KR20210040679A - Composition for hard tissue formation and, dentin or pulp regeneration containing compounds isolated from Eucommia ulmoides extract - Google Patents
Composition for hard tissue formation and, dentin or pulp regeneration containing compounds isolated from Eucommia ulmoides extract Download PDFInfo
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- KR20210040679A KR20210040679A KR1020190123251A KR20190123251A KR20210040679A KR 20210040679 A KR20210040679 A KR 20210040679A KR 1020190123251 A KR1020190123251 A KR 1020190123251A KR 20190123251 A KR20190123251 A KR 20190123251A KR 20210040679 A KR20210040679 A KR 20210040679A
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- South Korea
- Prior art keywords
- dentin
- geniposide
- composition
- pulp
- present
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
본 발명은 두충 추출물로부터 분리한 화합물을 포함하는 상아질 또는 치수조직 재생용 조성물에 관한 것으로 보다 상세하게는 제니포사이드 또는 제니포시딕산을 포함하는 치수줄기세포의 상아 전구세포로의 분화용 조성물에 관한 것이다.The present invention relates to a composition for regeneration of dentin or pulp tissue comprising a compound isolated from a cephalopathy extract, and more particularly, to a composition for differentiation of pulp stem cells comprising geniposide or genifosidic acid into dentin progenitor cells. .
충치, 즉 치아우식증은 플랙(치면세균막)내의 세균(S. mutans)이 배설하는 산에 의해서 치아 표면의 칼슘과 인 같은 무기질이 빠져나가고 그 속의 단백질과 같은 유기질이 용해되어 결국 치아의 파괴 현상을 초래하는 증상을 말한다. 이러한 산은 플랙(치면세균막)의 수소이온농도를 pH 4.0 내지 4.5까지 낮출 수 있다. 일반적으로 수소이온농도지수가 pH 5.0~5.5 정도 이하이면 치면의 탈회(Demineralization)가 일어나게 되는데 치면에 부착된 플랙의 세균이 배설하는 산에 의해 치면의 탈회가 일어나는 현상을 치아우식이라고 한다. 치아우식 발생에 작용하는 인자는 환자 자신의요인(치아의 위치, 형태, 타액의 양과 점조도, 유전, 질병, 임신), 세균요인(구강내 산성세균의 종류, 양, 활동성 등) 및 환경요인(구강 위생 상태, 음식의 종류, 식수의 불소농도 등) 이 3가지가 합쳐지는 곳에서 치아우식이 발생하며 여기에 시간 요인이 합쳐져서 치아우식이 진행되게 된다. Caries, or dental caries, is caused by the acid excreted by bacteria (S. mutans) in the flack (tooth surface bacteria), and minerals such as calcium and phosphorus on the tooth surface are removed, and organic substances such as proteins in the tooth are dissolved, resulting in the destruction of teeth. It refers to the symptoms that cause it. Such an acid can lower the hydrogen ion concentration of the flak (tooth surface bacteria film) to a pH of 4.0 to 4.5. In general, if the pH of the hydrogen ion concentration index is less than 5.0~5.5, demineralization of the tooth surface occurs, but the phenomenon that the demineralization of the tooth surface occurs due to the acid excreted by the bacteria of the flack attached to the tooth surface is called dental caries. Factors influencing the occurrence of dental caries include the patient's own factors (position, shape, amount and consistency of saliva, inheritance, disease, pregnancy), bacterial factors (type, amount, activity, etc. of acidic bacteria in the oral cavity) and environmental factors ( Oral hygiene conditions, type of food, fluoride concentration in drinking water, etc.) are combined where dental caries occurs, and time factors are added to this, causing dental caries to progress.
초기 우식 단계에는 치과에서 우식된 부위를 치과용 드릴로 긁어내고 그 자리에 아말감, 금 또는 레진(합성수지의 일종)과 같은 치과용 재료로써 채워주는 충전 치료가 필요하다. 이 경우에는 신경치료로 치료가 되는 경우도 있지만 대부분의 경우 치료가 불가능하여 치아를 뽑게 된다. 충치치료를 한 후 충전물이 깨지거나 충전물과 치질사이에 또다시 우식이 발생될 수 있게 된다. In the initial caries stage, filling treatment is required in which a dental drill is used to scrape the caries from the dentist and fill the place with a dental material such as amalgam, gold, or resin (a type of synthetic resin). In this case, it is sometimes treated with neurological treatment, but in most cases, treatment is impossible and the tooth is pulled out. After caries treatment, the filling may break, or caries may occur again between the filling and the hemorrhoids.
인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)는 치아의 내부에 있는 치수 조직에서 얻을 수 있다. 치수줄기세포는 중간엽 줄기세포 (MSC)와 같은 다분화능을 가지는 세포로써 상아질 이외에 뼈 조직, 혈관, 치수 결체조직 및 신경 등으로 분화될 수 있다. 한편 치아의 상아 전구세포에서 치아 발생(odontogenesis)을 통해 상아질을 만든다. 상아질은 법랑질, 백악질과 함께 치아를 구성하고 있는 고도로 석회화된 경조직들 중 하나이다. 상아질은 상아질 모세포라는 전구세포가 유리하는 신호분자와 단백질에 의해 주변 기질의 석회화를 유도함으로써 형성된다. 그러나 충치나 외상 등에 의해 이들 조직에 손상이 생길 경우 재생되기 매우 어려우므로 현재 치과적인 치료법은 치아 경조직과 유사한 물성을 지닌 치과용 재료로 치아를 충전하는 방식에 의하고 있다. 따라서 상아질로만 특정하게 분화될 수 있는 상아 전구세포를 치수줄기세포로부터 얻기 위한 최적의 분화 조건에 대한 연구의 필요성이 있다. Human dental pulp stem cells (HDPSCs) can be obtained from pulp tissue inside the tooth. Pulp stem cells are cells having a multipotency like mesenchymal stem cells (MSCs) and can be differentiated into bone tissue, blood vessels, pulp connective tissue, and nerves in addition to dentin. On the other hand, dentin is produced through odontogenesis in the dentin progenitor cells of the tooth. Dentin is one of the highly calcified hard tissues that make up teeth along with enamel and chalk. Dentin is formed by inducing calcification of the surrounding matrix by signaling molecules and proteins that are free from progenitor cells called dentin blasts. However, if these tissues are damaged due to tooth decay or trauma, it is very difficult to regenerate them, so the current dental treatment is based on a method of filling the teeth with dental materials having properties similar to those of hard tooth tissue. Therefore, there is a need for a study on the optimal differentiation conditions for obtaining from pulp stem cells dentin progenitor cells that can be specifically differentiated only into dentin.
두충나무(Eucommia ulmoides OLIV)는 두충과(Eucommiaceae)에 속하는 중국원산의 낙엽교목으로서, 건조한 근피(根皮)를 두충이라 지칭하여 혈압강하작용, 이뇨작용 등이 알려져 잇으며, 알려진 성분으로는 굽타-퍼르카(gutta-percha), 알칼로이드(alkaloid), 펙틴(pectin), 진방, 수지 등의 성분을 함유한 것으로 알려져 있으며,(정보섭외 향약대사전, 영림사, p605-606, 1998년) 또한, 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid) 등의 성분을 함유한 것으로 알려져 있다(The promoting effects of geniposidic acid and aucubin in Eucommia ulmoides Oliver leaves on collagen synthesis./ Li Y, Sato T, Metori K, Koike K,Che QM, Takahashi S /Biological & Pharmaceutical Bulletin [1998, 21(12):1306-1310] ).Eucommia ulmoides OLIV is a deciduous tree native to China that belongs to the Eucommiaceae family.Dried root bark is referred to as Eucommia ulmoides OLIV. -It is known to contain ingredients such as gutta-percha, alkaloid, pectin, Jinbang, and resin. It is known to contain ingredients such as geniposide or geniposidic acid (The promoting effects of geniposidic acid and aucubin in Eucommia ulmoides Oliver leaves on collagen synthesis./ Li Y, Sato T, Metori K, Koike K, Che QM, Takahashi S/Biological & Pharmaceutical Bulletin [1998, 21(12):1306-1310]).
