KR101164110B1 - Manufacturing method of glycolipoprotein gintonin from Ginseng - Google Patents

Abstract

The present invention relates to a method for producing glycolipid protein gintonin present in ginseng, and more particularly, physiological activity of mobilizing free calcium (free Ca ²⁺ ) in animal cytoplasm in the roots, stems and leaves of ginseng. The present invention relates to a method for mass separation of glycolipoprotein gintonin with high yield in a relatively simple manner.
The present invention is not harmful because it separates crude oil from the butanol extract of ginseng root, stem and leaf by using anion exchange resin method, does not generate industrial waste, saves time and cost, and utilizes waste resources. It works.

Description

Manufacturing method of glycolipoprotein gintonin from Ginseng

The present invention relates to a method for preparing glycolipoprotein (gintonin) present in ginseng, and more specifically to mobilizing free calcium (free Ca ²⁺ ) in the animal cytoplasm from the root, stem and leaf of ginseng ( The present invention relates to a method for mass separation of glycolipid protein, gintonin, which has an increasing physiological activity, in a high yield in a relatively simple manner.

Ginseng is generally used as an tonic (adaptogen) or tonic for life extension and improves biological function against stress, fatigue, disease, cancer and diabetes. This ginseng has been used for hundreds of years in China and Japan, as well as in Korea, and recently ginseng is one of the most famous herbs consumed in the world (Tyler, J. Pharm. Technol. 11 , 214-220, 1995).

Ginsenoside was isolated and characterized in the early 1960s and has been used as a representative component in physiological and pharmacological studies (Shibata, et al. Tetraheadron Letters 1962 , 1239-1245, 1963; Wagner-Jauregg and Roth, Pharm Acta Helv 37 , 352-357, 1962). In addition, recent studies have found that ginseng contains other unknown components such as polysaccharides, polyacetylenes, and proteins (Nah, Kor. J. Ginseng Sci. 21, 1-12, 1997).

The ginsenoside component of ginseng is low in quantity, complex in the separation process, and expensive for pure ginsenoside. Therefore, the crude ginseng total saponin fraction (CGSF) obtained from the extract of butanol from the root of ginseng has been used. (Kanzaki, et al. Br J Pharmacol. 125 (2), 255-262, 1998; Choi, et al. Br J. Pharmacol. 132 , 641-648, 2001; Choi, et al., J. Biol. Chem 276 , 48797-48802, 2001; Choi, et al. Eur J Pharmacol 468 , 83-92, 2003; Lee, et al, J Biol Chem. 279 , 9912-9921, 2004; Jeong, et al, Br J Pharmacol . 142, 585-593, 2004; Reay , et al, J Psychopharmacol 19, 357-365, 2005;. Lee, et al, Arch Pharm Res 28, 413-420, 2005;. Wei, et al J Ethnopharmacol 111, 613-618, 2007; Eriksson, et al J Ethnopharmacol. 119 , 17-23, 2008).

Crude saponin fraction (CGSF) is known to exhibit its activity through the signaling pathway of the cell membrane. For example, Choi et al. Linked to PLCb-IP3 when the total saponin fraction was treated in Xenopus oocytes. The PTX-insentive Gα _{q / 11} protein was found to activate the Ca ²⁺ activated Chloride Channel (CaCC) (Choi, et al., J. Biol. Chem . 276 , 48797-48802). , 2001). In addition, this report reported that spontaneous reduction of calcium-dependent chlorine ion channel currents (CaCC currents) activated by the ROS saponin fraction when continuous treatment of the ROS saponin fraction in frog eggs ( Xenopus oocytes) (Lee, et al. al, J Biol Chem. 279 , 9912-9921, 2004).

