KR20010039228A - Acidic polysaccharide fraction of ginseng root extract and The Process for Preparation Thereof - Google Patents
Acidic polysaccharide fraction of ginseng root extract and The Process for Preparation Thereof Download PDFInfo
- Publication number
- KR20010039228A KR20010039228A KR1019990047531A KR19990047531A KR20010039228A KR 20010039228 A KR20010039228 A KR 20010039228A KR 1019990047531 A KR1019990047531 A KR 1019990047531A KR 19990047531 A KR19990047531 A KR 19990047531A KR 20010039228 A KR20010039228 A KR 20010039228A
- Authority
- KR
- South Korea
- Prior art keywords
- acidic polysaccharide
- polysaccharide fraction
- ginseng
- acidic
- fractions
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Sustainable Development (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
본 발명은 위장질환 발병원인균으로 알려져 있는 헬리코박터의 위장내 점막세포 결합을 저해하는 인삼뿌리에서 분리된 산성다당류와 상기 산성다당류를 인삼에서 추출, 분리하는 제조방법에 관한 것이다.The present invention relates to an acidic polysaccharide isolated from a ginseng root inhibiting gastrointestinal mucosal cell binding of Helicobacter known as a pathogen causing gastrointestinal diseases and a method for extracting and separating the acidic polysaccharide from ginseng.
헬리코박터는 그램음성(Gram-negative) 박테리아로서 인체에 만성, 급성위염, 위궤양 및 위암 등을 유발하는 것으로 알려진 병원체이다. 산업사회의 경우 60세 이상의 인구 중 과반수 이상이 감염되어 있으며, 개발 도상국가에서는 대부분의 인구가 유년기부터 감염되어 있는 것으로 보고되어 있다. 2살부터 8살사이 유아의 경우 약 10%가 매년 감염되기 때문에 10대 청소년이 되면 거의 대부분이 감염되는 추세를 보이고 있다.Helicobacter is a Gram-negative bacterium known to cause chronic, acute gastritis, gastric ulcer and gastric cancer in the human body. In the industrial world, more than half of the population over 60 years of age is infected, and in developing countries, most of the population has been infected since childhood. About 10% of infants between the ages of 2 and 8 are infected every year, and most of the teenagers are infected.
이와 같은 헬리코박터는 위장내 점막상피세포에 결합하는데 그 결합기작이 주로 탄수화물과 이를 인식하는 박테리아 표면 단백질간의 상호작용에 의존하는 것으로 알려져 있고, 상기 인식 탄수화물로는 뮤신(mucin) 당단백질의 황산화당(sulfated carbohydrate), 사이알릴 락토스(sialyllactose)및 퓨코실 루이스 비 항원(fucosylated Lewis b antigen)등이 알려져 있다.Helicobacter binds to mucosal epithelial cells in the gastrointestinal tract, and its binding mechanism is known to depend mainly on the interaction between carbohydrates and bacterial surface proteins that recognize them, and the recognized carbohydrates include sulfated sugars of mucin glycoproteins. sulfated carbohydrate, sialyllactose, and fucosylated Lewis b antigen.
세포와 세포간 인식작용에 이용되어온 적혈구 세포의 표면은 위점막 상피세포와 같거나 비슷한 탄수화물 조성을 보유하고 있으며 헬리코박터와 결합함으로써 응집반응을 일으킨다. 대부분 박테리아 세포는 응집반응을 보이는데 이는 박테리아 표면에 존재하는 결합단백질이 적혈구와 같은 숙주세포 표면의 탄수화물을 인식함으로써 일어나는 반응이다.The surface of red blood cells, which have been used for cell-to-cell recognition, possesses the same or similar carbohydrate composition as gastric mucosal epithelial cells and causes aggregation by binding to Helicobacter. Most bacterial cells show agglutination, which occurs when the binding protein on the bacterial surface recognizes carbohydrates on the surface of host cells such as red blood cells.
이전에는 위염, 위궤양과 같은 위장 질환이 일차적으로 위산과다, 즉 과량의 위산 분비에 기인하며 그 원인으로 스트레스, 식품 및 유전적 요인 등에 의하여 위에 염증이 생긴다고 알려져 왔지만 최근에 와서 헬리코박터에 의하여 감염되었을 때 위장에 염증이 발생한다는 것이 정설로 되어 있다. 염증이 위장내 점막하층까지 퍼지게 되면 위궤양으로 발전하게 되고 위암으로 바로 진행되는 것은 아니지만, 헬리코박터에 감염되면 위암으로 진행될 가능성이 3∼5배 정도 높아지는 것으로 알려지고 있다.Previously, gastrointestinal diseases such as gastritis and gastric ulcer have been known to be primarily caused by excessive gastric acid secretion, ie, excessive gastric acid secretion, which causes inflammation of the stomach due to stress, food, and genetic factors. It is hypothesized that inflammation of the stomach occurs. When inflammation spreads to the submucosal layer of the stomach, it develops gastric ulcers and does not immediately progress to gastric cancer.
수년전까지 위장질환을 치료하기 위해서 제산제(antacid), 히스타민 수용체의 길항작용제 등이 사용되었을 뿐이었으며 미국 국립보건원은 90년대 중반에 와서야 헬리코박터와 위장질환과의 관계를 인정하여 테트라사이클린(tetracycline)이나 아목시실린(amoxicillin)과 같은 항생제의 사용을 다른 치료제와 병행하여 사용할 수 있도록 허용하였다.Antagonists and histamine antagonists have been used to treat gastrointestinal diseases many years ago. The National Institutes of Health recognized the relationship between Helicobacter and gastrointestinal diseases only in the mid-90s. The use of antibiotics such as or amoxicillin was allowed to be used in combination with other therapeutic agents.