이에 본 발명자들은 섭취가 용이하고 인체에 안전하며 구강건강을 증진시킬 수 있는 물질을 찾고자 연구 노력한 결과, 두충 열수 추출물 또는 이로부터 분리한 화합물을 치수줄기세포에 처리한 결과, 상아 전구세포로 분화가 증가하여 뼈형성, 치형성 및 기질의 석회화를 증가시키고, 이와 관련된 바이오마커의 발현을 증가시키는 것을 확인하였다. 또한 상기 화합물과 덱사메타손을 함께 치수줄기세포에 처리한 결과, 상아 전구세포로 분화가 증가하여 뼈형성, 치형성 및 기질의 석회화를 더욱 증가시키고, 뼈형성 또는 치형성 유전자 마커의 발현을 증가시키는 것을 확인하고 본 발명을 완성하였다. Accordingly, the present inventors have tried to find a substance that is easy to ingest, is safe for the human body, and can improve oral health. As a result of processing the pulp stem cells with the hot water extract or a compound isolated therefrom, differentiation into dentinal progenitor cells is possible. As a result, it was confirmed that bone formation, dentition, and calcification of the matrix were increased, and the expression of biomarkers related thereto was increased. In addition, as a result of treating pulp stem cells with the compound and dexamethasone, differentiation into dentinal progenitor cells further increased, further increasing bone formation, dentition formation, and calcification of the matrix, and increasing the expression of bone formation or dentition gene markers. Confirmed and completed the present invention.
본 발명의 목적은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for inducing differentiation of pulp stem cells into dentinal progenitor cells, including geniposide or geniposidic acid.
본 발명의 다른 목적은 치수줄기세포에 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 처리하는 단계; 를 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도 방법을 제공하는 것이다.Another object of the present invention is to treat the pulp stem cells with geniposide or geniposidic acid; It is to provide a method for inducing the differentiation of pulp stem cells to dendritic progenitor cells comprising a.
본 발명의 또 다른 목적은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for inducing differentiation of pulp stem cells into dentinal progenitor cells, including geniposide or geniposidic acid.
본 발명의 다른 목적은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating dentin-dimensional diseases, including geniposide or geniposidic acid.
본 발명의 또 다른 목적은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a health functional food composition for preventing or improving dentin-dimensional diseases, including geniposide or geniposidic acid.
본 발명의 다른 목적은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 개선용 건강식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a health food composition for preventing or improving dentin-dimensional diseases, including geniposide or geniposidic acid.
상기 목적을 달성하기 위하여,To achieve the above object,
본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도용 조성물을 제공한다.The present invention provides a composition for inducing differentiation of pulp stem cells into dentinal progenitor cells, including geniposide or geniposidic acid.
또한, 본 발명은 치수줄기세포에 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 처리하는 단계; 를 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도 방법을 제공한다.In addition, the present invention comprises the steps of treating geniposide or geniposidic acid on pulp stem cells; It provides a method for inducing the differentiation of pulp stem cells to dendritic progenitor cells comprising a.
나아가 본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도용 키트를 제공한다.Furthermore, the present invention provides a kit for inducing differentiation of pulp stem cells into dentinal progenitor cells, including geniposide or geniposidic acid.
또한, 본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating dentin-dimensional diseases, including geniposide or geniposidic acid.
더 나아가 본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.Furthermore, the present invention provides a health functional food composition for preventing or improving dentin-dimensional diseases including geniposide or geniposidic acid.
또한, 본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 개선용 건강식품 조성물을 제공한다.In addition, the present invention provides a health food composition for preventing or improving dentin-dimensional diseases including geniposide or geniposidic acid.
본 발명의 두충 추출물로부터 분리된 화합물인 제니포사이드 또는 제니포시딕산을 처리하는 경우 치수줄기세포의 상아 전구세포로의 분화 효율이 유의적으로 증가하고 기질의 석회화가 유도되며, 상아 전구세포의 분화능이 향상되며, 또한 뼈형성 또는 치형성 유전자 마커인 ALP, OSTEOCALCIN, BSP, DSPP, DMP-1 및 RUNX2의 발현을 각각 증가시키는 효과가 있고, 덱사메타손을 병용하여 사용하는 경우, 분화 효율, 기질의 석회화 및 유전자 마커의 발현을 더욱 증가시키는 효과가 있어, 치수줄기세포의 상아 전구세포로의 분화용 조성물 또는 키트에 사용될 수 있고, 상아질을 재생시켜 치수와 상아질의 손상을 회복시킬 수 있어, 상아질-치수질환의 예방, 치료 또는 개선용 조성물로 유용할 수 있다.In the case of treatment of geniposide or genifosidic acid, which is a compound isolated from the cervical plexus extract of the present invention, the efficiency of differentiation of pulp stem cells into dentinal progenitor cells is significantly increased, calcification of the matrix is induced, and the differentiation ability of the dentinal progenitor cells is increased. It has the effect of increasing the expression of ALP, OSTEOCALCIN, BSP, DSPP, DMP-1, and RUNX2, which are bone formation or dentition gene markers, respectively, and when dexamethasone is used in combination, differentiation efficiency, calcification of the substrate, and Since it has the effect of further increasing the expression of the gene marker, it can be used in a composition or kit for differentiation of pulp stem cells into dentin progenitor cells, and it can recover damage to pulp and dentin by regenerating dentin, and dentin-dental disease It may be useful as a composition for the prevention, treatment or improvement of.
도 1은 MC3T3E1 및 HDPSCs에서 두충 추출물, 제니포사이드 및 제니포시딕산 처리에 따른 세포생존도를 측정한 결과이다.
도 2는 MC3T3E1 및 HDPSCs에서 두충 추출물, 제니포사이드, 제니포시딕산 처리에 따른 Alizarin-red solution 염색을 통한 분화능을 확인한 결과이다.
도 3은 MC3T3E1 및 HDPSCs에서 두충 추출물, 제니포사이드, 제니포시딕산 처리에 따른 ALP, OSTEOCALCIN, BSP, DSPP, DMP-1 및 RUNX2의 발현을 확인한 결과이다.
도 4는 MC3T3E1 및 HDPSCs에서 제니포사이드 및 덱사메타손 병용사용에 따른 Alizarin-red solution 염색을 통한 분화능을 확인한 결과이다.
도 5A는 HDPSCs에서 제니포사이드 및 덱사메타손 병용사용에 따른 ALP 발현을 확인한 결과이다.
도 5B는 HDPSCs에서 제니포사이드 및 덱사메타손 병용사용에 따른 ALP 활성도를 확인한 결과이다.
도 5C는 MC3T3E1 및 HDPSCs에서 제니포사이드 및 덱사메타손 병용사용에 따른 DSPP, DMP-1의 발현을 확인한 결과이다.1 is a result of measuring the cell viability of MC3T3E1 and HDPSCs according to the treatment of Euphorbia chinensis extract, geniposide, and genifosidic acid.
Figure 2 is a result of confirming the differentiation ability of MC3T3E1 and HDPSCs through Alizarin-red solution staining according to the treatment of cephalic extract, geniposide, and genifosidic acid.