When calmodulin is directly injected into frog eggs or when the intracellular calcium reservoir is depleted, the activity of calcium-dependent chlorine channels by the crude total saponin fraction disappears (Lee, et al, Arch Pharm Res 28 , 413-420). , 2005). In addition, the treatment of co-saponins in frog eggs results in stored-operated Ca2 + entry (SOCE) (Jeong, et al, Br J Pharmacol. 142 , 585-593, 2004), and therefore calcium from outside the cell or in the intracellular calcium reservoir. It is known that CaCC is activated in frog eggs due to an increase in the concentration of (Dascal, CRC Crit Rev Biochem 22, 317-387, 1987).

We find that ginsenosides activate CaCC For the purpose of separating pure ginsenosides from crude gross saponin fractions (CGSF), ginsenoside-rich fractions than CGSFs were found to rapidly disappear or disappear. In other words, ginsenoside purely isolated from CGSF was found to have no CaCC activity effect in frog eggs, and CGSF had a novel glycolipid protein in addition to ginsenoside, which affects the activity of CaCC in frog eggs. It was confirmed that it exists, and named the glycolipid protein as gintonin, and applied for "a novel glycolipid protein isolated from ginseng and a method for preparing the same" (Patent Application 2009-0110662).

However, in the above method, silica gel column chromatography was first performed from a butanol extract of ginseng (that is, CGSF) using a mixed solvent of chloroform, methanol, and water, and then secondly, a mixed solvent of ethanol, ethyl acetate, and water was used. Since the crude gintonin is separated through silica gel column chromatography, there is a limitation in applying the product to food by using harmful organic solvents that cannot be used in the food manufacturing process.

In addition, by using silica gel column chromatography using an organic solvent, the production cost is high, and when applied to the industry, it is difficult to reuse the remaining organic solvent and silica gel, which can cause huge industrial waste, using the polarity of the solvent Silica gel column chromatography is limited in the application of a large amount of material because it does not have a large amount of material that can be loaded (load) in one column, it takes a long time to separate the material. In addition, since gintonin has a property of being adsorbed onto silica gel as a glycolipid protein, some kinds of gintonin may be lost along with saponin, which is a main component of butanol extract (CGSF) of ginseng.

After all, the main object of the present invention is to provide a method for separating gintonin, a new bioactive substance from ginseng, in a relatively simple manner in high yield.

In order to achieve the above object, the present invention provides a method for separating a glycolipoprotein (gintonin) with a high yield as a new bioactive substance present in ginseng.

Specifically, the present invention, (1) preparing a ginseng alcohol extract; (2) fractionating the alcohol extract into water and n-butanol; And (3) anion exchange resin (DEAE sehparose) chromatography using n-butanol fraction (crude saponin fraction, CGSF) in the fraction using a weak alkaline buffer (20 mM Tris-HCl, pH 8.2) containing NaCl. It provides a method for producing glycolipid protein gintonin present in ginseng comprising a.

Gintonin (gintonin) according to the present invention is a glycolipid protein identified from ginseng as a pentamer having a molecular weight of about 67 kDa and an apparent molecular weight of about 13 kDa, which is negatively charged in neutral or weakly alkaline buffer solution. There is a characteristic.

The gintonin is a butanol extract of ginseng, that is, ginseng saponin, which is a main component of crude gross saponin fraction (CGSF), is composed of sugar and triterpenoids, and has a molecular weight of 1,000 to 2,000, and is charged in a neutral or weakly alkaline buffer solution. It can be distinguished from ginseng saponin in that it does not have a functional group.

In the present invention, an anion exchange resin column using a weakly alkaline buffer containing NaCl is used instead of silica gel column chromatography using a conventional organic solvent to separate crude gintonin from crude ginseng saponin fraction (CGSF) of ginseng. to be.

Hereinafter, the present invention will be described in detail for each manufacturing step.