그러나, 항생제의 사용에 따른 인체내의 축적, 부작용이 생길 뿐 아니라 이를 사용함으로 인하여 세포파괴가 일어나고 항생제에 내성을 갖는 박테리아의 출현때문에 궁극적으로는 위장질환을 치료할 수 있는 대체약품의 개발의 필요성이 대두되고 있으며 이에 따라 최근에 진행되고 있는 국내,외 위장질환 치료제 및 예방용 제재의 개발현황을 보면 면역시스템이나 항균제를 이용하거나, 박테리아 세포외 배출효소를 이용한 키트의 제조가 주류를 이루고 있으며 아직까지 헬리코박터의 세포인식에 관여하는 당배합체를 이용한 제재는 개발되고 있지 않다.However, due to the use of antibiotics, not only does accumulation and side effects occur in the human body, but also due to the use of them, cell destruction and the emergence of antibiotic-resistant bacteria ultimately necessitate the development of alternative drugs to treat gastrointestinal diseases. As a result, the development of domestic and foreign gastrointestinal disease treatment and prevention agents has recently been made. The production of a kit using an immune system or an antimicrobial agent or bacterial extracellular excretion enzyme is still mainstream. There is no development of a drug using a glycomer that is involved in cell recognition.
그리고, 종래에는 인삼에서 다당류 혹은 탄수화물을 포함하는 조추출물 분획으로 생리활성 측정이 이루어져 왔다. 따라서 분획 중에서 어느 특정 성분이 활성을 갖고 있는지에 대한 분자적 수준에서의 연구가 기술적 한계에 부딪쳐 왔지만 현재 개발되고 있는 탄수화물 공학적 기술을 이용하여 인삼에서 다당류의 분리, 정제, 동정 및 구조적 분석을 할 수 있게 되었다.In addition, conventionally, the biological activity has been measured as a crude extract fraction containing polysaccharide or carbohydrate in ginseng. Therefore, while molecular studies on which specific components in fractions have activity have hit technological limits, carbohydrate engineering techniques that are currently being developed can be used to isolate, purify, identify, and structurally analyze polysaccharides in ginseng. It became.
이에, 본 발명자들은 헬리코박터의 위장내 증식 및 활동을 저해하는 생리활성 물질을 천연자원에서 탐색하던 중 인삼내 생리활성 물질 중에서 수용성 단백질 및 펩타이드를 탐색하여 단백질과 강한 결합을 하고 있는 탄수화물인 산성 다당류 당배합체를 분리하게 되었고, 산성 다당류 당 배합체의 생리활성을 측정한 결과 millimolar 이하의 수준에서 헬리코박터의 적혈구 응집반응에 대하여 강한 억제효과를 갖는 다는 것을 확인함으로써 위장질환 예방 및 치료제로서 높은 가능성이 입증되어 본 발명을 완성하게 되었다.Accordingly, the present inventors searched for a bioactive substance that inhibits the growth and activity of Helicobacter in the natural resources, while searching for a water-soluble protein and peptide among the bioactive substances in ginseng, an acidic polysaccharide sugar saccharide that is a carbohydrate strongly bound to the protein. As a result of measuring the physiological activity of the acidic polysaccharide sugar compound, it was confirmed that it had a strong inhibitory effect on the hemagglutination reaction of Helicobacter at sub-millimolar levels, proving a high possibility as an agent for preventing and treating gastrointestinal diseases. The present invention has been completed.
따라서, 본 발명의 목적은 위장질환의 원인되는 헬리코박터의 위장내 증식 및 활동을 저해하는 유로닌산을 함유하는 인삼뿌리 추출물의 산성다당류 분획을 제공하는데 있다.Accordingly, it is an object of the present invention to provide an acidic polysaccharide fraction of ginseng root extract containing uronic acid that inhibits gastrointestinal proliferation and activity of Helicobacter causing gastrointestinal diseases.
본 발명의 다른 목적은 인삼뿌리로 부터 헬리코박터의 위장내 증식 및 활동을 저해하는 산성다당류 분획을 분리 정제하는 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for separating and purifying an acidic polysaccharide fraction that inhibits gastrointestinal proliferation and activity of Helicobacter from ginseng root.
본 발명의 상기 목적은 인삼뿌리를 증류수로 추출하고 이온교환 크로마토그래피로 생리활성 산성다당류를 분리하여 이의 헬리코박터 적혈구의 응집반응을 저해하는 활성을 측정함으로서 달성하였다.The above object of the present invention was achieved by extracting ginseng root with distilled water and measuring the activity of inhibiting the aggregation reaction of Helicobacter erythrocytes by separating physiologically active acid polysaccharides by ion exchange chromatography.
이하, 본 발명의 구체적인 구성과 작용을 단계별로 상세히 설명한다.Hereinafter, the specific configuration and operation of the present invention will be described in detail step by step.
도 1은 인삼으로부터 당배합체의 분리정제를 설명하는 도면1 is a diagram illustrating the separation and purification of sugar mixture from ginseng
도 2는 아세틸화 유도체 단당류의 가스크로마토그래피 유출프로필Figure 2 shows the gas chromatography outflow profile of acetylated derivative monosaccharides.