3 is a result of confirming the expression of ALP, OSTEOCALCIN, BSP, DSPP, DMP-1 and RUNX2 in MC3T3E1 and HDPSCs according to treatment with cephalic extract, geniposide, genifosidic acid.
4 is a result of confirming the differentiation ability through Alizarin-red solution staining according to the combined use of geniposide and dexamethasone in MC3T3E1 and HDPSCs.
5A is a result of confirming the expression of ALP according to the combination use of geniposide and dexamethasone in HDPSCs.
5B is a result of confirming the ALP activity according to the combined use of geniposide and dexamethasone in HDPSCs.
5C is a result of confirming the expression of DSPP and DMP-1 according to the combined use of geniposide and dexamethasone in MC3T3E1 and HDPSCs.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
치수줄기세포의 상아 전구세포로의 분화 유도용 조성물Composition for inducing differentiation of pulp stem cells into dentinal progenitor cells
본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도용 조성물에 관한 것이다.The present invention relates to a composition for inducing differentiation of pulp stem cells into dentinal progenitor cells, including geniposide or geniposidic acid.
본 발명의 조성물에 있어서, 상기 조성물은 덱사메타손(dexamethasone)을 더 포함하여 사용할 수 있다.In the composition of the present invention, the composition may further include dexamethasone.
본 발명의 조성물에 있어서, 상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)은 두충(Eucommia ulmoides Oliv.) 추출물로부터 분리된 화합물일 수 있다.In the composition of the present invention, the geniposide or geniposidic acid may be a compound isolated from the Eucommia ulmoides Oliv. extract.
본 발명의 상기 조성물은 치수줄기세포가 상아 전구세포로의 분화 효율이 유의적으로 증가하고 기질의 석회화를 유도하며, 상아 전구세포의 상아질로의 분화능이 향상된다. 따라서 치수줄기세포의 상아 전구세포로의 분화용 조성물에 사용될 수 있고 상아질을 재생시켜 치수와 상아질의 손상을 회복시킬 수 있는 효과가 있다. The composition of the present invention significantly increases the differentiation efficiency of pulp stem cells into dentinal progenitor cells, induces calcification of the matrix, and improves the differentiation ability of dentinal progenitor cells into dentin. Therefore, it can be used in a composition for differentiation of pulp stem cells into dentin progenitor cells, and has the effect of recovering damage to pulp and dentin by regenerating dentin.
본 발명의 일실시예에 있어서, 상기 조성물을 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)에 처리한 결과, 분화제를 처리하지 않은 대조군(BM)에 비해 치수줄기세포의 세포 분화를 증가시키는 것을 확인하였다(도 2 참조).In one embodiment of the present invention, as a result of treatment of the composition on human dental pulp stem cells (HDPSCs), cell differentiation of pulp stem cells compared to a control group (BM) not treated with a differentiation agent It was confirmed that the increase was confirmed (see Fig. 2).
또한, 상기 조성물을 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)에서 상아모세포 분화 마커 유전자인 DSPP(Dentin sialophosphoprotein)의 유전자 발현수준을 증가시키고, 뼈모세포 및/또는 백악모세포 분화 마커 유전자인 DMP1(dentin matrix protein 1) 및 BSP(Bone sialoprotein) 유전자의 발현수준을 증가시켰으며, 뼈와 상아질에서 발견되는 비 콜라겐 성 단백질 호르몬으로써, 골 형성 촉진 또는 뼈 형성에 관여하는 Osteocalcin의 발현 또한 증가시켰다(도 3참조).In addition, the composition increases the gene expression level of the oblast differentiation marker gene DSPP (Dentin sialophosphoprotein) in human dental pulp stem cells (HDPSCs), and is an osteoblast and/or chalky blast differentiation marker gene. It increased the expression levels of DMP1 (dentin matrix protein 1) and BSP (bone sialoprotein) genes, and as a non-collagen protein hormone found in bone and dentin, it also increased the expression of osteocalcin, which is involved in promoting bone formation or bone formation. (See Fig. 3).
RUNX2은 골아 세포의 분화와 관련된 중요한 전사이다. RUNX2은 미성숙 골아 세포에서 상향 조절되고, 성숙 골아 세포에서는 하향 조절 되며, 미성숙 골아 세포로의 다능성 중간엽 세포의 분화를 유도하고 골아 세포 분화 및 골 기질 유전자를 유지하는 몇 가지 핵심 하류 단백질의 발현을 활성화한다. RUNX2가 증가하면 골아 세포의 분화가 활성화 되는데, 본 발명의 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 조성물은 RUX2의 유전자 발현 수준을 증가시켰다. 또한, DMP1 및 BSP 유전자의 mRNA 발현 수준이 증가되면, 뼈모세포 및/또는 백악모세포의 분화와, 뼈 및/또는 백악질의 재생이 촉진된다고 알려져 있으므로, 상기 DMP1 및 BSP 유전자의 mRNA 수준을 증가시키는 효과를 나타내는 본 발명의 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 조성물은 뼈모세포 및/또는 백악모세포의 분화와, 뼈 및/또는 백악질의 재생을 촉진하는 효과를 나타냄을 확인할 수 있다(도 3참조).RUNX2 is an important transcription involved in the differentiation of osteoblasts. RUNX2 is upregulated in immature osteoblasts, downregulated in mature osteoblasts, induces differentiation of pluripotent mesenchymal cells into immature osteoblasts, and expresses several key downstream proteins that maintain osteoblast differentiation and bone matrix genes. Activate. When RUNX2 increases, the differentiation of osteoblasts is activated, and the composition containing geniposide or geniposidic acid of the present invention increased the gene expression level of RUX2. In addition, when the mRNA expression level of the DMP1 and BSP genes is increased, it is known that the differentiation of osteoblasts and/or cretaceous cells and the regeneration of bone and/or chalk is promoted, so the effect of increasing the mRNA levels of the DMP1 and BSP genes. The composition comprising geniposide or geniposidic acid of the present invention showing an effect of promoting the differentiation of osteoblasts and/or chalky cells and regeneration of bone and/or chalk can be confirmed. Yes (see Fig. 3).
치계(dental origin)인 인간 치수세포에서 상아모세포, 뼈모세포 및/또는 백악모세포의 분화를 촉진한다. 이에 더해, 사람의 사랑니 등에서 분리할 수 있는 인간 치수줄기세포(humandental pulp stem cell; hDPSC)는 일반적으로 인간 치수세포에 혼재되어 있으며, 상아모세포, 뼈모세포 및/또는 백악모세포로 분화 가능하므로, 본 발명의 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 조성물은 인간 치수줄기세포의 상아모세포, 뼈모세포 및/또는 백악모세포로의 분화를 촉진하는 효과를 나타낼 수 있음을 알 수 있다.Promotes the differentiation of oblasts, osteoblasts and/or chalks in human pulp cells of dental origin. In addition, human dental pulp stem cells (hDPSC), which can be isolated from human wisdom teeth, are generally mixed with human pulp cells and can be differentiated into oblasts, osteoblasts, and/or cretaceous cells. It can be seen that the composition comprising geniposide or geniposidic acid of the present invention can exhibit the effect of promoting the differentiation of human pulp stem cells into oblasts, osteoblasts, and/or chalky stem cells. .