In order to prepare the ginseng alcohol extract of the present invention as a first step, first, dried ginseng powder is a volume of water, methanol, ethanol, butanol of about 1 to 20 times the sample weight, preferably about 1 to 10 times the sample weight. Polar solvents of C1 to C4 lower alcohols such as the like or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably water having a mixing ratio (v / v) of 1: 0.2 to 1: 5 As a mixed solvent of methanol and methanol, extraction methods such as stirring extraction, hot water extraction, cold leaching extraction, warming extraction, reflux cooling extraction or ultrasonic extraction at about 70 to 120 ° C. for about 0.1 to 48 hours, preferably 5 to 12 hours, are preferred. Preferably, the mixture is cooled to reflux and then filtered to recover the supernatant. The above procedure is repeated several times, preferably 2 to 5 times, and the supernatant is collected under reduced pressure to obtain an alcohol extract.

Next, as a second step, the alcohol extract is solvent fractionated with a mixed solvent in which water and n-butanol are mixed in the same amount to obtain a fraction of water and n-butanol.

In the third step, the n-butanol fraction corresponding to the crude total saponin (CGSF) fraction of the fractions is subjected to anion exchange resin chromatography packed with 20 mM Tris-HCl (pH 8.2), and has no negative charge. After removing other low molecular weight substances, Na3-containing Tris-HCl (pH 8.2) was passed through to recover the substance bound to the anion exchange resin column, and the substance recovered from the column was put in a dialysis membrane (pore size 6,000 ~ 8,000) and dialyzed. When the salt contained therein is removed and then freeze-dried or concentrated in vacuo, a crude extract of gintonin can be obtained.

Alternatively, the anion exchange resin chromatography may be performed using an anion exchange resin column filled with 20 mM Tris-HCl (pH 8.2) containing water, methanol, ethanol or a mixed solvent thereof. At this time, after removing the negatively charged ginsenosides and other low-molecular substances, the material bound to the anion exchange resin column is passed through NaCl-containing water, methanol, ethanol or a mixed solvent Tris-HCl (pH 8.2). Recover.

In the present invention, ginseng such as wild ginseng, camphor ginseng, cultivated ginseng, Western ginseng, and Chinese ginseng may be used for the production of gintonin, and the ginseng may be used in the form of red ginseng, ginseng, white ginseng, and the like. Preferably, red ginseng, 4-6 years old, made from Korean ginseng ( Panax ginseng CA Meyer) is recommended, and the roots of ginseng as well as the leaves, stems, fruits or / and flowers of ginseng. It is possible to use

In particular, to separate gintonin from the leaves or stems of ginseng, n-butanol fraction (crude saponin fraction) is further fractionated with 90% methanol or a solvent mixed with ethanol and hexane in the same amount to increase extraction efficiency. It is then preferred to perform anion exchange chromatography on the methanol or ethanol fractions.

In addition, the crude gintonin obtained as described above was dissolved in phosphate buffer saline (PBS) containing NaCl, and subjected to anion exchange chromatography and gel filtration chromatography to obtain two gintonin fractions. Gintonin fractions may be subjected to step gradient anion exchange chromatography using NaCl-containing Tris-HCl (pH 8.2) and NaCl-containing PBS (pH 7.2) to obtain individual gintonin.

The gintonin prepared by the above method is a pentamer having a molecular weight of about 67 kDa and an apparent molecular weight of about 13 kDa. and aspartic acid, glutamine and glutamic acid, serine, glycine, arginine, arginine, threonine, alanine, proline, valine, Isoleucine, leucine, phenylalanine, tryptophan and lysine; Carbohydrate composition as glucose (galcose), galactose (galactose), galactosamine (galactosamine), glucuronic acid (glucuronic acid); And a glycolipid protein comprising linoleic acid, palmitic acid, stearic acid, and other small amounts of other compounds as lipid components.

The present invention is effective to provide a process for present and separating the animal cytoplasm within the free calcium mobilization ^{(free Ca 2 +) (mobilization} ) with the physiological activity of increasing glycolipid protein dust Nin (gintonin) in bulk in high yield in carrot There is.

