KR101629687B1 - Composition for prevention or treatment of gastrointestinal motility disorders - Google Patents

Abstract

The present invention relates to a composition for preventing or treating gastrointestinal motility disorders.
According to the composition of the present invention as described above, the electrical activity of khaki interstitial cells, which is known to contain gintonin as an active ingredient and plays an important role in gastrointestinal motility and contractility in digestive organs, , Which promotes intestinal motility, diabetes mellitus and other diseases such as diarrhea caused by the slowing of the absorption of nutrients to prevent the delay and control the gastrointestinal motility is effective.

Description

Technical Field [0001] The present invention relates to a composition for preventing or treating gastrointestinal motility disorders,

The present invention relates to a composition for preventing or treating gastrointestinal motility disorder disorder, and more particularly, to a composition for preventing or treating gastrointestinal motility disorder disorder containing ginseng-derived gintonin as an effective ingredient.

Studies have shown that the role of pacemaker in smooth muscle, which exhibits dynamic motion in the stomach, small intestine, and large intestine, originates from interstitial cells of Cajal (ICS) (Huizinga JD, Zarate N, Farrugia G. Physiology, injury, and recovery of interstitial cells of Cajal: basic and clinical science. Gastroenterology. 2009 Nov; 137 (5): 1548-56). In the absence or absence of carotid stromal cells, the digestive tract causes an abnormal electrical wave of the intestinal smooth muscle and is known to suppress the contractile nature of the intestinal smooth muscle and to reduce the intestinal transit time of the food. Resulting in abnormalities of the digestive system.

A morphological study of carotid stromal cells clinically showed a decrease in the number of carotid stellate cells in intestinal obstruction, achalasia, Hirschsprung's disease, chronic constipation and diabetic gastrointestinal disorders (Ohlsson B, Veress B, Lindgren S, and Sundkvist G. Enteric ganglioneuritis and abnormalities in the development of gastrointestinal dyskinesia and obsessive-compulsive disorder. Interstitial cells of Cajal: Inflammatory Bowel Disease 2007 Jun; 13 (6): 721-6 / Ordog T. Interstitial cells of Cajal in diabetic gastroenteropathy Neurogastroenterol Motil 2008 Jan; 20 (1): 8-18.).

Recently, transient receptor potential melastatin (TRPM) 7 has been reported to act as a non-selective cation channels (NSCCs) or a Ca ²⁺ -activated Cl ^- channel in the mouse small intestine ICC pacemaking was reported to play a ^{role, anoctamin 1 (ANO1) Cl -} channel is Ca ²⁺ -activated Cl ^- were reported to be the channel role. Therefore, TRPM7 or ANO1 is currently being targeted for the development of therapeutic agents for diseases of the digestive organs (Yang et al., Nature 455: 1210-1215, 2008; Zhu et al., J Physiol 2009; 587: 4905-4918 ; 2009).

Korean Patent No. 10-0500372 discloses a bicyclic benzamide of a novel substance 3- or 4-substituted 4- (aminomethyl) -piperidine derivative having gastrointestinal motility, Japanese Patent No. 10-0320771 discloses a preparation containing clevofred or a salt thereof which has swellability and a water-soluble polymer having an adhesive force to the gastrointestinal mucosa. However, related prior arts for improving the gastrointestinal motility disorder include gastrointestinal motility control effect There is a problem that side effects such as constipation and diarrhea occur.

Meanwhile, the main physiological and pharmacologically active ingredient of ginseng has been known to be ginseng saponin. However, in recent studies, the inventors of the present invention have found that the characteristics of ginseng are not a new ginsenoside but a new glycolipoprotein ), And the glycolipid protein was separated and purified from ginseng and named as gintonin (refer to Korean Patent No. 10-0973202). In addition, the present inventors have recently found that gin tonin is coupled to GTP-binding protein (G protein) via LPA receptor activation, particularly Gαq / 11 → phospholipase C → Inositol 1,4,5-trisphosphate (IP ₃ ) → Ca ²⁺ pathway → protein kinase C (PKC) activity leads to an increase in intracellular free calcium (Ca ²⁺ ) ²⁺ ] _i ), resulting in activation of calcium-dependent ion channels and exert various physiological and pharmacological effects (Nah, Curr Drug Targets. 213 (13): 1659-1664, 2012).

LPA receptors have been reported to be expressed in the digestive system as well as in the nervous system (Mori et al., Can J Physiol Pharmacol 78 (9): 729-736, 2000; Kraichely et al., Am J Physiol Gastrointest Liver Physiol 296 ): G833-839, 2009). However, the gastrointestinal efficacy of LPT receptor activity by gintonin is not yet known.

It is an object of the present invention to provide a new composition which is effective for preventing or treating gastrointestinal motility disorder disease without side effects and provides a new use of gingotin, a glycolipid protein isolated from ginseng.

In order to achieve the above object, the present invention provides a composition for preventing or treating gastrointestinal motility disorder disease containing gintonin as an active ingredient.

The gintonin is distinguished from ginseng, red ginseng, ginseng, white ginseng, cultivated ginseng, camellia ginseng or wild ginseng.

The composition is characterized by containing gin tonin in an amount of 0.0001 to 50% by weight based on the total weight of the composition.