상아 전구세포(odontoblast)는 상아질(dentin)과 닿아 있으며, 상아질을 형성하는 원주형 세포로 기능적으로 치아의 주된 구성성분인 상아질을 직접적으로 만드는 역할을 하며, 이 세포자체는 상아질 내측에 존재하는 치수(pulp) 조직의 한 구성성분으로 치아 내부의 치수강 벽에 배열되어 존재한다. 치아의 대부분을 차지하는 상아질은 상아 전구세포로부터 형성된다. 상아질은 치아의 중요한 구성 성분으로서, 치관과 치근의 대부분을 구성하며, 치아의 맹출 후에는 외부 자극에 반응하여 2차 상아질을 형성함으로 치수(pulp)를 보호하는 작용을 한다. The odontoblast is in contact with the dentin and is a columnar cell that forms the dentin. Functionally, it plays a role in directly making the dentin, the main component of the tooth, and this cell itself is a pulp that exists inside the dentin. (pulp) As a component of tissue, it exists arranged in the wall of the pulp cavity inside the tooth. Dentin, which occupies most of the tooth, is formed from dentin progenitor cells. Dentin is an important constituent of teeth, and constitutes most of the crown and roots, and acts to protect pulp (pulp) by forming secondary dentin in response to external stimuli after eruption of the tooth.
본 발명에서 치수줄기세포(dental pulp stem cell)는 치계 (dental origin) 중간엽 줄기세포의 일종으로, 상기치계 중간엽 줄기세포는 ‘치계상피세포의 영향을 받은 (일부분의) 줄기세포들을 의미하며, 중배엽성 기원으로 치아내부의 치수와 치아 주위조직에 분포하는 줄기세포를 말한다. 줄기세포의 예로는 치수줄기세포(dental pulp stem cell (DPSC)), 탈락 유치 줄기세포(stem cell from exfoliated deciduous teeth (SHED)), 치주 인대 줄기세포 (periodontal ligament stem cells (PDLSC)), 치근단 유두 줄기세포(stem cell from the apical papilla (SCAP), 및 치배 줄기세포(dental follicle precursor cells (DFPC))가 있다.In the present invention, dental pulp stem cells are a type of dental origin mesenchymal stem cells, and the dental mesenchymal stem cells refer to'(part of) stem cells affected by dental epithelial cells. It is a mesodermal origin and refers to stem cells distributed in the pulp and tissues around the tooth. Examples of stem cells include dental pulp stem cells (DPSC), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSC), and apical papilla. There are stem cells (stem cells from the apical papilla (SCAP), and dental follicle precursor cells (DFPC)).
치수줄기세포의 상아 전구세포로의 분화 유도용 키트Kit for inducing the differentiation of pulp stem cells into dentinal progenitor cells
본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도용 키트에 관한 것이다.The present invention relates to a kit for inducing differentiation of pulp stem cells into dentinal progenitor cells, including geniposide or geniposidic acid.
본 발명의 상기 분화 유도용 키트에 있어서, 상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)에 덱사메타손(dexamethasone)을 더 포함하여 사용할 수 있다.In the kit for inducing differentiation of the present invention, dexamethasone may be further included in the geniposide or geniposidic acid.
상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)은 두충(Eucommia ulmoides Oliv.) 추출물로부터 분리된 화합물일 수 있다.The geniposide or geniposidic acid may be a compound isolated from an extract of Eucommia ulmoides Oliv.
본 발명의 상기 키트는 치수줄기세포가 상아 전구세포로의 분화 효율이 유의적으로 증가하고 기질의 석회화를 유도하며, 상아 전구세포의 상아질로의 분화능이 향상된다. 따라서 치수줄기세포의 상아 전구세포로의 분화용 키트에 사용될 수 있고 상아질을 재생시켜 치수와 상아질의 손상을 회복시킬 수 있는 효과가 있다. The kit of the present invention significantly increases the differentiation efficiency of pulp stem cells into dentinal progenitor cells, induces calcification of the matrix, and improves the differentiation ability of dentinal progenitor cells into dentin. Therefore, it can be used in a kit for differentiation of pulp stem cells into dentin progenitor cells, and has the effect of recovering damage to pulp and dentin by regenerating dentin.
치수줄기세포의 상아 전구세포로의 분화 유도 방법Method for inducing the differentiation of pulp stem cells into dentinal progenitor cells
본 발명은 치수줄기세포에 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 처리하는 단계; 를 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도 방법에 관한 것이다.The present invention comprises the steps of treating a pulp stem cell with geniposide or geniposidic acid; It relates to a method for inducing the differentiation of pulp stem cells to dendritic progenitor cells comprising a.
본 발명의 상기 분화 유도 방법에 있어서, 상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)에 덱사메타손(dexamethasone)을 더 포함하여 사용할 수 있다.In the method of inducing differentiation of the present invention, dexamethasone may be further included in the geniposide or geniposidic acid.
상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)은 두충(Eucommia ulmoides Oliv.) 추출물로부터 분리된 화합물일 수 있다.The geniposide or geniposidic acid may be a compound isolated from an extract of Eucommia ulmoides Oliv.
본 발명의 분화 유도 방법에 있어서, 상기 처리는 7일 내지 14일 동안 수행할 수 있다. In the method of inducing differentiation of the present invention, the treatment may be performed for 7 to 14 days.
상아질-치수질환의 예방 또는 치료용 약학적 조성물Dentin-Pharmaceutical composition for prevention or treatment of dimensional disease
본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for the prevention or treatment of dentin-dimensional diseases comprising geniposide or geniposidic acid.
본 발명의 조성물에 있어서, 상기 조성물은 덱사메타손(dexamethasone)을 더 포함하여 사용할 수 있다.In the composition of the present invention, the composition may further include dexamethasone.
본 발명의 조성물에 있어서, 상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)은 두충(Eucommia ulmoides Oliv.) 추출물로부터 분리된 화합물일 수 있다.In the composition of the present invention, the geniposide or geniposidic acid may be a compound isolated from the Eucommia ulmoides Oliv. extract.
본 발명의 조성물에 있어서, 상기 조성물은 상아질, 뼈 및 백악질을 포함하는 경조직 또는 치수조직의 재생을 촉진시킬 수 있으며, 치수세포에서 상아모세포, 뼈모세포 또는 백악모세포의 분화를 촉진시킬 수 있다.In the composition of the present invention, the composition may promote the regeneration of hard tissues or pulp tissues including dentin, bone, and chalk, and may promote differentiation of odontoblasts, osteoblasts, or cretaceous cells from pulp cells.
본 발명의 용어 "상아질-치수질환"이란, 상기 치수조직의 손상으로 인하여, 치수조직과 이에 결합된 상아질이 손상되어 발병되는 질환을 의미한다The term "denin-dental disease" of the present invention means a disease caused by damage to the pulp tissue and the dentin associated therewith due to the damage to the pulp tissue.
본 발명의 조성물에 있어서, 상기 상아질-치수질환의 예로는 상아질 지각과민증, 치수충혈, 치수염, 치수변성, 치수의 괴사 또는 괴저증일 수 있다.In the composition of the present invention, examples of the dentin-dimensional disease may be dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis or gangrene.
본 발명의 용어 "예방"이란, 본 발명의 약학 조성물의 투여로 상아질-치수질환의 발생을 저해 또는 지연시키는 모든 행위를 의미한다.The term "prevention" of the present invention means any action that inhibits or delays the occurrence of dentin-dental disease by administration of the pharmaceutical composition of the present invention.
본 발명의 용어 "치료"란, 본 발명의 약학 조성물을 상아질-치수질환의 치료가 요구되는 개체에 투여하여, 상아질 또는 치수조직의 재생을 촉진함으로써 치수질환의 치료가 수행되도록 하는 모든 행위를 의미한다.The term "treatment" of the present invention refers to any act of administering the pharmaceutical composition of the present invention to an individual in need of treatment of dentin-dental disease, thereby promoting the regeneration of dentin or pulp tissue, thereby performing treatment of pulp disease. do.