도 3은 갈락토스의 매스스펙트라 프로필Figure 3 is a mass spectra profile of galactose
도 4는 젤여과 크로마토그래피(실선:효소처리선, 점선:효소처리후)4 is gel filtration chromatography (solid line: enzyme treatment line, dotted line: after enzyme treatment)
도 5a는 산성다당류 분획의 헬리코박터 적혈구 응집반응 저해활성의 현미경사진Figure 5a is a micrograph of the Helicobacter erythrocyte aggregation inhibitory activity of the acidic polysaccharide fraction
도 5b는 펙티네이스 처리된 산성다당류분획의 헬리코박터 적혈구 응집반응 저해활성의 현미경사진Figure 5b is a micrograph of the Helicobacter erythrocyte aggregation inhibitory activity of pectinase treated acidic polysaccharide fraction
도 5c는 음성 응집반응(적혈구만 있을 때의 결과)을 나타낸 사진Figure 5c is a photograph showing the negative aggregation reaction (results with red blood cells only)
도 5d는 양성응집반응(적혈구와 헬리코박터가 같이 있을 때의 결과)을 나타낸 사진Figure 5d is a photograph showing a positive aggregation reaction (results of the combination of red blood cells and Helicobacter)
본 발명은 (1) 인삼 뿌리 부분을 증류수로 추출하는 단계와,The present invention (1) extracting the ginseng root portion with distilled water,
(2) 이온교환 크로마토그래피로 생리활성 산성다당류를 분리하는 단계,(2) separating the bioactive acid polysaccharide by ion exchange chromatography,
(3) 상기 산성다당류를 분석하는 단계,(3) analyzing the acidic polysaccharide,
(4) 그리고, 헬리코박터 적혈구의 응집반응을 저해하는 활성측정 단계로 이루어져 있다.(4) And it consists of the activity measurement step to inhibit the aggregation reaction of Helicobacter erythrocytes.
상기 (1)단계에서 사용되는 인삼의 뿌리는 인산(phosphate) 완충용액으로 균질화시킨 다음 원심분리하여 얻은 침전물을 증류수로 3번 추출한 후 상등액을 얻었다. 이경우 사용되는 상기 인산완충용액의 pH는 6∼8이고 바람직하게는 pH는 7.2이다.The root of ginseng used in step (1) was homogenized with a phosphate buffer solution, and the precipitate obtained by centrifugation was extracted three times with distilled water to obtain a supernatant. In this case, the pH of the phosphate buffer solution used is 6-8, preferably pH 7.2.
기존의 인삼 생리활성물질 추출은 주로 유기용매 추출법에 의존하고 있는데, 본원발명에서는 인삼의 생리활성 물질 탐색에 좀 더 실제적인 접근을 하기 위해서 60∼100℃의 증류수에 의하여 추출한다.Existing ginseng bioactive material extraction mainly depends on organic solvent extraction method, in the present invention is extracted by distilled water of 60 ~ 100 ℃ in order to take a more practical approach to search for bioactive substances of ginseng.
상기 (2)단계에서는 상기 (1)단계에서 추출된 상등액(불용성분획)을 모아 농축한 다음 증류수에 투석한 후 7배 부피의 에탄올로 침전시키고, 침전물을 인산 완충용액에 녹여 투석후 이온 교환 크로마토그래피를 이용하여 결합성 분획을 분리한다. 이경우에 있어서 사용되는 인산완충용액의 pH는 6∼8이고 바람직하게는 pH는 7.6이다.In step (2), the supernatant (insoluble component fraction) extracted in step (1) is collected, concentrated, and then dialyzed in distilled water and precipitated with 7 times the volume of ethanol, and the precipitate is dissolved in phosphate buffer solution and dialyzed after ion exchange chromatography. The binding fraction is separated using chromatography. The pH of the phosphate buffer solution used in this case is 6-8, Preferably pH is 7.6.
그리고, 산성다당류는 0∼1M 염화나트륨 용액 농도기울기로 유출하였으며 분리된 다당류는 투석후 동결건조한다. 본 발명의 산성다당류를 인삼에서 분리, 정제하는 과정은 도1에 나타난 바와 같다.In addition, the acidic polysaccharides flowed out at a concentration gradient of 0-1 M sodium chloride solution, and the separated polysaccharides were lyophilized after dialysis. The process of separating and purifying the acidic polysaccharide of the present invention from ginseng is as shown in FIG.
상기 침전물에서 인산 완충용액에 녹여 투석후 이온 교환 크로마토그래피를 이용하여 결합성 분획을 분리한 후 남은 비결합성 분획은 주로 중성다당류이며, 상기 결합성 분획에서는 산성다당류가 특히 활성이 있는 분획임을 확인하였다.After dissolving in the precipitate in phosphate buffer and dialysis to separate the binding fractions by ion exchange chromatography, the remaining non-binding fractions are mainly neutral polysaccharides, the acidic polysaccharides in the binding fractions was found to be particularly active fractions. .
상기 (3)단계에서는 본 발명의 효과를 나타내는 산성다당류를 분석하기 위해서 인삼에서 분리, 정제된 산성다당류를 구성하고 있는 단당류를 분석하였다. 2M 트라이플로로 초산(trifluoroacetic acid)에 녹여 밀폐된 유리관에서 질소기체하에서 반응시킨 다음 회전 농축기(rotary evaporator)로 농축시켜 가수분해 한후 표준시료와 함께 환원반응시킨후 환원된 다당류를 아세틸화반응시키고 농축하고 건조된 남은 물질은 클로로포름/물에 녹여 vortex 하고난 다음, 원심분리를 하여 유기용매층과 수용액층으로 분리하였다. 건조된 시료에 메탄올 염산을 첨가하여 밀폐된 유리관내 80℃에서 반응시킨 다음 진공상태에서 농축하고 건조되고 남은 물질을 다이클로로메탄(dichloromethane), 트라이플로로 초산(trifluoroacetic acid)에 의하여 트라이플르오로 아세틸화 반응을 수행하였다. 그리고, 가스 크로마토그래피를 이용하여 분석하였으며, 그 결과는 도2에 나타난 바와같다. 인삼에서 분리, 정제된 다당류의 동정은 매스 스펙트라(mass spectra)로 확인하였으며 그 결과는 도3에 나타난 바와 같다.In step (3), monosaccharides constituting the acidic polysaccharide isolated and purified from ginseng were analyzed in order to analyze the acidic polysaccharide showing the effect of the present invention. Dissolved in 2M trifluoroacetic acid (trifluoroacetic acid) and reacted in a closed glass tube under nitrogen gas, concentrated with a rotary evaporator, hydrolyzed and reduced with a standard sample. The remaining dried material was dissolved in chloroform / water, vortexed, and then centrifuged to separate the organic solvent layer and the aqueous layer. Methanol hydrochloric acid was added to the dried sample, and the reaction was carried out at 80 ° C. in a closed glass tube. The resultant was concentrated in vacuo, and the remaining material was dried by dichloromethane and trifluoroacetic acid. The reaction was carried out. And analyzed using gas chromatography, the results are as shown in FIG. Identification of polysaccharides isolated and purified from ginseng was confirmed by mass spectra, and the results are shown in FIG. 3.