The composition is characterized by increasing pacemaking activity of interstitial cells of Cajal.

The gastrointestinal motility disorder disorder is characterized by functional dyspepsia, irritable bowel syndrome, diabetic gastrointestinal motility disorder, gastrointestinal motility disorder caused by chemotherapy, constipation, intestinal obstruction or myotonic dystrophy.

The ginsenoside is characterized in that the constitutive protein is composed of ginseng major latex-like protein (MLP151) and ginseng ribonuclease-like major storage protein.

At this time, the ginseng major latex-like protein comprises the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and has three N-glycosylation sites Wherein the ginseng ribonuclease-like major storage protein comprises the amino acid sequence of SEQ ID NO: 6 to SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 11.

The present invention also provides a health functional food for preventing or ameliorating a gastrointestinal motility disorder disease containing gintonin as an active ingredient.

The gintonin is distinguished from ginseng, red ginseng, ginseng, white ginseng, cultivated ginseng, camellia ginseng or wild ginseng.

The health functional food is characterized by containing gin tonin in an amount of 0.0001 to 50% by weight based on the total weight of the health functional food.

The health functional food is characterized by increasing pacemaking activity of interstitial cells of Cajal.

The gastrointestinal motility disorder disorder is characterized by functional dyspepsia, irritable bowel syndrome, diabetic gastrointestinal motility disorder, gastrointestinal motility disorder caused by chemotherapy, constipation, intestinal obstruction or myotonic dystrophy.

The ginsenoside is characterized in that the constitutive protein is composed of ginseng major latex-like protein (MLP151) and ginseng ribonuclease-like major storage protein.

At this time, the ginseng major latex-like protein comprises the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and has three N-glycosylation sites Wherein the ginseng ribonuclease-like major storage protein comprises the amino acid sequence of SEQ ID NO: 6 to SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 11.

According to the composition of the present invention as described above, it is possible to provide a caraway epidermal cell, which is known to play an important role in intestinal motility and gastrointestinal motility and contractility in digestive organs, which contains ginseng- , It is possible to control the gastrointestinal motility disorder by preventing the delay of absorption of nutrients due to the decrease of intestinal motility due to diseases such as diabetes.

Figure 1 shows that treatment with gin tonin in ICC clusters of mice resulted in depolarization of pacemaking activity (A to D) and decrease of pacemaking amplitudes (E) by administration concentration (Bars represent means ± SEs. ** P <0.01, significantly different from the control.).
FIG. 2 shows that treatment of the LPA1 / 3 receptor antagonist Ki16425 before treatment with gin tonin in mouse ICC clusters blocked depolarization of pacemaking activity induced by gin tonin (A) and statistically significant inhibition (B) (Bars represent means ± SEs. ** P <0.01, significantly different from the control.).
Fig. 3 shows the pacemaking activity of mouse ICC clusters. When perfusion of GDPβS (1 mM) into the cells was carried out in the pipette, the depolarization of pacemaking activity induced by gin tonin was blocked (A) (B) (Bars represent means ± SEs. ** P <0.01, significantly different from the control.).
Figure 4 shows that the depolarization induction of pacemaking activity by gin tonin treatment in mouse ICC clusters is blocked by PLC inhibitor U73122 (A), PKC inhibitors Chelerythrine (D) and calphostin (E) (Bars represent means ± SEs. ** P <0.01, significantly different from the control.).
Figure 5 shows that depolarization of pacemaking activity by gin tonin treatment in mouse ICC clusters requires extracellular calcium (A) and intracellular calcium mobilization (B) (Bars represent means ± SEs. ** P <0.01, significantly different from the control.).
FIG. 6 shows that gin tonin treatment in mouse ICC clusters induces depolarization of pacemaking activity depending on the administration concentration. As a result, gin tonin has no effect on TRPM7 ion channel (A and B), ANO1 Cl ^- channel activation (C and D).
FIG. 7 shows that gutonin increases ITR by administration concentration in the intestinal transit rate of normal and STZ-induced diabetic mice, and particularly increases ITR to normal mouse level in STZ-induced diabetic mice (Significant difference (* p <0.05 and ** p <0.01) compared with control.
Figure 8 shows the signaling pathway for the action of inducing depolarization of pacemaking activity by gin tonin in mouse ICC clusters. Gentonin induces the process of LPA receptor activation → G protein → PLC activity → PKC activity → Ca ²⁺ → ANO1 channel activity → pacemaking activity depolarization → increase of intestinal smooth muscle roots → GI motility, Indicating that the movement delay can be prevented or remedied.
9 is a graph showing the results of Coomassie blue staining (lane 1: Molecular mark, lane 2; before extraction of methanol, lane 3, after extraction with methanol) after electrophoresis on components present inside and outside the dialysis membrane before and after methanol extraction of gintonin Dialysis solution, lane 4; dialyzer external dialysis solution after methanol extraction).

Hereinafter, the present invention will be described in detail.

The present inventors examined the effect of gentonin on the pacemaking activity of interstitial cells of cajal, which is known to play an important role in digestive motility, and the ion channel associated with pacemaking activity. As a result, pacemaking activity and related ANO1 Cl ^- channel.