본 발명의 약학 조성물은, 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체(자연적 또는 비자연적 담체), 부형제 또는 희석제를 추가로 포함하는 상아질-치수질환의 치료용 약학 조성물의 형태로 제조될 수 있다. 구체적으로, 상기 약학 조성물은, 각각 통상의 방법에 따라 상아질-치수질환이 유발된 부위에 투여할 수 있는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 약학조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조 에이트, 탈크, 마그네슘 스테아레이트, 광물유, 콜라겐 등을 들 수 있다. 제제화 할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 특히, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제, 연고제(예를 들어, 치수이장재 등) 등이 포함될 수 있다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for the treatment of dentin-dimensional diseases, further comprising an appropriate carrier (natural or non-natural carrier), an excipient, or a diluent commonly used in the manufacture of a pharmaceutical composition. have. Specifically, the pharmaceutical composition may be formulated and used in the form of a sterile injectable solution that can be administered to a site where a dentin-dental disease is caused, respectively, according to a conventional method. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and collagen. In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In particular, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, ointments (for example, pulp paste, etc.) may be included. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol,
상기 약학 조성물은 약제학적으로 유효한 양으로 투여될 수 있는데, 본 발명의 용어 "약제학적으로 유효한 양"이란 의학적 치료 또는 예방에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료 또는 예방하기에 충분한 양을 의미하며, 유효 용량 수준은 질환의 중증도, 약물의 활성, 환자의 연령, 체중, 건강, 성별, 환자의 약물에 대한 민감도, 사용된 본 발명 조성물의 투여 시간, 투여 경로 및 배출 비율 치료기간, 사용된 본 발명의 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학 조성물은 단독으로 투여하거나 공지된 상아질-치수질환을 치료용 약학 조성물과 병용하여 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하다.The pharmaceutical composition may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. The effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion, treatment period, use. It may be determined according to factors including drugs used in combination or co-use with the composition of the present invention and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or may be administered in combination with a pharmaceutical composition for treating known dentin-dimensional diseases. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects.
본 발명의 약학 조성물의 투여량은 사용목적, 질환의 중독도, 환자의 연령, 체중, 성별, 기왕력, 또는 유효성분으로서 사용되는 물질의 종류 등을 고려하여 당업자가 결정할 수 있다. 예를 들어, 본 발명의 약학 조성물은 성인 1인당 약 0.1 ng 내지 약 100 mg/kg, 바람직하게는 1 ng 내지 약 10 mg/kg로 투여할 수 있고, 본 발명의 조성물의 투여빈도는 특별히 이에 제한되지 않으나, 1일 1회 투여하거나 또는 용량을 분할하여 수회 투여할 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the age, weight, sex, history, or type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention can be administered in an amount of about 0.1 ng to about 100 mg/kg, preferably 1 ng to about 10 mg/kg per adult, and the frequency of administration of the composition of the present invention is specifically Although not limited, it may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.
본 발명의 약학 조성물에 포함된 두충 추출물의 농도는 10 내지 500 μg/ml 일 수 있으며 바람직하게는 100 내지 500 μg/ml일 수 있다. 두충 추출물로부터 분리된 화합물인 제니포사이드(geniposide, GPS) 또는 제니포시딕산(geniposidic acid, GPA)의 농도는 10 내지 100 μg/ml일 수 있으며, 바람직하게는 5 내지 100 μg/ml일 수 있다. The concentration of the cephalopathy extract contained in the pharmaceutical composition of the present invention may be 10 to 500 μg/ml, and preferably 100 to 500 μg/ml. The concentration of geniposide (GPS) or geniposidic acid (GPA), which is a compound isolated from the Euphorbia extract, may be 10 to 100 μg/ml, preferably 5 to 100 μg/ml.
상기 두충 추출물 또는 두충 추출물로부터 분리된 화합물인 제니포사이드(geniposide, GPS) 또는 제니포시딕산(geniposidic acid, GPA)의 함량은 특별히 이에 제한되지 않으나, 최종 조성물 총 중량을 기준으로 0.0001 내지 50 중량%, 보다 바람직하게는 0.01 내지 20 중량%의 함량으로 포함될 수 있다.The content of geniposide (GPS) or geniposidic acid (GPA), which is a compound isolated from the Cordyceps extract or Cordyceps extract, is not particularly limited thereto, but 0.0001 to 50% by weight based on the total weight of the final composition, More preferably, it may be included in an amount of 0.01 to 20% by weight.
본 발명의 약학 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다. 본 발명의 약학 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라 구강내 투여 또는 구강내 주사 등의 경로를 통해 투여될 수 있다.The route of administration of the pharmaceutical composition of the present invention may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered through a route such as intraoral administration or intraoral injection, depending on the purpose.
상아질-치수질환의 예방 또는 개선용 건강기능식품 또는 건강식품 조성물Dentin-health functional food or health food composition for preventing or improving dimensional disease
본 발명은 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 포함하는 상아질-치수질환의 예방 또는 개선용 건강기능식품 또는 건강식품 조성물에 관한 것이다.The present invention relates to a health functional food or a health food composition for preventing or improving dentin-dimensional disease, including geniposide or geniposidic acid.
본 발명의 조성물에 있어서, 상기 조성물은 덱사메타손(dexamethasone)을 더 포함하여 사용할 수 있다.In the composition of the present invention, the composition may further include dexamethasone.
본 발명의 조성물에 있어서, 상기 상아질-치수질환의 예로는 상아질 지각과민증, 치수충혈, 치수염, 치수변성, 치수의 괴사 또는 괴저증일 수 있다.In the composition of the present invention, examples of the dentin-dimensional disease may be dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis or gangrene.
본 발명의 용어, "개선"이란, 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term "improvement" means any action that at least reduces the severity of a parameter related to the condition being treated, for example a symptom.
식품의 종류에는 특별한 제한은 없다. 본 발명의 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 첨가할 수 있는 식품의 예로는 드링크제, 육류, 소시지, 빵, 비스킷, 떡, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 알코올 음료 및 비타민 복합제, 유제품 및 유가공 제품 등이 있으며, 통상적인 의미에서의 건강식품 및 건강기능식품을 모두 포함한다.There are no special restrictions on the type of food. Examples of foods to which geniposide or geniposidic acid of the present invention can be added include drinks, meats, sausages, breads, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, etc. There are dairy products including noodles, gums, ice creams, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and include all health foods and health functional foods in the usual sense.
본 발명에 따른 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 함유하는 건강식품 및 건강기능식품 조성물은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강식품 및 건강기능식품 중의 상기 조성물의 양은 전체 식품 중량의 0.1 내지 90 중량부로 가할 수 있다. 그러나 건강 유지를 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)은 상기 범위 이상의 양으로도 사용될 수 있다.The health food and health functional food composition containing geniposide or geniposidic acid according to the present invention can be added to food as it is or can be used with other foods or food ingredients, and is appropriate according to a conventional method. Can be used. The mixing amount of geniposide or geniposidic acid may be appropriately determined according to the purpose of use (for prevention or improvement). In general, the amount of the composition in health foods and health functional foods may be added in an amount of 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of maintaining health or controlling health, the amount may be less than the above range, and there is no problem in terms of safety, so geniposide or geniposidic acid is used. It may be used in an amount more than the above range.