그리고, 본 발명의 산성다당류의 항균성을 나타내는 특성을 보다 정확하게 분석하게 위해서 인삼에서 분리된 다른 당배합체 분획들(조추출물, 에탄올침전물, 중성다당류)과 구성성분을 비교하였으며 그 결과는 다음의 표 1과 같다.In addition, in order to more accurately analyze the antimicrobial properties of the acidic polysaccharides of the present invention, the components of different sugar copolymers (crude extract, ethanol precipitate, neutral polysaccharide) isolated from ginseng were compared and the results are shown in Table 1 below. Same as
상기 표1에 나타난 바와 같이, 본 발명의 산성다당류 분획은 유로닌산의 함량이 높을 뿐만 아니라 다른 당배합체 분획에 비하여 아라비노스(arabinose)와 갈락토스(galactose)의 함량이 매우 높았다. 이에 반하여 비결합성 분획에서 얻어진 중성다당류 분획은 유로닌산의 함량이 상대적으로 매우 적고, 단당류의 조성물도 대부분 포도당임을 확인하였다.As shown in Table 1, the acidic polysaccharide fraction of the present invention not only had a high content of uronic acid but also had a very high content of arabinose and galactose as compared to other sugar copolymer fractions. In contrast, the neutral polysaccharide fraction obtained from the non-binding fraction was found to have a relatively low content of uronic acid, and most of the monosaccharide composition was glucose.
그리고, 산성다당류는 유로닌산의 함량이 25∼35%으로 다른 다당류 분획에 비하여 유로닌산 함량이 제일 높은데 이는 헬리코박터의 숙주세포 인식을 저해하는 중요한 요인으로 판단된다.In addition, acidic polysaccharides have a content of uronic acid of 25-35%, the highest content of uronic acid compared to other polysaccharide fractions, which is considered to be an important factor that inhibits host cell recognition of Helicobacter.
즉, 유로닌산은 중성 다당류의 C6 알코올기 탄소가 카르복실기로 치환되어 중성 pH에서 마이너스전하를 띠고 있는 특징을 갖고있으며, 헬리코박터의 숙주세포에 결합하는 기작에 참가하는 탄수화물의 특성은 주로 황산화(sulfated) 탄수화물이나 특이적인 탄수화물으로 이중 황산화 탄수화물도 마이너스 전하를 띠고 있는 점을 알 수있다. 따라서 분리된 산성다당류가 유로닌산의 함량이 높은 점이 헬리코박터의 숙주세포 인식을 저해하는 중요한 요인이 되는 것으로 판단되었다.In other words, uronic acid is characterized by a neutral polysaccharide C6 alcohol carbon is substituted with a carboxyl group and has a negative charge at neutral pH, and the properties of carbohydrates participating in the mechanism of binding to host cells of Helicobacter are mainly sulfated. ) Carbohydrates and specific carbohydrates, the double sulfated carbohydrate also has a negative charge. Therefore, the high content of euronic acid in the isolated acidic polysaccharides was considered to be an important factor to inhibit the host cell recognition of Helicobacter.
상기 (4)단계는 본 발명의 산성다당류가 헬리코박터 적혈구의 응집반응의 저해 정도를 측정하는 것이며, 여러 분획 중에서도 본 발명의 산성다당류 분획은 0.25 mg/ml의 낮은 농도에서 저해 활성을 보임으로써 기존에 보고된 다당류들이 대부분 mg/ml 이상의 농도에서 저해활성을 보이는데 반하여 상대적으로 높은 활성을 지닌 것을 확인할 수 있었다.In step (4), the acidic polysaccharide of the present invention measures the degree of inhibition of the aggregation reaction of Helicobacter erythrocytes. Among the various fractions, the acidic polysaccharide fraction of the present invention exhibits inhibitory activity at a low concentration of 0.25 mg / ml. Most of the reported polysaccharides showed inhibitory activity at concentrations of mg / ml or higher, but relatively high activity.
따라서, 본 발명은 산성다당류를 유효성분으로 하여 위장병 치료제 내지 위장병 치료식이 등을 제조할 때 본 발명의 산성다당류가 0.25mg/ml이상의 양이 첨가되는 것이 바람직하다.Therefore, in the present invention, the acidic polysaccharide of the present invention is preferably added in an amount of 0.25 mg / ml or more when preparing a gastrointestinal therapeutic agent or gastrointestinal disease treatment diet and the like as an active ingredient.
그리고, 이외에도 위장병치료에 통상 첨가되는 성분들이 본 발명의 위장병 치료제에 첨가될 수 있으며, 본 발명의 위장병 치료식이에도 산성다당류외에 치료용식이에 통상적으로 첨가되는 성분들이 첨가될 수 있다.And, in addition to the components that are usually added to the treatment of gastrointestinal diseases can be added to the gastrointestinal treatment of the present invention, in addition to the acidic polysaccharides in the gastrointestinal disease treatment of the present invention may be added to the components commonly added to the therapeutic diet.