The composition and the health functional food according to the present invention contain gintonin as an active ingredient to prevent or ameliorate intestinal dysfunction and increase intestinal motility, and particularly to prevent gastrointestinal motility disorder caused by delayed intestinal motility due to diseases such as diabetes, Prevention and treatment.

The gintonin has a molecular weight of about 67 kDa and is separated from roots, stems or leaves of ginseng, red ginseng, fresh ginseng, white ginseng, cultivated ginseng, camellia ginseng or wild ginseng, preferably 4 to 6 years old Panax ginseng CA Meyer It is preferable to use the red ginseng produced, but is not limited thereto.

The composition and the health functional food for gastrointestinal motility disorder disease according to the present invention contain gin tonin in an amount of 0.0001 to 50% by weight, preferably 0.01 to 50% by weight, based on the total weight, Parenteral or oral administration of 0.0001 to 100 mg / kg, preferably 0.1 to 100 mg / kg, more preferably 10 to 100 mg / kg per day is the most effective.

The ginsenoside according to the present invention comprises a ginseng major latex-like protein (MLP151) and a ginseng RNAse-like major storage protein.

The ginseng major latex-like protein (MLP151) comprises the amino acid sequence of SEQ ID NO: 1 to 4, the amino acid sequence of SEQ ID NO: 5, and has three N-glycosylation sites.

SEQ ID NO: 5;

mgltgklicq tgiksdgdvf helfgtrphh vp nit paniq gcdlhegefg kvgsvviw ny

s idgnamia k eeivaideed ks vtfkvveg hlfeefksiv fsvhvdtkge dnlvtwsidy

k kl nes vkdp tsyldfllsv trdieahhlp k

Herein, the underlined portion of the sequence corresponds to the peptide fragment (SEQ ID NOS: 1 to 4) obtained through the proteomic analysis, and the italic amino acid portion is the N-glycosylation site.

Also, the ginseng ribonuclease-like major storage protein comprises the amino acid sequence of SEQ ID NO: 6 to 10, and the amino acid sequence of SEQ ID NO: 11.

SEQ ID NO: 11;

mraiyiisvi ivslsifswg gna rsdypwa m falrlqwpa gfcevnnacd t ksllntfti

hglypynakg tpalycdgta fdvnsvsdfl aemhlawpsh etntediqfw ehewkkhgrc

seallkqtdy f rtalafrka fdivgllnqe giypnndlyr pkm ikeaik k hlnavpeidf

t kn enseyvl tdinvcvnqq atrfvdcptd datddyrlkf vrlpskmkfa dprtnsii

At this time, the underlined portion of the sequence corresponds to the peptide fragment (SEQ ID NOS: 6 to 10) obtained through the proteomic analysis.

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 embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Preparation of gin tonin

80-100% ethanol (w / v) or 16 L of methanol was added to 8 kg of 6-year old red ginseng ( Panax ginseng CA Meyer) purchased from Korea Ginseng Corporation (Daejeon, Korea) Next, the supernatant was recovered by vacuum filtration. This procedure was repeated three times. The supernatant was collected and concentrated under reduced pressure (1.3 kg of each of the ethanol / methanol extracts). To each of the ethanol and methanol extracts, a mixed solvent of water and n-butanol (1: v), and the n-butanol fraction in the fraction of water and n-butanol obtained was concentrated in vacuo to obtain 300 g of the crude saponin fraction (CGSF).

The grape saponin fraction was dissolved in 20 mM Tris-HCl (pH 8.2) or 20 mM Tris-HCl (pH 8.2) containing 50% ethanol or methanol and then DEAE sepharose CL-6B (GE Healthcare, Uppsala, Sweden) with 20 mM Tris-HCl (pH 8.2) or 20 mM Tris-HCl (pH 8.2) containing 50% ethanol or methanol Substances which are not bound to the column, that is, ginsenosides and other low-molecular substances which do not negatively charge are removed and then 50% ethanol or methanol Tris-HCl (pH 8.2) containing 1 M NaCl or Tris- HCl (pH 8.2) to recover the bound material to the anion exchange resin column.

The recovered material was poured into a dialysis membrane (pore size 6,000 ~ 8,000), dialyzed to remove salts, and lyophilized or vacuum concentrated to obtain 16 g of crude tonnone (yield: 0.20%).

Preparation Example 1. Cell preparation and cell culture

Male Balb / c mice weighing approximately 20 g were used. The mouse was sacrificed and then about 1 cm from the pyloric ring to the cecum was cut out first, followed by incision along the mecentric border and the intestinal contents were washed with Krebs-Ringer bicarbonate solution. After removing the intestinal mucosa, the tissues obtained were cultured in ICCs or single cells according to the method of Kim et al. (Kim et al., J Smooth Muscle Res 42: 1-7, 2006).

Preparation example 2. Patch-clamp experiment

The physiological solution for culturing ICCs clusters is as follows.

(Na ⁺ -Tyrode) (in mM): KCl 5, NaCl 135, CaCl ₂ 2, glucose 10, MgCl ₂ 1.2 and HEPES 10 (pH 7.4 adjusted with NaOH).

The pipette solution for measuring the pacemaking activity of the ICCs cluster is as follows.