The present invention is not harmful because it separates crude oil from the butanol extract of ginseng using anion exchange resin method, and does not generate industrial waste, but also saves time and money.

In addition, the present invention can utilize the waste resources, as well as ginseng root (ginseng root), can be separated from the stem or leaves of ginseng that was normally discarded after harvesting ginseng, reducing the cost of manufacturing Price can be competitive.

Figure 1 A is a diagram illustrating a method of separating crude gin toner from the root of ginseng, B is a chromatogram confirming the CaCC activity in the frog eggs of gintonin fraction.
FIG. 2A is a diagram illustrating a method for separating crude gintonin from the stem of ginseng, and B is a chromatogram confirming CaCC activity in frog eggs of gintonin fraction.
FIG. 3A is a diagram illustrating a method for separating crude ginnin from leaves of ginseng, and B is a chromatogram confirming CaCC activity in frog eggs of gintonin fraction.
FIG. 4A shows SDS-PAGE results of ginzintonin isolated from ginseng roots, stems and leaves, and B shows gel staining SDS-PAGE results of carbohydrate components of ginzintonin (R: root; S: stem; L: leaf).
Figure 5 shows the elution pattern of zogintonin isolated from ginseng root (A), stem (B), and leaf (C) by gel filtration chromatography.
Figure 6 shows the carbohydrate component analysis of individual gintonin by HPAED-PAD chromatogram, A is the result of the analysis of the composition of the neutral sugar (standard: 1. L-fructose; 2. L-rhamnose; 3. D-arabinose; 4. D-galactose; 5. D-glucose; 6. D-mannose; 7. D-xylose), B is the result of component analysis of amino sugars (standard Substances: 1. D-galactosamine; 2. D-glucosamine).
FIG. 7A is a GC-MS spectrum analysis result of lipid components of crude gintonin and individual gintonin isolated from ginseng root, stem, and leaf.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1. Ginseng Root ( root )in Chozintonin ( crude gintonin ) detach

Add 80-100% (w / v) ethanol or methanol 16 ℓ to 8 ㎏ of 6-year-old red ginseng ( Panax ginseng CA Meyer) powder purchased from Korea Ginseng Corporation (Daejeon, Korea), and extract it at 80 ℃ for 8 hours under reflux cooling. The supernatant was collected by vacuum filtration. This process was repeated three times, and the supernatant was collected and concentrated under reduced pressure (1.3 kg of ethanol / methanol extracts were obtained). For each ethanol and methanol extract, a mixed solvent of water and n-butanol (1: 1) (v / A total of two solvent fractions of v) and n-butanol fractions in water and n-butanol fractions were concentrated in vacuo to obtain 300 g of crude total saponin fraction (CGSF).

The crude total saponin fraction was dissolved in 20 mM Tris-HCl (pH 8.2) or 20 mM Tris-HCl (pH 8.2) containing 50% (w / v) ethanol or methanol, followed by DEAE Sepharose (pre-filled with the same solvent). sepharose) 20 mM Tris-HCl (pH 8.2) or 20 mM Tris containing 50% (w / v) ethanol or methanol loaded on anion exchange chromatography (GE Healthcare, Uppsala, Sweden) equipped with CL-6B column -Pass enough HCl (pH 8.2) to remove any non-bonded material, such as non-negative ginsenosides and other low molecular weight substances, then 50% (w / v) ethanol with 1 M NaCl. Alternatively, the material bound to the anion exchange resin column was recovered by passing through methanol Tris-HCl (pH 8.2) or Tris-HCl (pH 8.2) containing 1 M NaCl.

The recovered material in the column was put in a dialysis membrane (pore size 6,000 ~ 8,000) to remove the salt by dialysis, and then freeze-dried or concentrated in vacuo to give 16 g of crude gintonin (yield 0.20%).

