46 ± 0.42 / g / g).

<64>

<65> (A)

<66> (B)

<67> Fig. 2. HPLC analysis {(A): 3 standard, (B): ephedra fraction according to the present invention}

<68>

Example 4 Structural Analysis of Useful Bioactive Components

<70>

Experiment 1.IR Analysis

<72> Characterization and structure of molecules using FT / IR (Fourier Transform Infraed spectrometer, UMA-500, BIO-RAD, Cambridge, USA) for analysis of the compound According to the vibration transmission absorption spectrum was analyzed. Fractional powder samples in solid phase were dissolved in KBr and the structure was analyzed by IR energy of 100 Hz. IR analysis uses the principle that infrared light in the region from 780 nm to 1,000 produces an absorption spectrum by vibratory or rotational movements present in the sample and provides information on functional groups present in the molecule. As a result of analyzing the peaks of the useful material contained in the TLC spot of the ephedra fraction obtained by the process according to the present invention, it can be seen that the chemical structure of ephedrine based on the results shown in 3,000, 1500, 1,000 wavenumber cn ¹ (Fig. 3).

<73>

JECROXS XICOLET205X FT-BR

WA \ TE> rmiBERS

<74>

<? 5> Fig. 3. IR analysis

<76>

<77> Experiment 2. NMR Analysis

<78> Using Bruker Avance DPX 600 Mhz spectrometer (Germany) for structural analysis of the TLC spot of ephedra fraction obtained by the process according to the present invention — NMR (Magnetic resonance imaging) nuclear magnetic resonance (NMR) , Varian UI 500 MHz) spectra were obtained and the peaks were resolved and integrated using Win NMR software. Ephedra preparative samples were used as DMS0-i »solvent with TMS. NMR analysis confirmed that the ephedra fraction, which had undergone ultra high pressure, produced a compound similar to ephedrine (Fig. 4).

<79>

Fig 4. NMR Analysis

<82>

<83> Experiment 3. MASS Analysis

<84> For specific mass spectrometry for ephedra useful materials, Autospec. M363 series

(Micromass, Euroscience, Manchester.UK) were used to form ions from neutral molecules, revealing the molecular weight of the material through a continuous decomposition process occurring at the ions. Fractional aliquots were dissolved in acetic acid 1 ^ to obtain the correct molecular weight of the eluted material and analyzed using vacuum MASS. Analysis of the peaks revealed that the mass and structure of the unknown isolate was ephedrine. Ephedrine MASS analysis showed an intensity of 163% at 170 m / z (Fig. 5).

<85>

<86> [ι <87> Fig. 5. MASS Analysis <88>

Example 5 Activity Test of Isolation

<90>

<91> Experiment 1. Measurement of immune cell growth

The effect of enhancing immune function was verified using B cell (Raji), a human immune cell. The growth of the cells was incubated ^at 5¾ C0 ₂ , 37 ^° C using RPMI 1640 medium containing 10% FBS. Immunostimulatory effect of the cells was 1 ~ 2><10 ⁴ eel ls / After adjusting the concentration, the sample was administered and cultured for 6 days, the cells of each well were counted by a hemacytometer to measure the number of cells. As a result, when the ultra-high pressure extraction process and fraction of the present invention were introduced, it showed high immune cell growth of 178% (Fig. 6).

<93>

1 2 3 4 5 6

<95> Fig. 6. Measurement of immune B cell growth

<96>

<97> Experiment 2. Measurement of normal cytotoxicity

<8> SRB assay was used for the cytotoxicity measurement. Sulforhodamine B (SRB) assay is a cellular protein by staining the experiment by measuring cell proliferation or toxicity of the target cells of human kidney normal cells HEK293 (in 10% FBS media) 4 ~ 5><10 4 , the concentration of 100 ^ of each well of 96 well piate with eel Is / ^ was incubated for 24 hours (37 V, 5% C0 ₂ ), and then the respective concentrations were 0.2, 0.4, 0.6, 0.8, 1.0 rag. / By 100

^ Incubated for 48 hours. After the incubation was completed, add SRB solution 100 ^ each, and dye for 30 minutes at room temperature. Unbound SRB stain was washed 4-5 times with 1% acetic acid, dried and then 10 mM Tris buffer 100 ^ After dissolving the dye solution by the addition of the absorbance was measured using a microplate reader at 540 ran. The cytotoxicity against human normal cell HEK293 was examined.

<99> The toxicity of HEK293 cells of the isolated material by the process of the present invention

It was lower than 15% at 1.0 mg / m.

<100>

1 2 3 4 5

Concentration (rag / ml)

<101>

<i02> Fig. 7. Measurement of normal cell toxicity

<103>

<104> Experiment 3. Measurement of NK cell growth

After adding the ephedra isolate to B cells, the culture was added to NK-cells to measure activity. In the ephedrine-added group, which was separated by the second fraction and the supercritical process after the ultra-high pressure extraction process, the NK cell density was 15.94X10 ⁴ cells / ii ^.

Through this measure of ephedrine's various immune activities, we have examined the general applicability of it as a new immune enhancing material.

Day

Fig. 8. NK Cell Growth

Claims

[Range of request]

[Claim 1]

Ultra-high pressure extraction of ephedra at 300 Mpa;

Extracting the above extract with a 0.5M ¾SO ₄ and 99% ethyl alcohol (1: 1, v / v) at a temperature of 30 ~ 40 ^° C for 1CKL 4 hours to obtain a soluble fraction;

Drying the above fractions under reduced pressure to obtain a powder;

Dissolve the above powder in methane, 99% C0 ₂ , extraction temperature 30-40 ^° C, pressure 3,000

Performing supercritical extraction at a condition of ˜3,500 psi;

6M NaOH was added to the obtained product to adjust the pH to 11 13, followed by removal of salt by adding NaCl to obtain pure ephedrine.

A high yield separation of ephedrine from ephedra.

[Claim 2]

An adjuvant for adjuvant containing as an active ingredient ephedrinol obtained by the separation method of claim 1.