(in mM): KCl 140, MgCl ₂ 5, K ₂ ATP 2.7, NaGTP 0.1, creatine phosphate disodium 2.5, HEPES 5 and EGTA 0.1 (pH 7.2 adjusted with KOH).

ICCs were performed in the following solutions for TRPM7 channel recording in single ICCs.

(in mM): KCl 2.8, NaCl 145, CaCl ₂ 2, glucose 10, MgCl ₂ 1.2 and HEPES 10 (pH 7.4 adjusted with NaOH).

The pipette included the following solutions.

(in mM): Cs-glutamate 145, NaCl 8, Cs-2-bis (2-aminophenoxy) -ethane- N, N, N =, N = -tetraacetic acid 10 , and HEPES-CsOH 10 (pH 7.2 adjusted with CsOH ).

For recording of Ca ²⁺ activated Cl ^- (ANO1 / TMEM16A) currents in single ICCs, ICCs were run in the following solutions.

(in mM): HCl 146, HEPES 10, glucose 10, MgCl ₂ 1, CaCl ₂ 1 and N-methyl-D-glucamine (NMDG) 150 (pH 7.4).

The pipette solution contained the following solutions.

NMDG-Cl 150, MgCl ₂ 1, MgATP 3, EGTA 10, CaCl ₂ 5 and HEPES 5 (pH 7.2, titrated with NMDG).

The calcium-free solution was run with 200 nM using WEBMAX-C software. Patch-clamp techniques were performed using the method of Kim et al., And the experiment was performed at 30 to 33 ° C (Kim et al., J Smooth Muscle Res 42: 1-7, 2006).

Preparation Example 3. Measurement of intestinal transit rate (ITR) using Evans blue

Intestinal transit was measured using Evans blue solution (5%, w / v, in DW) for intestinal propulsion.

First, 30 minutes after oral administration of gin tonin in normal mice, 0.1 ml of Evans blue solution was orally administered as gin tonin. Steptozotocin (STZ) -induced diabetic animal models were also used in this manner. Subsequently, the experimental animals were sacrificed 30 minutes after administration of Evans blue. The distance traveled by Evans blue (ITR) for 30 minutes was determined by measuring the distance from the pyloru to the farthest point of the field.

Intestinal transit was expressed as ITR (%), and all experiments were performed on the same day to minimize the statistical change of measurement date in ITR measurement (Lyu and Lee, Arch Pharm Res. 36 (5): 641-648 , 2013).

Preparation Example 4. STZ-induced diabetic model mouse

Five-week-old male ICR mice were randomly divided into two groups. That is, the control group and the STZ-induced diabetes-induced mouse group were divided. To induce diabetes, STZ (Sigma) was dissolved in 0.1 M ice-cold citrate buffer (pH 4.0) and injected into the abdominal cavity at 200 mg / kg in mice. Control group injected only buffer. Two months later, blood was collected from the tail vein of the animals and the animals were tested for blood glucose levels above 16 mM using a one-touch blood glucose monitoring system (Johnson & Johnson Medical Company).

Preparation Example 5. Drug Preparation

All drugs except Ki16425 (Cayman Chemicals) were purchased from Sigma.

Preparation Example 6. Experimental statistical analysis

Data were expressed as mean ± standard errors. Statistical significance was considered statistically significant when P -values were <0.05 using Student's t- test. n means the number of cells used in the experiment or the number of ICC clusters.

Experimental Example 1. Effect of gin tonin on pacemaking activity in cultured ICC clusters

The resting membrane potential of ICC clusters incubated at 30 ° C in current clamp mode showed an average of -51 ± 2 mV and the frequency of electrical pacing activity was -14 ± 2 cycles / min. The amplitude for electrical pacing activity was -18.3 ± 3 mV (n = 45). Gin tonin decreased the amplitude at 0.1 to 10 μg / ml and induced membrane depolarization of pacemaking activity in a concentration-dependent manner (FIGS. 1A-1C). Mean depolarizations were 3.8 ± 0.9 mV (n = 5), 8.1 ± 1.4 mV (n = 4), and 13.2 ± 0.8 mV (n = 6) at 1 μg / ml and 0.1 μg / 1D), and the corresponding amplitudes were 5.1 ± 1.3 mV (n = 5), 2.3 ± 0.5 mV (n = 4) at 1 μg / ml and 0.5 ± 1.5 mV at 10 μg / 0.3 mV (n = 6; Fig. 1E). These results indicate that gin tonin reduces the amplitude dependent on treatment concentration and stimulates depolarization to pacemaking activity.

EXPERIMENTAL EXAMPLE 2 In the pacemaking activity of cultured ICC clusters, the induction of depolarization by gin tonin was carried out through the LPA1 / 3 receptor

Gintonin has been shown to activate LPA receptors in cells expressing LPA receptors (Hwang et al., Mol Cells 33: 151-162, 2012). Therefore, we investigated the LPA receptor activity to induce depolarization of pacemaking activity by gintonin. First, Ki16425, a LPA1 / 3 receptor antagonist for the depolarization of pacemaking activity by gintonin, was examined, suggesting that Ki16425 blocked the action of gintonin on pacemaking activity (FIG. 2A). The mean depolarization was 0.2 ± 0.3 mV (Fig. 2B) in the presence of Ki16425. These results indicate that depolarization of pacemaking activity induced by gin tonin is mediated through the LPA1 / 3 receptor.