Example  2. Ginseng stem ( stem ) In Chozintonin  detach

For 38 g of Panax ginseng CA Meyer stem, 2.0 g of crude total saponin fraction (CGSF) was obtained by the same method as in Example 1, followed by n-hexane and 90% (v / v) methanol or ethanol. N-hexane and 90% (v / v) methanol or ethanol fraction of 90% (v / v) methanol or ethanol, fractionated two or three times with a solvent mixed in equal amounts (methanol or ethanol: water = 9: 1) Fractions were concentrated under reduced pressure.

The concentrated 90% (v / v) methanol or ethanol fraction was concentrated in 20 mM Tris-HCl (pH 8.2) or 20 mM Tris-HCl (pH 8.2) containing 50% (v / v) ethanol or methanol. Then loaded onto anion exchange chromatography (GE Healthcare, Uppsala, Sweden) equipped with a DEAE sepharose CL-6B column pre-filled with the same solvent, 20 mM containing 50% (v / v) ethanol or methanol A sufficient passage of Tris-HCl (pH 8.2) or 20 mM Tris-HCl (pH 8.2) removed material not bound to the column, ie, negatively charged ginsenosides and other low molecular weight materials.

Finally, 50% (v / v) ethanol or methanol Tris-HCl (pH 8.2) containing 1 M NaCl or Tris-HCl (pH 8.2) containing 1 M NaCl was passed to the anion exchange resin column. The material was recovered, and the material recovered in the column was put in a dialysis membrane (pore size 6,000-8,000), and dialyzed to remove salt, followed by freeze-drying or vacuum concentration to obtain 0.11 g of zozintonin (yield 0.29%).

Example  3. Ginseng leaves ( leaves ) In Chozintonin  detach

0.3 g of ginzintonin was obtained from 37.3 g of leaves of Panax ginseng CA Meyer in the same manner as in Example 2 (yield 0.81%).

Example 4. Individual Gintonin Isolation

In order to separate individual gintonins from crude gintonin, the crude gintonin obtained in Examples 1 to 3 was dissolved in phosphate buffer saline (PBS) (pH 7.2) and equipped with a DEAE Sepharose CL-6B column. Anion exchange resin chromatography (GE Healthcare, Uppsala, Sweden) was performed and two major peaks were obtained. At this time, the solvent used was 0 to 2 M NaCl dissolved in PBS, each of the two main peaks obtained by separating in a linear gradient method while monitoring at 280 nm at a flow rate of 1 ml / min each Gintonin E (early eluted gintonin) and gintonin L (late eluted gintonin).

In addition, the gintonin E and gintonin L fractions were concentrated using a CentriVap DNA vacuum concentrator (Labconco, Missouri, USA), and then PBS (pH 7.2) as an eluent, 0.5 mL using a Superdex 75 gel filtration column. Chromatography was performed while monitoring at 280 nm at a flow rate of / min to obtain major and minor peaks with large molecular weights.

It was confirmed that the gintonin E and gintonin L thus obtained show CaCC activity in frog eggs. First, the gintonin E fraction was again step gradient using 20 mM Tris-HCl (pH 8.2) containing 100, 150, 200, 250 mM NaCl, and anion exchange column (HiTrap ^™ DEAE FF). , 1 ml) was loaded at a flow rate of 1 ml / min to obtain four peaks, of which fractions of 100, 150, and 200 mM NaCl eluents were concentrated with extensive dialysis and again from 0 to 1 M. Step gradient DEAE anion exchange column chromatography was performed using NaCl containing 20 mM Tris-HCl (pH 8.2) followed by Superdex 75 gel filtration chromatography to obtain the final fraction (individual gintonin).

Next, the gintonin L fraction was obtained using PBS (pH 7.2) containing 150, 200, 250, 300, 350 mM and 400 mM NaCl in the same manner as above to obtain the final fraction (individual gintonin).