본 발명의 건강식품 및 건강기능식품 조성물은 지시된 비율로 필수 성분으로서 본 발명 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트라이톨 등의 당알코올이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 건강기능 식품 조성물 100 당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health food and health functional food composition of the present invention is not particularly limited in other ingredients except for containing geniposide or geniposidic acid of the present invention as an essential ingredient in the indicated ratio, and various Eggplant flavors or natural carbohydrates, etc. may be contained as additional ingredients. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the health functional food composition of the present invention.
상기 외에 본 발명의 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 함유하는 건강식품 및 건강기능식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 건강식품 및 건강기능식품 조성물은 천연 과일쥬스 및 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition to the above, the health food and health functional food composition containing geniposide or geniposidic acid of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors, and natural flavors. Agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages And the like. In addition, the health food and health functional food composition of the present invention may contain pulp for the manufacture of natural fruit juice and fruit juice beverages and vegetable beverages.
이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)을 함유하는 건강식품 및 건강기능식품 조성물 100 중량부 당 0.1 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.These components may be used independently or in combination. The ratio of these additives is not so important, but it is selected from 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing geniposide or geniposidic acid of the present invention. It is common.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기의 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
<준비예 1> 과잉치 발치 및 치수줄기세포의 채취<Preparation Example 1> Excess tooth extraction and collection of pulp stem cells
치수줄기세포를 채취하기 위하여, 전신질환이 없는 성인의 상악 정중 매복 과잉치를 발치하여 오염과 감염이 되지 않도록 즉시 항생제와 소혈청 20%를 함유한 α-MEM media (GIBCO LifeTechnologies, Grand Island, NY, USA)에 보관하였다. 당일 치수 세포 채취를 위해 실험실로 옮겼고, 백악법랑경계 (cementoenamel junction, CEJ) 부분에서 치수가 노출되지 않도록 치질만 삭제한 후 멸균된 파일을 이용하여 치수줄기세포를 채취하였다.In order to collect pulp stem cells, α-MEM media containing antibiotics and 20% bovine serum (GIBCO LifeTechnologies, Grand Island, NY, USA). On the same day, the pulp cells were transferred to a laboratory for collection, and pulp stem cells were collected using a sterilized file after removing only the hemorrhoids so that the pulp was not exposed at the cementoenamel junction (CEJ).
<준비예 2> 두충 추출물(Eucommia ulmoides extract, EUE), 제니포사이드(geniposide, GPS) 및 제니포시딕산(geniposidic acid, GPA) 준비<Preparation Example 2> Preparation of Eucommia ulmoides extract (EUE), geniposide (GPS) and geniposidic acid (GPA)
실험에 사용된 건조상태의 두충 추출물은 한국생명공학연구원 한국 식물추출물은행((한국식물추출물은행 http://extract.pdre.re.kr, 대전)으로부터 구입하였다. 분쇄된 두충시료 30~40g을 HPLC 메탄올 용액 200mL으로 50℃에서 초음파분쇄기 (BRAONSON Ultrasonication corporation, ultrasonic cleaner, BRANSON)를 이용하여 추출하였고, 상기 두충 추출액을 무형광 솜을 사용하여 여과시킨 후, 농축시켰다. 상기 농축시킨 시료를 동결건조하여, 건조물 21.18g을 수득하여(이하, "EUE"라 함) 하기 실험에 두충 추출물(EUE) 21.18 mg/mL의 원액을 만든 후에 300μg/mL농도로 실험에 사용하였다. The dried headworm extract used in the experiment was purchased from Korea Research Institute of Bioscience and Biotechnology, Korea Plant Extract Bank ((Korea Plant Extract Bank http://extract.pdre.re.kr , Daejeon). Crushed headworm sample 30-40g was purchased. 200 mL of an HPLC methanol solution was extracted using an ultrasonic grinder (BRAONSON Ultrasonication Corporation, ultrasonic cleaner, BRANSON) at 50° C., and the extract was filtered using a non-fluorescent cotton, and then concentrated. , To obtain 21.18 g of the dried product (hereinafter referred to as "EUE"), a stock solution of 21.18 mg/mL of the Eucalyptus extract (EUE) was prepared in the following experiment, and then used in the experiment at a concentration of 300 μg/mL.
또한, 두충 추출물 유래 화합물인 제니포사이드(geniposide, GPS) 및 제니포시딕산(geniposidic acid, GPA)을 각각 구입하여 DMSO(D2650,sigma)에 원액 20 mg/mL를 만든 후 이 원액을 희석하여 10μg/mL농도로 실험에 사용하였다.In addition, geniposide (GPS) and geniposidic acid (GPA), which are compounds derived from the cervical plexus extract, were purchased respectively to make 20 mg/mL of the stock solution in DMSO (D2650, sigma), and then dilute this stock solution to 10 μg/mL. It was used in the experiment in mL concentration.
<준비예 3> 세포배양<Preparation Example 3> Cell culture
인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)의 분화능을 확인하기 위해 골분화를 유도하였다. 각 계대에서 줄기세포의 골모세포로의 분화유도를 평가하기 위해 HDPSCs를 분화제를 처리하지 않은 군과 분화제를 처리한 군으로 나누어 배양하였다. 골분화를 유도하기 위해, α-MEM (Gibco, LifeTechnologies, Grand Island, NY, USA)에 10% fetal bovine serum (FBS; Gibco, Life Technologies Corporation, Carlsbad, Calif, USA)에 5mM β-glycerophosphate (Sigma-Aldrich Co., St. Louis, MO, USA), 100 nM Dexamethasone (Sigma-Aldrich Co., St. Louis, MO, USA) 그리고 100 μM Ascorbic acid (Sigma-Aldrich Co., St. Louis, MO, USA) 를 혼합하여 제작한 골분화 배지에서 배양하였다. 배양액은 2일에 1회씩 교환해주며 20배 광학현미경 하에서 세포의 형태학적 특성을 7일, 14일 두 번 관찰하였다.Bone differentiation was induced to confirm the differentiation ability of human dental pulp stem cells (HDPSCs). In order to evaluate the induction of differentiation of stem cells into osteoblasts at each passage, HDPSCs were cultured by dividing into a group not treated with a differentiation agent and a group treated with a differentiation agent. To induce bone differentiation, α-MEM (Gibco, LifeTechnologies, Grand Island, NY, USA) in 10% fetal bovine serum (FBS; Gibco, Life Technologies Corporation, Carlsbad, Calif, USA) in 5mM β-glycerophosphate (Sigma). -Aldrich Co., St. Louis, MO, USA), 100 nM Dexamethasone (Sigma-Aldrich Co., St. Louis, MO, USA) and 100 μM Ascorbic acid (Sigma-Aldrich Co., St. Louis, MO, USA) USA) was cultured in bone differentiation medium prepared by mixing. The culture medium was exchanged once every 2 days, and the morphological characteristics of the cells were observed twice on the 7th and 14th days under a 20x optical microscope.