Experimental Example 3: Gastonin-induced depolarization of pacemaking activity in cultured ICC clusters was performed through GTP-binding protein activity

To study the role of GTP-binding proteins in signaling depolarization of pituitary-induced pacemaking activity, a non-hydrolysable guanosine 5-diphosphate analogue (GDPβS), known to permanently inactivate GTP- et al., Neurogastroenterol Motil 11: 311-338, 1999) were placed in patch pipettes and the action of gin tonin was examined. As a result, the action of gintonin was significantly lower than in the case of not including GDPss (Fig. 3A, n = 7). The average depolarization was 3.2 ± 0.8 mV when GDPβS was present. This suggests that the induction of depolarization of pituitary-induced pacemaking activity requires the activation of GTP-binding protein.

EXPERIMENTAL EXAMPLE 4 The gonotanin-induced apoptosis-inducing action on pacemaking activity in cultured ICC clusters was achieved through the activity of phospholipase C (PLC) and protien kinase C (PKC)

Because the action of gutonin on the pacing action of gin tonin is mediated through the activation of LPA receptors and GTP-binding proteins, we first investigated whether PLC activity is involved (Sakamoto et al., J Pharmacol Sci 100: 215-226, 2006). For this, the action of gintonin in the presence of the active PLC inhibitor U73122 (5 uM) was shown to inhibit the action of gintonin (Fig. 4A), i.e. depolarization to pacemaking activity by gintonin 1.1 ± 0.6 mV, whereas treatment with the inactive PLC inhibitor U73343 did not inhibit the depolarization induction of pacing action by gin tonin (FIGS. 4B and C).

In addition, both PKC inhibitors chelerythrine (1 μM) and calphostin (10 μM) both inhibited the action of gentonin, as a result of experimentation of gentonin-induced depolarization of ICCs pacemaking activity through PKC activity (Aiello et al., Am J Physiol 271: H109-119, 1996) (Figures 4D and E). In the presence of chelerythrine or calphostin, the depolarization rate of the pacemaking activity was reduced to 2.1 ± 1.9 mV or 2.2 ± 2.1 mV (n = 6, Fig. 4F). This shows that the action of gintonin acts in a manner that depends on PLC and PKC activity.

EXPERIMENTAL EXAMPLE 5 The induction of gonotinergic depolarization on pacemaking activity in cultured ICC clusters is dependent on intracellular calcium concentration

Intracellular inflow of calcium outside of cells is essential for pacmaking acivity activity and GI muscle contraction of ICCs. In addition, the activity of paccmaking acivity of ICCs requires intracellular calcium mobilization (Ward et al., J Physiol 525: 355-361, 2000). Non-dust conditions to remove calcium outside the cell to induce depolarization of the pacemaking activation of ICCs to test whether extracellular or intracellular calcium mobilization induced by the cells by (Ca ²⁺ -free condition) and Ca ²⁺ from the endoplasmic reticulum of the cell- ATPase inhibitor thapsigargin (Koh et al., J Physiol 540: 803-814, 2002; Choi et al., Mol Cells 27: 307-312, 2009). As a result, the induction of depolarization of pacemaking activity by gin tonin in the state of removing calcium was remarkably decreased as compared with the presence of calcium (Fig. 5A, n = 5). In addition, the action of gintonin was completely blocked when thapsigargin was present (Fig. 5B, n = 6). The mean depolarization of ICCs was 3.1 ± 1.1 mV when no calcium was present outside the cells and 1.1 ± 0.5 mV when thapsigagin was present (FIG. 5C). This suggests that mobilization of extracellular calcium and intracellular calcium by gintonin plays an important role in inducing depolarization by pacemaking activity activity.

EXPERIMENTAL EXAMPLE 6 The gonotanin-induced depolarization of pacemaking activity in cultured ICC clusters was induced by an increase in ANO1 channel currents

TRPM7 and ANO1 channels are known to be involved in pacemaking induction in ICC clusters (Huizinga et al., Gastroenterology 2002; 123: 1627-1636; Zhu et al., J Physiol 587: 4905-4918, 2009). In the present invention, it was experimentally determined which pathway was involved in the depolarization induction of pacemaking activity by gin tonin in a single ICC in the whole cell current measurement using the patch-clamp technique. When ramp pulses were applied from -100 mV to +100 mV for 2 s on the current-voltage curve, gin tonin showed no effect on a single ICC (Figs. 6A and B, n = 6). However, gin tonin in a single ICC has been shown to induce an inward current (Figures 6C and D). The inward current induced by gin tonin was blocked by the Cl ^- channel blocker NPPB (not shown). This shows that gin tonin activates the ANO1 Cl ^- channel present in ICCs.