Example 5 Determination of Molecular Weight of Gintonin

Electrophoresis (SDS-PAGE) of gintonin determined using standard proteins revealed that six gintonins showed a broad but single major band and their apparent molecular weight. weight) was found to be about 13 Dka (see Figure 6A).

Example 6.Amino Acid Composition Analysis of Gintonin

For general amino acid analysis, 30 μg of gintonin was hydrolyzed at 110 ° C. for 24 hours with 6 N HCl in vacuo . In addition, for analysis of cysteine, gintonin was hydrolyzed at 110 ° C. for 24 hours with 6 N HCl after peroxidation, and then treated with formic acid: hydrogen peroxide (10: 1) (v / v) solution. For analysis of tryptophan, gintonin was hydrolyzed with 4 M methanesulfonic acid followed by 4 M KOH.

In addition, amino acids hidden in phenylisothiocyanate (PITOC) derivatives were commissioned by the Korea Institute of Basic Science, Daejeon, Korea, and were equipped with a Waters Nova-Pak C18 column (3.9 * 300 mm). It was analyzed by HPLC (Hewlett Packard 1100 Series).

Protein components were determined by the Bradford method (Bradford, MM, Anal, Biochem . 72 , 248-254, 1976) using bovine serum albumin (BSA) as a standard.

As a result, hydrophobic amino acid was higher in gintonin than hydrophilic amino acid, but histidine (His), tyrosine (Tyr) and methionine (Met) were not detected. Of these, methionine was not detected because the N-terminus of gintonin was blocked.

Contents (%) Root stem leaf CYA * 5.36 8.74 10.31 ASX ** 11.26 4.98 6.71 GLX *** 7.15 5.20 5.24 Ser 5.46 4.60 5.17 Gly 10.99 15.83 18.93 His 0.00 0.00 0.00 Arg 2.57 1.32 1.38 Thr 4.46 1.76 2.25 Ala 6.71 3.87 3.86 Pro 6.48 5.14 5.24 Tyr 1.31 0.99 0.84 Val 5.06 4.70 3.49 Met 0.89 1.19 0.70 Ile 5.39 8.15 6.78 Leu 7.98 5.39 5.34 Phe 11.45 17.42 14.89 Trp 1.10 1.86 1.65 Lys 6.38 8.85 7.22

Wherein CYA * is the sum of cysteine and cystine, ASX ** is the sum of asparagine and aspartic acid, and GLX *** is the sum of glutamine and glutamic acid.

Example 7 Carbohydrate Composition Analysis of Gintonin

As shown in FIG. 6A, the band of gintonin in SDS-PAGE was single but broad, and may contain carbohydrates because it was not strongly stained with Comassie Brilliant blue straining.

In the present invention, in order to confirm the carbohydrate composition of gintonin, after hydrolysis of gintonin with 2 M trifluoroacetic acid at 100 ℃ for 4 hours to obtain a neutral sugar (ginutin) In a glass ampule (6 N HCl) hydrolyzed at 100 ℃ for 4 hours to obtain an amino sugar (acid sugar) and acid sugar (acid sugar).

The carbohydrate composition of gintonin was commissioned by Sejong University's Carbohydrate Materials Research Institute in Seoul, which was dyed using PAS staining (periodic acid-schiff based staining) and then HPAEC-PAD system equipped with CarboPac ^TM PA1 column (high performance anion exchange). Analysis was performed using a chromatography-plused amphermetric dectection system (Dionex, California, USA), and the molar weight of the monosaccharides was calculated from the peak area.

Carbohydrate content is also determined by the phenol-sulfonic acid method (Hounsell, EF, Davies, MJ, and Smith, KD, Protein protocol handbood , Humanna press , Totawa, 803-804, 1997) for neutral sugars. Determined by Anthrone method (Scott, TA, and Melvin, EH, Anal. Biochem. , 25 , 1656-1660, 1953).

As a result, gintonin has two kinds of neutral sugars, such as glucose and galactose, one type of amino sugar such as galactosamine, and one type such as glucoronic acid. It was confirmed that it was composed of a kind of acidic sugar.