또한, 제약분야에서 조제시에 통상적으로 사용하는 적절한 부형제를 사용하여 정제, 캡슐제, 산제, 액제, 현탁제 등의 제형으로 이용될 수 있다.In addition, it can be used in the formulation of tablets, capsules, powders, solutions, suspensions and the like using appropriate excipients commonly used in the pharmaceutical field.
이하, 본 발명의 구체적인 구성 및 작용을 실시예에 의해 더욱 상세히 설명한다. 단, 하기의 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 권리범위가 이들에만 한정되는 것은 아니다.Hereinafter, the specific configuration and operation of the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.
실시예Example
재 료material
본 발명에 이용된 DEAE-Sepharose CL-6B 이온교환 수지는 아마샴-파마시아 바이오텍(Amersham Phamacia Biotech, Sweden)에서 구입하였으며, 효소 펙티네이스(pectinase), 트립신(trypsin), 글루구로닌산(glucuronic acid), 갈락튜로닌산(galacturonic acid), 람노스(rhamnose), 아라비노스(arabinose), 자일로스(xylose), 포도당(glucose), 갈라고오스(galactose) 등은 시그마(Sigma, USA)에서 구입하였다. 적혈구 응집반응에 이용된 AB형 적혈구는 조치원 내과에서 구입하여, 항응고제와 트립신효소 처리를 한 다음 이용하였다. 실험에 이용된 헬리코박터 파이로라이 박테리아는 10% 소혈청, 브루셀라(Brucella) 배지에서 37℃, 12% 이산화탄소 조건에서 72시간 배양하였으며, 인산 완충용액을 이용하였다.DEAE-Sepharose CL-6B ion exchange resin used in the present invention was purchased from Amersham Phamacia Biotech, Sweden, enzyme pectinase, trypsin, glucuronic acid Galacturonic acid, rhamnose, arabinose, albino, xylose, glucose, galactose and the like were purchased from Sigma, USA. Type AB erythrocytes used for erythrocyte agglutination were purchased from Jochiwon Internal Medicine, and treated with anticoagulants and trypsin enzymes. Helicobacter pyrolai bacteria used in the experiment was incubated for 72 hours at 37 ℃, 12% carbon dioxide conditions in 10% bovine serum, Brucella (Brucella) medium, using a phosphate buffer solution.
단백질 및 탄수화물 정량방법Protein and Carbohydrate Determination
전체 탄수화물, 유로닌산(uronic acid) 및 단백질의 양은 페놀-황산방법, 카바졸(carbazole) 및 로우리(Lowry) 방법을 각각 포도당, 갈락튜로닌산 (galacturonic acid), 소혈청 알부민을 표준으로 사용하여 결정하였다.Total carbohydrate, uronic acid, and protein levels were measured using glucose, galacturonic acid, and bovine serum albumin as standard, respectively, using the phenol-sulfuric acid method, the carbazole, and Lowry methods. Decided.
실시예 1 : 산성다당류의 분리Example 1 Separation of Acidic Polysaccharides
산성 다당류는 6년근 인삼의 뿌리를 50mM 인산(phosphate) 완충용액(pH 7.2)으로 균질화시킨 다음 원심분리하여 얻은 침전물을 뜨거운 증류수로 3번 추출한 후 상등액을 얻은 후 이들 상등액을 모아 농축한 다음 증류수에 투석한 후 7배 부피의 에탄올로 침전시키고, 이를 최소부피의 10mM 인산 완충용액 (pH 7.5)에 녹여 투석후 이온 교환 크로마토그래피를 이용하여 분리하였다. 산성다당류는 이 때 0∼1M 염화나트륨 농도기울기로 유출하였고, 분리된 다당류는 투석후 동결건조하였다. 비결합성 분획은 주로 중성다당류로 이루어져 있음을 확인하였고, 결합성 분획에서는 산성다당류, 특히 활성이 있는 분획임을 확인하였다.Acidic polysaccharide is homogenized the root of 6-year-old ginseng with 50mM phosphate buffer (pH 7.2), centrifuged and the precipitate obtained is extracted three times with hot distilled water, and then the supernatant is collected. After dialysis, the precipitate was precipitated with 7 times the volume of ethanol, which was dissolved in a minimum volume of 10 mM phosphate buffer (pH 7.5), and separated by dialysis and ion exchange chromatography. At this time, the acidic polysaccharides flowed out at a concentration of 0-1 M sodium chloride, and the separated polysaccharides were lyophilized after dialysis. It was confirmed that the non-binding fraction was mainly composed of neutral polysaccharides, and the binding fraction was an acidic polysaccharide, particularly an active fraction.
실시예 2 : 다당류 분석Example 2 Polysaccharide Analysis
가) 가수분해A) hydrolysis
2M 트라이플로로 초산(trifluoroacetic acid)에 시료를 녹여 밀폐된 유리관에서 질소기체하에서 반응시킨 다음 회전 농축기(rotary evaporator)로 농축하여 가수분해시켰다.The sample was dissolved in trifluoroacetic acid with 2M trifluoro, reacted under a nitrogen gas in a closed glass tube, and then concentrated by a rotary evaporator to hydrolyze.
나) 환원반응B) reduction reaction
시료와 표준시료를 0.05M 소듐하이드록사이드(NaOH)와 소듐보로하이드라이드 (NaBH4)에 각각 가하고 실온에서 환원반응시켰다. 환원 반응후 남은 소듐보로하이드라이드 물질은 빙초산으로 제거하였다.The sample and the standard sample were added to 0.05M sodium hydroxide (NaOH) and sodium borohydride (NaBH 4 ), respectively, and reduced at room temperature. The sodium borohydride material remaining after the reduction reaction was removed with glacial acetic acid.