Experimental Example 7. Gintonin Increases Intestinal Movement Rates (ITRs) in Normal and Diabetic Model Mice

ITRs (%) were measured 30 minutes after the administration of gentianin in mice that had induced diabetes with normal mice and STZ (FIG. 7). ITR was 63.1 ± 1.8% in mice not treated with gentonin in normal mice, but ITR was 63.3 ± 2.1, 64.2 ± 1.4 and 67.5 ± 1.5% when oral gentonin was administered at 10, 50 and 100 mg / kg (Fig. 7A, p < 0.05). Diabetes mellitus has been shown to reduce gastrointestinal motility by damaging the autonomic nervous system of the digestive system, thus delaying the digestive absorption of diabetic patients due to ingestion of food (Yin et al., 2002) G563-70, 2010). In diabetic mellitus, the effect of GI motility by gin tonin was confirmed. ITR was 53.3 ± 1.4% when treated with gentonin in STZ-diabetic mice (FIG. 7B), but oral administration of gentonin significantly increased GI motility in a dose-dependent manner . At concentrations of gin tonin treated mice, the mice did not exhibit any abnormal behavior or symptoms, suggesting that gin tonin is safe at the above oral dose.

Experimental Example 8. Determination of protein sequence of gin tonin

The lipid component was separated from the protein component of gintonin through methanol dialysis, and the protein band was cut out after electrophoresis and the composition of the band was analyzed by proteomic analysis method.

(1) Methanol dialysis of gintonin

After dissolving gintonin in ginseng (refer to Korean Patent Application No. 10-2010-0052913), 200 mg of gintonin was dissolved in 10 to 15 mL of 100% (w / v) methanol, and the dissolved gintonin was dissolved in the pore size: 6,000 ~ 8,000, Sigma-Aldrich, St. Louis, USA) and dialyzed with 30-50 volume of 100% methanol for 48 hours.

During dialysis, methanol was exchanged two times. After dialysis, inner dialysate and outer dialysate remaining in dialysis membrane were separately concentrated and dried.

(2) In-gel tryptic digestion carried out

The component (dialysis membrane internal dialysis solution) remaining in the obtained dialysis membrane was dried and then 100 μg / ml was obtained and subjected to 10% SDS-PAGE.

Fig. 9 shows the result of Coomassie blue staining after electrophoresis of gin tonin before and after methanol dialysis.

The gin tonin band before the methanol dialysis appears to be broad and not stained with the band (2 lane in FIG. 9), but after the methanol dialysis, the gin tonin electrophoresis was followed by Coomassie blue staining , The band spread widely without changing the molecular weight, but the dyeing was stained much stronger than before the methanol dialysis (3 lane in FIG. 9). On the other hand, the components (dialysate external dialysate) that exited outside the dialysis membrane after methanol dialysis were not stained with Kumasi blue (4 lane in FIG. 9). This suggests that the fat content of gintonin affects the protein staining by Kumashibul.

Coomassie blue staining was performed and the stained protein portion was cut from the gel and washed in a solution containing 25 mM ammonium bicarbonate and 50% (w / v) acetonitrile. The gel pieces were again placed in 40 mM ammonium bicarbonate, 10% (w / v) acetonitrile and 25 μg / ml trypsin (Promega, Madison, WI, USA) and incubated at 37 ° C. for 18-20 hours Lt; / RTI &gt;

Peptides digested by trypsin treatment were extracted twice with 50 mM ammonium bicarbonate, 50% acetonitrile and 5% (w / v) trifluoroacetic acid (TFA). The resulting peptide extracts were combined and lyophilized in a vacuum centrifuge and stored at 4 ° C prior to use.

(3) Ion-Mobility Spectrometry (IM-MS) Analysis

In-gel tryptic digestion Identification of protein components in hujintonin was carried out at the Korea Basic Science Research Institute in Seoul (Ono et al., Cell Proteomics Sci., 5, 1338-1347, 2006; Gao et al ., Mol cell Proteomics Sci 7, 2399-2409, 2008).

The lyophilized peptide was dissolved in 20 μl of 0.1% formic acid, and the mixture was subjected to nanoACQUITY ultrapressure liquid chromatography (UPLC) equipped with a NanoLockSpray ion source and SynapQ-TOF (Time of fight) mass And analyzed by LC / MS ^E using a mass spectrometer (Waters, Manchester, UK).

Specifically, the resuspended peptides (5 μl) were subjected to a Waters Symmetry C18 trapping column (180 μm id × 20 mm length) in a 0.1% formic acid solution for 5 minutes at a flow rate of 10 μl / with 5 ㎛ particle size). The peptides were eluted from the pre-column and eluted at 75 占 퐉 i.d. × 250 mm length column packed with BEH130 3 to 45% (w / v) linear gradient for 55 min under conditions of resin and 1.7 μm particle size (elution rate 280 nl / min) B (0.1% (w / v) formic acid in water and 0.1% (w / v) formic acid in acetonitrile). The column was washed at 90% (w / v) B for 25 minutes.

The used column was again allowed to parallel for 20 minutes at 3% B before the next experiment. The temperature of all the columns was adjusted to 35 ° C and the mass accuracy of the peptide [glu1] -fibrinopeptide delivered to the auxiliary pump of the NanoACQUITY NanoLockSpray maintained at 400 fmol / ㎛ and the flow rate of 500 nl / min B (fibrinopeptide B).