Carbohydrate contents (%) Root stem leaf  GalN 3.59 1.66 1.89  Gal 19.72 12.13 53.09  Glc 70.74 79.43 45.02  Glu 5.95 6.78 -

Example 8. Lipid Composition Analysis of Gintonin

Since the gintonin of the present invention was in the same fraction as ginsenoside by n-butanol extraction, it is expected that the gintonin also contains a lipid moiety.

To confirm this, gintonin was hydrolyzed with 100 N HCl for 4 hours at 100 ° C. or digested with lipoprotein lipase to identify lipid and hydrophobic moieties.

Specifically, the hydrolyzed or digested crude total saponin fraction (CGFS) was concentrated and further fractionated with water and n-hexane, and then the n-hexane fraction was separated into a DB5-MS capillary column (30 cm × 250 μm). Lipid and hydrophobic moieties were analyzed at the Korea Institute of Basic Science and Support using an Agilent 6890N GC-MS system (California, USA) equipped with a. At this time, the GC (6890N) was equipped with a flame ionization detector and a split injection system, and fixed a supelo SPB-1 capillary column (inner diameter: 15 m × 0.32 mm / thickness: 0.25 m). It was.

As a result, as shown in Table 3 and Figure 7, the gintonin of the present invention is a lipid composition component linoleic acid (linoleic acid), palmitic acid (palmitic acid), stearic acid (stearic acid) and other small amounts of other compounds, etc. It was confirmed that it contained.

Taken together, the results suggest that gintonin of the present invention is a glycolipoprotein.

Lipids Content (%) Root stem leaf  Palmitic acid (C16; 0) 38.79 35.78 40.33  Stearic acid (C18; 0) 2.29 2.077 2.33  Linoleic acid (C18; 2) 52.66 57.34 57.34  Others 6.26 4.81 -

Measurement Example 1. Measurement of CaCC activity in Xenopus laevis oocytes

1- (1). Oocyte Preparation

To store and process Xenopus laevis from Xenopus I (Ann Arbor, MI, USA) according to the best standards, and to isolate oocytes (oocytes), frogs were used as 3-amino benzoic acid ethyl esters. After anesthesia with an aerated solution, the cells were treated with collagenase, followed by 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl ₂ , 5 mM HEPES, 2.5 mM sodium pyruvate, It was isolated by stirring for 2 hours in calcium free (Ca ²⁺ free) medium containing 100 units / ml penicillin and 100 μg / ml streptomycin.

Collect eggs from V-VI and in ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl ₂ , 1.8 mM CaCl ₂ , and 5 mM HEPES; pH 7.4) to which 50 μg / ml of gentamicin was added. The solution containing the egg was kept at 18 ° C. with gentle shaking with succession and exchanged daily.

1- (2). Measurement of CaCC

Two-electrode voltage clamp recordings were obtained from individual eggs placed in a small plexiglass net chamber (5 ml), and electrophysiological experiments were performed with a microelectrode filled with 3 M KCl ( Resistance: 0.2-0.7 kHz) and an Oocyte Clamp amplifier (OC-725C, Warner Instrument, CT, USA) were used at room temperature and CaCC was recorded at -80 ㎷ holding potential.

In addition, gintonin is a method such as Lee, JH, Jeong, SM, Lee, BH, Noh, HS, Kim, BK, Kim, JI, Rhim, H., Kim, HC, Kim, KM, and Nah, SY, J Boi Chem 279 , 9912-9921, 2004).

Experimental Example 1. Confirmation of gintonin effect on CaCC activity endogenously present in Xenopus laevis oocytes

In order to confirm the effects of ginogentonin and individual gintonin on CaCC activity endogenously present in frog eggs (Xenopus laevis oocytes), gingintonin obtained in Examples 1 to 3 and individual gintonin isolated from ginogentonin According to the method of Measurement Example 1, the support potential recorded as the increase of CaCC activity was measured.