다) 아세틸화 반응C) acetylation reaction
환원된 다당류는 피리딘(pyridine), 아세틱 안하이드라이드(acetic anhydride)를 가하여 아세틸화 시켰다. 실온에서 시료가 건조상태가 될 때까지 농축하고, 톨루엔 용액에 첨가하여 완전히 증발할 때까지 농축하였다. 건조된 남은 물질은 클로로포름/물에 녹여 vortex 하고난 다음, 1,000g에서 원심분리를 하여 유기용매층과 수용액층으로 분리하였다. 유기용매층을 진공에서 다시 농축하여 다음 실험에 사용하였다. 건조된 시료와 표준시료에 메탄올 염산을 첨가하여 밀폐된 유리관내 80℃에서 반응시킨 다음 진공상태에서 농축하였다.The reduced polysaccharide was acetylated by adding pyridine and acetic anhydride. At room temperature the sample was concentrated to dryness, added to toluene solution and concentrated until complete evaporation. The remaining material was dissolved in chloroform / water, vortexed, and then centrifuged at 1,000 g to separate the organic solvent layer and the aqueous layer. The organic solvent layer was concentrated again in vacuo and used for the next experiment. Methanol hydrochloric acid was added to the dried sample and the standard sample, and the resultant was reacted at 80 DEG C in a closed glass tube, and then concentrated in vacuo.
라) 트라이플로로 아세틸화 반응(trifluoroacetylation)D) trifluoroacetylation
농축후 건조되고 남은 물질은 다이클로로메탄(dichloromethane), 트라이플로로 초산(trifluoroacetic acid)에 의하여 트라이플르오로 아세틸화 반응을 수행하였다.After drying, the remaining material was subjected to acetylation with trifluoro by dichloromethane and trifluoroacetic acid.
마) 가스 크로마토그래피에 의한 분석E) analysis by gas chromatography
시료는 가스 크로마토그래피를 이용하여 측정하였다. 시료는 CBP5 25 미터 융합 실리카 미세관 컬럼(fused silica capillary column)에 가하고. 다당류 유도체를 프래임-이온 측정기(flame-ionization detector)을 이용하여 측정하였다. 오븐온도는 처음 온도 140℃ 그리고 점차 증가하여 분당 4℃의 기울기로 270℃까지 높여 사용하였으며 그 결과는 도2에 나타난 바와 같았다.Samples were measured using gas chromatography. Samples were added to a CBP5 25 meter fused silica capillary column. Polysaccharide derivatives were measured using a flame-ionization detector. The oven temperature was initially increased to 140 ° C. and gradually increased to 270 ° C. with a slope of 4 ° C. per minute. The results were as shown in FIG. 2.
사) 매스 스펙트라 측정(mass spectra)G) mass spectra measurement;
다당류의 동정은 매스 스펙트라로 동정 확인하였으며 그 결과는 도3에 나타난 바와 같았다.Identification of polysaccharides was confirmed by mass spectra and the results were as shown in FIG.
실시예 3 : 적혈구 응집반응Example 3 Hemagglutination
배양된 헬리코박터를 인산 완충용액으로 묽게 만든후 동량의 적혈구 용액과 혼합하여 실온에서 30분간 반응시켰으며, 이를 양성 적혈구 응집반응으로 하였다. 분리된 당배합체 분획들을 이용하여 응집반응에 대한 저해활성은 우선 헬리코박터 용액과 당배합체 용액을 혼합하여 15분간 반응시켰다. 다시 적혈구 용액을 혼합하여 2시간 동안 반응시킨 후 응집반응을 측정하였으며, 반응 측정은 현미경을 이용하였으며 그 결과는 다음의 표2와 같다.The cultured Helicobacter was diluted with phosphate buffer solution, mixed with the same amount of red blood cell solution, and reacted for 30 minutes at room temperature. The inhibitory activity for the aggregation reaction using the separated sugar copolymer fractions was first reacted for 15 minutes by mixing the Helicobacter solution and the sugar copolymer solution. Again, the red blood cell solution was mixed and reacted for 2 hours, and then the aggregation reaction was measured. The reaction was measured using a microscope, and the results are shown in Table 2 below.
상기 표 2에서 보는 바와 같이 여러 분획 중에서도 산성다당류 분획은 0.25 mg/ml의 낮은 농도에서 저해 활성을 보임으로써 기존에 기 보고된 다당류들이 대부분 mg/ml 이상의 농도에서 저해활성을 보이는데 반하여 상대적으로 높은 활성을 지닌 것을 확인할 수 있었다.As shown in Table 2, among the various fractions, the acidic polysaccharide fraction showed an inhibitory activity at a low concentration of 0.25 mg / ml, whereas most of the previously reported polysaccharides showed inhibitory activity at a concentration of mg / ml or more, and relatively high activity. It could be confirmed that with.
또한, 산성다당류 분획은 표 1에서 보인 바와 같이 일부 단백질을 포함하기 때문에 응집저해 활성이 단백질에서 비롯되었을 가능성을 배제할 수 없었다. 따라서 펙티네이스(pectinase) 효소를 처리한 다음 시료의 일부는 슈퍼덱스 75고압액체 크로마토그래피를 사용하여 젤 여과 크로마토그래피를 수행하여 작은 분자량의 올리고당을 얻었으며(제 4도), 시료의 다른 일부는 투석후 저해활성을 측정하였다. 결과적으로 효소처리된 산성다당류의 두 경우 모두 저해활성이 없다는 것을 도 5a 내지 도 5d를 통해서 확인하였다. 이는 단백질이 아닌 산성 다당류가 응집반응에 대한 저해활성을 보유하고 있다는 것을 증명하는 것이다.In addition, since the acidic polysaccharide fraction includes some proteins as shown in Table 1, the possibility of aggregation inhibitory activity originated from the protein could not be excluded. Therefore, after treating the pectinase enzyme, a part of the sample was subjected to gel filtration chromatography using Superdex 75 high pressure liquid chromatography to obtain a small molecular weight oligosaccharide (FIG. 4). Inhibitory activity after dialysis was measured. As a result, it was confirmed in FIGS. 5A to 5D that both of the enzyme-treated acidic polysaccharides had no inhibitory activity. This proves that acidic polysaccharides, not proteins, have inhibitory activity against aggregation.