The peptides were analyzed in positive ion mode and allowed to operate in v-mode with a typical resolving power of 10,000 fwhm. Prior to the analysis, the TOF analyzer calibrated the standard [glu1] -fibrinopeptide B fragments obtained at 30 eV impingement energy and mass range 50 ~ 1990 m / z as standard. The Q-TOP was operated in the LC / MS ^E acquisition mode and the MS ^E mode was programmed to acquire data according to the appropriate dual exact mass protocol.

ProteinLynx Global SERVER version 2.4 (PLGS 2.4) integrated into ProteinLynx Global SERVER version 2.4 (PLGS 2.4) processes all raw data files obtained through analysis. Each processed data file is stored in an Apials protein database (Apiales) obtained from UniProt (www.uniprot.org, May 3, 2011 Released version, number of entries 3,213) protein database).

Identification of the protein from the low / high collision spectra of each sample indicates that at least one peptide matches three fragment ions, one protein matches seven fragment ions, and one protein matches two peptides (Hierarchical Approach).

As shown in Table 1, IM-MS analysis after tryptic digestion of the protein extracted from the protein band resulting from the electrophoresis of gin tonin revealed that two types of proteins contained in gin tonin.

Results Discussion.

(1) induction of depolarization for activation of pacemaking activity in mouse ICCs cluster by gin tonin

As shown in FIG. 1, when gin tonin was treated with ICC clusters, the amplitude of the pacemaking activity was decreased but it induced depolarization (FIG. 1A-C). In addition, gintonin was shown to increase depolarization of ICCs by the administration concentration but to decrease the amplitude (Figs. 1D and E), which stimulated typical pacemaking activity. These results show that gintonin induces depolarization of pancreatic activity of mouse small intestinal ICCs cluster and enhances intestinal motility.

(2) The induction of depolarization for the activation of pacemaking activity in mouse ICCs cluster by gin tonin was confirmed by the involvement of LPA receptor

It has been reported that gintonin contains lysophosphatidic acids (LPAs) as an active ingredient and activates cell membrane signaling through the activation of LPA receptors (Hwang et al., Mol Cells 33: 151-162, 2012). Experiments on the induction of depolarization for the activation of pacemaking activity in mouse ICCs cluster by gin tonin revealed that LPA1 / 3 receptor antagonist Ki16425 was pretreated and treated with gin tonin There was no effect on depolarization and amplitude change of pacemaking activity of ICCs cluster. This shows that LPA receptors are also present in the small intestine, and pacemaking activity activity of gintonin is mediated through the activation of LPA1 / 3 receptors present in the small intestine (Fig. 2).

(3) Induction of depolarization for the activation of pacemaking activity in mouse ICCs cluster by gin tonin revealed that GTP-binding protein was involved in LPA receptor signaling

The signaling activity of gin tonin through LPA receptor activity has been shown to involve GTP-binding proteins that are sensitive to pertussis toxins and GTP-binding proteins that are not sensitive to pertussis toxins (Hwang et al., Mol Cells 33: 151-162 , 2012). In order to confirm whether GTP-binding protein is involved in the depolarization induction for the activation of pacemaking activity in the ICCs cluster, the GTP-binding protein is inactivated by incorporating GDPβS inactivating GTP-binding protein into a patch pipette, The results showed that the action of gentonin was significantly inhibited by the activation of pacemaking activity in ICCs cluster. These results show that GTP-binding protein is involved in the activity of ICC clusters by gin tonin (Fig. 3).

(4) The induction of depolarization of pacemaking activity by gin tonin in mouse ICCs cluster confirmed PLC and PKC activity involved in LPA receptor signaling

The LPA receptor activity by gintonin has been reported to be linked to the activities of GTP-binding protein and phospholipase C (PLC) and protein kinase C (Hwang et al., Mol Cells 33: 151-162, 2012) ). We confirmed that the induction of depolarization for the activation of pacemaking activity was mediated by PLC and PKC activity in ICCs cluster by gentonin. First, U73122 inhibited the action of gintonin in the treatment of gin tonin after treatment with the active PLC inhibitor U73122 and the inactive PLC inhibitor U73343. However, U73343 did not have gin tonin inhibitory effect. In addition, chellythythrine and calphostin, which are known as inhibitors of PKC activity, inhibit gingontinin treatment before treatment with gin tonin, suggesting that gadolinin induces depolarization of ICCs cluster pacemaking activity by the action of gin tonin PLC and PKC (Fig. 4).

(5) Induction of depolarization for the activation of pacemaking activity by gin tonin in mouse ICCs cluster confirmed that the intracellular calcium concentration was involved in LPA receptor signaling

The induction of depolarization for the activation of pacemaking activity in mouse ICCs cluster by gentonin confirmed PLC and PKC activity involved in LPA receptor signal transduction. Since the signal transduction pathway through LPA receptor activation is ultimately related to changes in intracellular calcium concentration, induction of depolarization for the activation of pacemaking activity in ICCs cluster by gentonin induces PLC and PKC activity and intracellular calcium . First, we investigated the effect of gintonin on the depolarization induction for the activation of pacemaking activity in calcium - free solution, and found that the action of gintonin was significantly reduced. This suggests that calcium present outside the ICCs cluster is required to enter the ICCs in order for gingitonin action to occur (see Figures 5A and C). In addition, pretreatment of thapsigargin, which inhibits calcium release in the cytoplasm, inhibits the action of gin tonin, suggesting that depolarization induction for the activation of pacemaking activity in mouse ICCs cluster by gin tonin is mediated by intracellular calcium mobilization Also show the need for influx of calcium (Figures 5B and C).