As a result, in the group treated with ginjintonin, an inwardly Cl ⁻ current was induced at a −80 kV support potential (see FIGS. 1B, 2B, and 3B).

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

(1) preparing a ginseng alcohol extract;
(2) fractionating the alcohol extract into water and n-butanol; And
(3) performing anion exchange resin chromatography on the n-butanol fraction (crude saponin fraction, CGSF) in the water and n-butanol fractions using 20 mM Tris-HCl at pH 8.2 containing 1 M NaCl; Method for producing glycolipid protein gintonin present in ginseng comprising a. The method of claim 1,
In addition to the above steps,
(4) the glycolipid protein gintonin present in ginseng, comprising the step of removing the salt contained by dialysis of the material recovered from the anion exchange resin column in a dialysis membrane having a pore size of 6,000 to 8,000; Manufacturing method. The method of claim 2,
In addition to the above steps,
(5) dissolving the fraction obtained in anion exchange resin chromatography in 7.2 phosphate buffered saline (PBS) containing 0 to 2 M NaCl, performing anion exchange chromatography and gel filtration chromatography to separate into two fractions. ; And
(6) The two fractions were subjected to anion exchange chromatography using Tris-HCl at pH 8.2 containing 100 to 250 mM NaCl and PBS at pH 7.2 containing 150 to 400 mM NaCl, respectively. A method for producing glycolipid protein gintonin present in ginseng comprising the step of separating. The method of claim 1,
The ginseng alcohol extract of step (1) is water, methanol, ethanol, butanol or a method for producing a glycolipid protein gintonin present in ginseng, characterized in that extracted with a mixed solvent thereof. The method of claim 1,
The anion exchange resin chromatography of step (3) is a glycolipid present in ginseng characterized by using 20 mM Tris-HCl of pH 8.2 containing 1 M NaCl containing water, methanol, ethanol or a mixed solvent thereof. Method for producing protein gintonin. The method of claim 1,
The ginseng is a ginseng, ginseng, cultivated ginseng, Western ginseng, Chinese ginseng, red ginseng, ginseng, and white ginseng, the method for producing glycolipid protein gintonin, which is present in ginseng. The method of claim 1,
The ginseng is a method for producing glycolipid protein gintonin present in ginseng, characterized in that at least one selected from the roots, leaves, stems, fruits, and flowers of ginseng. (a) preparing an alcohol extract from the leaves or stems of ginseng;
(b) fractionating the alcohol extract into water and n-butanol;
(c) fractionating the n-butanol fraction (crude saponin fraction, CGSF) in the water and n-butanol fractions with 90% methanol or a solvent in which ethanol and hexane are mixed in equal amounts; And
(d) performing anion exchange resin chromatography on the 90% methanol or ethanol fraction and the methanol or ethanol fraction in the n-hexane fraction using 20 mM Tris-HCl at pH 8.2 containing 1 M NaCl; Method for producing glycolipid protein gintonin present in ginseng. The method of claim 8,
In addition to the above steps,
(e) the glycolipid protein gintonin present in ginseng, comprising the step of removing the salt contained by dialysis of the material recovered from the anion exchange resin column in a dialysis membrane having a pore size of 6,000 to 8,000; Manufacturing method. The method of claim 9,
In addition to the above steps,
(f) dissolving the fraction obtained in anion exchange resin chromatography in phosphate buffered saline (PBS) containing 2 M NaCl, performing anion exchange chromatography and gel filtration chromatography to separate into two fractions; And
(g) The two fractions were subjected to anion exchange chromatography using Tris-HCl at pH 8.2 containing 100 to 250 mM NaCl and PBS at pH 7.2 containing 150 to 400 mM NaCl, respectively, to individual gintonins. A method for producing glycolipid protein gintonin present in ginseng comprising the step of separating.

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