이상 발명의 상세한 설명에서 확인할 수 있는 바와 같이, 본 발명의 인삼뿌리 추출물의 산성다당류는 위장질환을 일으키는 헬리코박터의 위장내 점막세포 결합을 저해하여 위장질환을 예방하고 이를 치료하는 우수한 효과가 있으며,As can be seen in the detailed description of the present invention, the acidic polysaccharide of the ginseng root extract of the present invention has an excellent effect of preventing and treating gastrointestinal diseases by inhibiting gastrointestinal mucosa binding of Helicobacter causing gastrointestinal diseases,
본 발명의 인삼뿌리 추출물의 산성다당류를 포함하는 조성물은 제약분야에서 공지된 방법에 의해서 조제되어 위장질환 치료제로서 사용될 수 있을 뿐만 아니라 위장질환의 치료보조제로 사용될 수 있고 식품에 첨가되어 건강식품이나 위장질환의 치료식이로 사용될 수 있다는 효과가 있다.The composition comprising the acidic polysaccharide of the ginseng root extract of the present invention can be used as a therapeutic agent for gastrointestinal disorders as well as prepared by a method known in the pharmaceutical field and can be used as a therapeutic aid for gastrointestinal disorders and is added to foods for health food or stomach There is an effect that can be used as a therapeutic diet of the disease.
그리고, 기존의 항생제나 화학의약품의 경우 부작용 뿐만 아니라 개발기술이 세포 파괴적인 반면, 본 발명은 숙주세포 표면의 탄수화물 인식 및 상호작용을 저해하는 신물질을 이용하여 세포간 결합을 저해하여 위장 밖으로 유출시켜 부작용을 최소화하고 위염증을 최소화하는 비파괴적 기술이라는 점에서 매우 중요한 미래지향적인 의약품의 의미를 갖고 있다. 뿐만 아니라 향후에는 인체내 세균의 침입에 따른 감염을 예방 혹은 치료하는데 본 발명의 당배합체를 이용할 것이므로 넓은 시장성을 보유하고 있다는 효과를 가지므로 본 발명은 의약산업 및 건강식품산업상 매우 유용한 발명인 것이다.In addition, in the case of conventional antibiotics or chemical drugs, as well as side effects as well as development technology is cell-destructive, the present invention inhibits intercellular binding by using a novel substance that inhibits carbohydrate recognition and interaction on the surface of the host cell to leak out of the stomach It is a future-oriented medicine that is very important in that it is a non-destructive technology that minimizes side effects and minimizes gastritis. In addition, the present invention is very useful invention in the pharmaceutical industry and health food industry because it will have the effect of having a wide marketability because the sugar mixture of the present invention will be used in the future to prevent or treat infection caused by the invasion of bacteria in the human body.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019990047531A KR100330138B1 (en) | 1999-10-29 | 1999-10-29 | The acidic polysaccaride extracted from ginseng root with inhibition of Helicobacter pylori-cell adhesion and a composition for the use of preventive and therapeutic againt a gastroenteric disorder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019990047531A KR100330138B1 (en) | 1999-10-29 | 1999-10-29 | The acidic polysaccaride extracted from ginseng root with inhibition of Helicobacter pylori-cell adhesion and a composition for the use of preventive and therapeutic againt a gastroenteric disorder |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20010039228A true KR20010039228A (en) | 2001-05-15 |
| KR100330138B1 KR100330138B1 (en) | 2002-03-27 |
Family
ID=19617663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1019990047531A KR100330138B1 (en) | 1999-10-29 | 1999-10-29 | The acidic polysaccaride extracted from ginseng root with inhibition of Helicobacter pylori-cell adhesion and a composition for the use of preventive and therapeutic againt a gastroenteric disorder |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100330138B1 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100415591B1 (en) * | 2001-03-08 | 2004-01-16 | 남양유업 주식회사 | Yoghurt formular to inhibit Helicobacter pylori growth in gastrointestinal tract |
| KR100423521B1 (en) * | 2001-08-29 | 2004-03-18 | 네비온 주식회사 | Cosmetic compositions containing panax ginseng polysaccharides |
| KR20040046382A (en) * | 2002-11-27 | 2004-06-05 | (주)서림식품 | Method of separating the polysaccharide fractions from ginseng residues |
| KR20040081932A (en) * | 2003-03-17 | 2004-09-23 | 설혜영 | Quick-Extraction & Condensed Liquid Enlargement from the steamed Red |
| KR100797016B1 (en) * | 2007-01-25 | 2008-01-22 | 주식회사 코인텍 | Purified substances of panax ginseng polysaccharides high-concentrate having immunostimulating activity, and the manufacturing method |
| KR101042903B1 (en) * | 2004-12-03 | 2011-06-20 | 주식회사 대우일렉트로닉스 | Hinge structure united door switch for the refrigerator |
| KR101434471B1 (en) * | 2012-10-24 | 2014-08-27 | (주)에스.앤.디 | The preventing gastritis and cancer composion containing natural extract, and therof manufacturing method |
| CN115651088A (en) * | 2022-08-23 | 2023-01-31 | 辽宁成大生物股份有限公司 | Preparation method and application of ginseng total polysaccharide, ginseng total polysaccharide vaccine adjuvant and vaccine composition thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100379189B1 (en) * | 2000-12-14 | 2003-04-08 | 학교법인고려중앙학원 | Agents for gastroenteric disorder by Helicobacter pylori and screening method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR970008129B1 (en) * | 1993-11-05 | 1997-05-21 | 박명규 | Method for extracting acid polysaccharide from ginseng |
-
1999
- 1999-10-29 KR KR1019990047531A patent/KR100330138B1/en active IP Right Grant
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100415591B1 (en) * | 2001-03-08 | 2004-01-16 | 남양유업 주식회사 | Yoghurt formular to inhibit Helicobacter pylori growth in gastrointestinal tract |