<실험예 1> 세포생존도 실험<Experimental Example 1> Cell viability experiment
세포생존도를 측정하기 위해, 조골세포 (MC3T3E1)과 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)를 96 well plate에 4Х103cells/well의 농도로 분주한 후, 24시간 동안 배양하였고, 두충 추출물(Eucommia ulmoides extract, EUE), 제니포사이드(geniposide, GPS), 제니포시딕산(geniposidic acid, GPA), 덱사메타손(dexamethasone, Dex)을 각각 1, 5, 10, 50, 100, 500nM의 농도로 처리하고 72시간 배양하였다. 그 후 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 각 well당 300 μL씩 처치하고 37 ℃, 4시간 동안 반응시켰다. 배지를 조심스럽게 제거하고 MTT를 환원시켜 생성된 formazan 결정을 dimethylsulfoxide (DMSO)로 용해시킨 후, 570 nm에서 microplate 측정기(Tecan Infinite M200 PRO, USA)를 이용하여 흡광도를 측정하고, 그 결과를 도 1에 나타내었다. To measure the cell viability, osteoblasts (MC3T3E1) and human dental pulp stem cells (HDPSCs) were dispensed into a 96 well plate at a concentration of 4Х10 3 cells/well, and cultured for 24 hours. , Eucommia ulmoides extract (EUE), geniposide (GPS), geniposidic acid (GPA), dexamethasone (D e xamethasone, Dex) 1, 5, 10, 50, 100, 500 nM, respectively It was treated at a concentration of and incubated for 72 hours. Thereafter, 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was treated with 300 μL per well and reacted at 37° C. for 4 hours. After removing the medium carefully and reducing MTT to dissolve the resulting formazan crystal with dimethylsulfoxide (DMSO), absorbance was measured using a microplate meter (Tecan Infinite M200 PRO, USA) at 570 nm, and the result is shown in FIG. 1 Shown in.
도 1에 나타낸 바와 같이, 두충 추출물(Eucommia ulmoides extract, EUE), 제니포사이드(geniposide, GPS), 제니포시딕산(geniposidic acid, GPA), 덱사메타손(dexamethasone, Dex)을 처리하고 세포생존도를 측정한 결과, 24시간에서 저농도 (1-100)에서 세포의 생존도에는 영향을 주지 않았지만 고농도 (500-1000) 처리시 세포 생존도에 영향을 줌을 확인하였다.As shown in Figure 1, Eucommia ulmoides extract (EUE), geniposide (GPS), geniposidic acid (GPA), dexamethasone (d e xamethasone, Dex) was treated and cell viability was measured. As a result of the measurement, it was confirmed that there was no effect on the viability of the cells at a low concentration (1-100) at 24 hours, but the treatment at a high concentration (500-1000) had an effect on the cell viability.
<실험예 2> Alizarin-red solution 염색을 통한 분화능 확인<Experimental Example 2> Confirmation of differentiation ability through staining with Alizarin-red solution
조골세포 (MC3T3E1)와 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)의 분화여부를 시각적으로 확인하기 위하여, Alizarin-red solution (ARS, pH 4.5) 염색을 시행하여, 세포 밖에 형성된 석회화 결절을 시각적으로 비교하였다. To visually confirm the differentiation of osteoblasts (MC3T3E1) and human dental pulp stem cells (HDPSCs), Alizarin-red solution (ARS, pH 4.5) staining was performed and calcified nodules formed outside the cells. Were compared visually.
10 mM β-glycerophosphate와 100 ug/ml ascorbic acid 가 함유된 기본 배양액 (IM)에 조골세포 (MC3T3E1)와 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)를 분주한 후, 제니포사이드(geniposide, GPS), 제니포시딕산(geniposidic acid, GPA)을 각각 5, 10, 50, 100μg/ml의 농도로 처리하고, 두충 추출물(Eucommia ulmoides extract, EUE)을 10, 50, 100, 500μg/ml의 농도로 처리하고, 덱사메타손(dexamethasone, Dex), BMP-2(bone morphogenic protein-2)를 각각 1, 5, 10, 50μg/ml의 농도로 7일 또는 14일 동안 처리하였다. 그 후 세포를 70% 열수에 5분간 고정하고 2% Alizarin-red solution (Sigma-Aldrich Co., St. Louis, MO, USA)으로 45분간 염색한 다음 증류수로 세척한 후 건조하고, 그 결과를 도 2에 나타내었다. After dispensing osteoblasts (MC3T3E1) and human dental pulp stem cells (HDPSCs) into a basic culture medium (IM) containing 10 mM β-glycerophosphate and 100 ug/ml ascorbic acid, geniposide , GPS), geniposidic acid (GPA) were treated at concentrations of 5, 10, 50, and 100 μg/ml, respectively, and Eucommia ulmoides extract (EUE) was treated with 10, 50, 100, 500 μg/ml At concentrations, dexamethasone (Dex) and BMP-2 (bone morphogenic protein-2) were treated for 7 days or 14 days at concentrations of 1, 5, 10 and 50 μg/ml, respectively. After that, the cells were fixed in 70% hot water for 5 minutes, stained with 2% Alizarin-red solution (Sigma-Aldrich Co., St. Louis, MO, USA) for 45 minutes, washed with distilled water, dried, and the result was It is shown in Figure 2.
조골세포 (MC3T3E1)와 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)의 분화여부를 확인한 결과, 도 2에 나타낸 바와 같이, 분화제를 처리하지 않은 대조군 (BM)에서는 분화능에서는 효과가 없었지만, 제니포사이드(geniposide, GPS)를 처리할 경우 다른 처리군에 비해 색상이 진해짐을 확인하였다(도 2 참조). 상기 결과는 제니포사이드(geniposide, GPS) 처리군에서 osteogenic 및 odontogenic mineralization이 증가함을 의미한다. As a result of confirming the differentiation of osteoblasts (MC3T3E1) and human dental pulp stem cells (HDPSCs), as shown in FIG. 2, there was no effect on differentiation ability in the control group (BM) not treated with a differentiation agent. , When the geniposide (GPS) was treated, it was confirmed that the color became darker compared to other treatment groups (see FIG. 2). The above results indicate that osteogenic and odontogenic mineralization are increased in the geniposide (GPS) treatment group.
<실험예 3> RP-PCR을 통한 골형성 또는 치형성 유전자 마커의 발현 확인<Experimental Example 3> Confirmation of expression of bone formation or tooth formation gene marker through RP-PCR
제니포사이드(geniposide, GPS), 제니포시딕산(geniposidic acid, GPA), 두충 추출물(Eucommia ulmoides extract, EUE)의 골형성 또는 치형성 유전자 마커의 발현에 미치는 영향을 확인하고자, 두충 추출물(Eucommia ulmoides extract, EUE) (100 ug/ml)과 제니포사이드(geniposide, GPS)와 제니포시딕산(geniposidic acid, GPA)을 각각 50ug/ml농도로 조골세포 (MC3T3E1)와 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)에 7일과 14일 동안 처리하였다. Total RNA는 trizol extraction method (invitrogen light tech)를 사용하여 실험군과 대조군에서 모두 채취하였다. 채취된 RNA는 cDNA 합성을 위하여 사용하였으며, RT-PCR premix (Bioneer, 대전, 한국)를 이용하여 RT-PCR을 수행하였다. 분리한 total RNA를 cDNA로 역전사한 후, primer 쌍을 이용하여 증폭하였다. ALP(인산분해 효소, Alkaline phosphatase), 오스테오칼신(OSTEOCALCIN), BSP(뼈 시알로단백질, bone sialoprotein), DSPP(상아질 시알로포스포프로틴, dentin sialophosphoprotein), DMP-1(상아질 매트릭스 산성 단백질, Dentin matrix acidic phosphoprotein-1)과 RUNX2(Runt-related transcription factor-2)의 발현을 측정하고, 대조군으로 GAPDH를 사용하여, 유전자 발현을 확인한 결과를 도 3에 나타내었다.To determine the effect of geniposide (GPS), geniposidic acid (GPA), and Eucommia ulmoides extract (EUE) on the expression of osteogenesis or dentition markers, Eucommia ulmoides extract , EUE) (100 ug/ml) and geniposide (GPS) and geniposidic acid (GPA) at a concentration of 50 ug/ml, respectively, in osteoblasts (MC3T3E1) and human dental pulp stem cells. cells; HDPSCs) were treated for 7 and 14 days. Total RNA was collected from both the experimental group and the control group using the trizol extraction method (invitrogen light tech). The collected RNA was used for cDNA synthesis, and RT-PCR was performed using an RT-PCR premix (Bioneer, Daejeon, Korea). The isolated total RNA was reverse transcribed with cDNA, and then amplified using a primer pair. Alkaline phosphatase (ALP), OSTEOCALCIN, BSP (bone sialoprotein), DSPP (dentin sialophosphoprotein), DMP-1 (dentin matrix acidic protein, Dentin matrix) The expression of acidic phosphoprotein-1) and RUNX2 (Runt-related transcription factor-2) was measured, and the result of confirming gene expression using GAPDH as a control is shown in FIG. 3.