(6) Identification of ion channel associated with induction of depolarization for activation of pacemaking activity in mouse ICCs cluster by gin tonin

The depolarization of the pacemaking activity of small intestinal ICC clusters has been reported to occur through the activation of two ion channels. One is TRPM7 ion channel and the other is ANO1 Cl ^- channel. It was confirmed that the intestinal activity stimulation by depolarization of pacemaking activity of small intestine ICCs clusters by gintonin is linked to ion channel activity. After isolating the single ICC and confirming the action of gin tonin, gin tonin activated the ANO1 Cl ^- channel. It is shown that the intracellular calcium increase in ICCs cluster by gin tonin is associated with calcium dependent ANO1 Cl ^- channel activity (Fig. 6).

(7) Effect of gin tonin on GI motility

Induction of depolarization for the activation of pacemaking activity in mouse ICCs cluster by gin tonin is linked to the depolarization of LPA receptor activity by gentonin → G protein → PLC activity → PKC activity → Ca ²⁺ → ANO1 channel activity → pacemaking activity Respectively. Because depolarization of pacing activity is associated with increased intestinal smooth muscle contraction and increased GI motility, we conducted experiments on the change of intestinal motility in diabetic state which is known to inhibit steady state and intestinal motility. As a result, gintonin induced a significant increase in GI motility at 100 mg / kg in normal mice (Fig. 7A). Interestingly, in the STZ-induced diabetes mellitus, GI motility was found to be less than in normal mice (Yin et al., Am J Physiol Gastrointest Liver Physiol. 298 (4): G563-70, 2010) In the diabetic group, administration of 100 mg / kg showed GI motility to the normal mouse level. This shows that the gin tonin efficacy is significantly increased in patients with a decrease in intestinal motility due to diseases such as diabetes (Fig. 7B).

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (16)

A composition containing gintonin as an active ingredient,
A composition for preventing or treating gastrointestinal motility disorder disease characterized by activating and depolarizing chlorine ion channels of a cell membrane to increase pacemaking activity of interstitial cells of Cajal.
The method according to claim 1,
Wherein the gintonin is isolated from ginseng, red ginseng, ginseng, white ginseng, cultivated ginseng, camellia ginseng or wild ginseng.
The method according to claim 1,
Wherein the composition contains gin tonin in an amount of 0.0001 to 50% by weight based on the total weight of the composition.
delete The method according to claim 1,
The composition for preventing or treating gastrointestinal motility disorders according to claim 1, wherein the gastrointestinal motility disorder disease is functional dyspepsia, irritable bowel syndrome, diabetic gastrointestinal motility disorder, gastrointestinal motility disorder caused by chemotherapy, constipation, intestinal obstruction or myotonic dystrophy .
The method according to claim 1,
Wherein the composition comprises ginseng major latex-like protein (MLP151) and ginseng ribonuclease-like major storage protein. A composition for preventing or treating a disease.
The method according to claim 6,
The ginseng major latex-like protein comprises the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and has three N-glycosylation sites Or a pharmaceutically acceptable salt thereof.
The method according to claim 6,
Wherein the ginseng ribonuclease pseudo-major storage protein comprises the amino acid sequence of SEQ ID NO: 6 to SEQ ID NO: 10, and the amino acid sequence of SEQ ID NO: 11.
It is a health functional food containing gintonin as an active ingredient,
Wherein the health functional food activates the chlorine ion channel of the cell membrane and depolarizes the cell membrane to increase pacemaking activity of interstitial cells of Cajal. Functional foods.
10. The method of claim 9,
Wherein the gintonin is isolated from ginseng, red ginseng, ginseng, white ginseng, cultivated ginseng, camellia ginseng or wild ginseng.
10. The method of claim 9,
Wherein the health functional food comprises 0.0001 to 50% by weight of gin tonin relative to the total weight of the health functional food, wherein the health functional food is for preventing or improving gastrointestinal motility disorders.
delete 10. The method of claim 9,
The gastrointestinal motility disorder disease is characterized in that the gastrointestinal motility disorder disease is functional dyspepsia, irritable bowel syndrome, diabetic gastrointestinal motility disorder, gastrointestinal motility disorder caused by chemotherapy, constipation, intestinal obstruction or myotonic dystrophy. Functional foods.
10. The method of claim 9,
Wherein the composition comprises ginseng major latex-like protein (MLP151) and ginseng ribonuclease-like major storage protein. Health functional food for prevention or improvement of disease.
15. The method of claim 14,
The ginseng major latex-like protein comprises the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and has three N-glycosylation sites A health functional food for preventing or improving gastrointestinal motility disorders.
15. The method of claim 14,
Wherein the ginseng ribonuclease pseudo-major storage protein comprises the amino acid sequence of SEQ ID NO: 6 to SEQ ID NO: 10, and the amino acid sequence of SEQ ID NO: 11.

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