| KR100423521B1 (en) * | 2001-08-29 | 2004-03-18 | 네비온 주식회사 | Cosmetic compositions containing panax ginseng polysaccharides |
| KR20040046382A (en) * | 2002-11-27 | 2004-06-05 | (주)서림식품 | Method of separating the polysaccharide fractions from ginseng residues |
| KR20040081932A (en) * | 2003-03-17 | 2004-09-23 | 설혜영 | Quick-Extraction & Condensed Liquid Enlargement from the steamed Red |
| KR101042903B1 (en) * | 2004-12-03 | 2011-06-20 | 주식회사 대우일렉트로닉스 | Hinge structure united door switch for the refrigerator |
| KR100797016B1 (en) * | 2007-01-25 | 2008-01-22 | 주식회사 코인텍 | Purified substances of panax ginseng polysaccharides high-concentrate having immunostimulating activity, and the manufacturing method |
| KR101434471B1 (en) * | 2012-10-24 | 2014-08-27 | (주)에스.앤.디 | The preventing gastritis and cancer composion containing natural extract, and therof manufacturing method |
| CN115651088A (en) * | 2022-08-23 | 2023-01-31 | 辽宁成大生物股份有限公司 | Preparation method and application of ginseng total polysaccharide, ginseng total polysaccharide vaccine adjuvant and vaccine composition thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100330138B1 (en) | 2002-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jin et al. | Structure characterization of a polysaccharide extracted from noni (Morinda citrifolia L.) and its protective effect against DSS-induced bowel disease in mice | |
| KR100602841B1 (en) | Acidic polysaccharide with cell-binding inhibition activity isolated from green tea and its composition for preventing and treating gastrointestinal diseases | |
| Scoparo et al. | Chemical characterization of heteropolysaccharides from green and black teas (Camellia sinensis) and their anti-ulcer effect | |
| KR100330138B1 (en) | The acidic polysaccaride extracted from ginseng root with inhibition of Helicobacter pylori-cell adhesion and a composition for the use of preventive and therapeutic againt a gastroenteric disorder | |
| US4855284A (en) | Calcium and magnesium complexes of phytohemagglutinin-polyheteroglycans, their preparation and pharmaceutical formulations | |
| CN103382229B (en) | A kind of preparation method and Structural Identification with the novel SEP-1 of immunoregulation effect | |
| JPH0539305A (en) | Immuno suppressive polysaccharide extracted from astragalus membranaceous and pharma- ceutical composition containing same | |
| Zhang et al. | A glycoprotein from mountain cultivated ginseng: Insights into their chemical characteristics and intracellular antioxidant activity | |
| CN101306012A (en) | Use of modified polysaccharide rich in mannose in preparing medicine for preventing the colitis cancer | |
| CN115414379B (en) | Application of notoginseng polysaccharide SQP20 in preparation of medicine for treating intestinal injury and inflammatory infiltration | |
| CN101862349B (en) | Effective part of centipede and application thereof | |
| Ingólfsdóttir | Bioactive compounds from Iceland moss | |
| KR100379189B1 (en) | Agents for gastroenteric disorder by Helicobacter pylori and screening method thereof | |
| CN105770869B (en) | Pharmaceutical composition for treating psoriasis | |
| JP2005526142A (en) | Preparation and standardization of immunomodulating peptide-binding glucan with verifiable oral absorption obtained from Coriolis versatile | |
| Hui et al. | Gut microbiome-based thiamine metabolism contributes to the protective effect of one acidic polysaccharide from Selaginella uncinata (Desv.) Spring against inflammatory bowel disease | |
| EP2905026B1 (en) | Peyer's patch activator | |
| KR100573631B1 (en) | Acidic polysaccharide extracted from ginseng root and its composition for the use of preventive and therapeutic agents against periodontal diseases | |
| KR100468649B1 (en) | Anti-complementary biopolymer from fruiting body of Coriolus versicolor and method for isolation thereof | |
| KR100630479B1 (en) | Composition for inhibition of Helicobacter pyroli infection containing acharan sulfate as an active component | |
| Lin et al. | Pharmacological bioactivity of enzymatically bio-transformed ginsenosides | |
| CN114316081B (en) | A sulfated polysaccharide of Botrytis longipedicularis with SARS-CoV-2 inhibiting activity, and its preparation method and application | |
| US7015313B2 (en) | Lectin protein prepared from Korean marine crab Philyra pisum, process for preparing the same and the use thereof | |
| CN111647095B (en) | Polysaccharide of fraxinus chinensis, preparation method and application thereof | |
| CN113476497B (en) | Prunella vulgaris extract with HSV (herpes Simplex Virus) resisting activity as well as preparation method and medical application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20130123 Year of fee payment: 12 |
|
| FPAY | Annual fee payment |
Payment date: 20140312 Year of fee payment: 13 |
|
| FPAY | Annual fee payment |
Payment date: 20160311 Year of fee payment: 15 |
|
| FPAY | Annual fee payment |
Payment date: 20170107 Year of fee payment: 16 |
|
| FPAY | Annual fee payment |
Payment date: 20180313 Year of fee payment: 17 |
|
| FPAY | Annual fee payment |
Payment date: 20190302 Year of fee payment: 18 |