그 결과, 도 3에 나타낸 바와 같이, 두충 추출물(Eucommia ulmoides extract, EUE)과 제니포사이드(geniposide, GPS)를 처리할 경우, 다른 처리군에 비해 골형성 또는 치형성 유전자 마커인 ALP, OSTEOCALCIN, BSP, DSPP, DMP-1와 RUNX2의 발현이 증가함을 확인하였다.As a result, as shown in Figure 3, when treated with Eucommia ulmoides extract (EUE) and geniposide (GPS), compared to other treatment groups, ALP, OSTEOCALCIN, BSP , DSPP, DMP-1 and RUNX2 expression was confirmed to increase.
<실험예 4> Alizarin-red solution 염색을 통한 덱사메타손 병용사용에 대한 분화능 확인<Experimental Example 4> Confirmation of differentiation ability for combined use of dexamethasone through Alizarin-red solution staining
조골세포 (MC3T3E1)와 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)에서 제니포사이드(geniposide, GPS)와 덱사메타손(dexamethasone, Dex) 병용사용에 관한 분화능을 확인하기 위해, Alizarin-red Solution을 염색통해 확인한 결과를 도 4에 나타내었다.Alizarin-red Solution was used to confirm the differentiation ability of the combination of geniposide (GPS) and dexamethasone (Dex) in osteoblasts (MC3T3E1) and human dental pulp stem cells (HDPSCs). The results confirmed through dyeing are shown in FIG. 4.
그 결과, 도 4에 나타낸 바와 같이, 제니포사이드(geniposide, GPS) 또는 덱사메타손(dexamethasone, Dex)을 각각 처리한 군에 비해 제니포사이드(geniposide, GPS)와 덱사메타손(dexamethasone, Dex)을 병용사용하였을 때 Alizarin-red Solution의 염색이 더 많이 증가하는 것을 확인하였다. As a result, as shown in FIG. 4, when geniposide (GPS) and dexamethasone (dexamethasone, Dex) were used in combination compared to the group treated with geniposide (GPS) or dexamethasone (Dex), respectively. It was confirmed that the dyeing of Alizarin-red Solution increased more.
<실험예 5> 덱사메타손 병용사용에 대한 골형성 또는 치형성 유전자 마커의활성도 및 유전자 발현 확인<Experimental Example 5> Confirmation of the activity and gene expression of bone formation or dentition formation gene markers for the combined use of dexamethasone
제니포사이드와 덱사메타손의 병용사용에 대한 골형성 또는 치형성 유전자 마커인 ALP 발현 정도를 확인하기 위해 제니포사이드(geniposide, GPS) (50 μg/ml)와 덱사메타손(dexamethasone, Dex) (10 nM)을 HDPSCs에 7일과 14일 동안 처리하였다. HDPSCs에 RIPA Buffer 완충액 (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4)에 단백질 분해효소 억제제(P3100-001, GenDEPOT)를 처리하여 단백질을 추출하였다. 각 시료를 완충액과 섞어 5분간 끓인 후 Sodium dodecyl sulfate-polyacrylamide 겔(0227, AMRESCO)에서 전기영동 하여 단백질은 크기 별로 분리한 후 차단 완충액(blocking buffer(REF232100, BD Difco)으로 차단하고 소포체 스트레스 관련 항체의 발현을 확인하고자 4℃에서 하룻 밤 동안 반응시킨 후, 이차 항체로 상온에서 1시간 반응시켰다. ECL 용액 (Dae Myung Science Co., Ltd, Korea)를 사용하여 단백질 발현을 확인하였다. Membrane을 actin 항체와 다시 반응시켜 일정한 양의 단백질을 사용하였는지 확인하였다. Geniposide (GPS) (50 μg/ml) and dexamethasone (Dex) (10 nM) were used in HDPSCs to confirm the expression level of ALP, which is a marker for bone formation or dentition for the combined use of geniposide and dexamethasone. It was treated for 7 days and 14 days. In HDPSCs, RIPA Buffer buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4) was treated with a protease inhibitor (P3100-001, GenDEPOT) to extract the protein. Each sample is mixed with a buffer and boiled for 5 minutes, and then subjected to electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel (0227, AMRESCO) to separate proteins by size, and then block them with a blocking buffer (REF232100, BD Difco), and vesicle stress-related antibodies. In order to confirm the expression of, the reaction was carried out overnight at 4° C., and then reacted with a secondary antibody for 1 hour at room temperature, and protein expression was confirmed using an ECL solution (Dae Myung Science Co., Ltd, Korea). It was confirmed whether a certain amount of protein was used by reacting with the antibody again.
그 결과, 도 5A에 나타낸 바와 같이, 제니포사이드(geniposide, GPS) 또는 덱사메타손(dexamethasone, Dex)을 각각 사용한 경우보다, 제니포사이드(geniposide, GPS) 및 덱사메타손(dexamethasone, Dex)을 병용사용한 경우 ALP 유전자 발현이 더욱 증가한 것을 확인하였다.As a result, as shown in FIG. 5A, compared to the case of using geniposide (GPS) or dexamethasone (Dex), respectively, when using geniposide (GPS) and dexamethasone (Dex) in combination, the ALP gene It was confirmed that the expression was further increased.
또한, 제니포사이드와 덱사메타손의 병용사용에 대한 ALP활성도를 측정하기 위해, HDPSCs에 제니포사이드(geniposide, GPS) (50 μg/ml)와 덱사메타손(dexamethasone, Dex) (10 nM)을 각각 단독 또는 병용처리하고, 7일 또는 14일 동안 처리하였다. 그 후 7일과 14일 배양한 세포를 PBS로 세척하였다. PBS를 제거한 뒤 alkaline phosphatase substrate (alkaline phosphatase yellow (pNPP) liquid substrate system for ELISA (sigma)) 0.5 mL를 넣고 incubation 한 뒤 3 N NaOH로 반응을 중단시켰다. 405 nm에서 흡광도를 측정한 후 standard curve (p-nitrophenol standard solution, sigma)를 그려 ALP 활성도를 측정한 결과를 도 5B에 나타내었다. In addition, in order to measure the ALP activity for the combination use of geniposide and dexamethasone, geniposide (GPS) (50 μg/ml) and dexamethasone (dexamethasone, Dex) (10 nM) were treated alone or in combination in HDPSCs. And treated for 7 or 14 days. After that, the cells cultured on the 7th and 14th days were washed with PBS. After removing the PBS, 0.5 mL of alkaline phosphatase substrate (alkaline phosphatase yellow (pNPP) liquid substrate system for ELISA (sigma)) was added and incubated, and the reaction was stopped with 3 N NaOH. After measuring the absorbance at 405 nm, a standard curve (p-nitrophenol standard solution, sigma) was drawn to measure the ALP activity, and the results are shown in FIG. 5B.
그 결과, 제니포사이드(geniposide, GPS) 또는 덱사메타손(dexamethasone, Dex)을 각각 사용한 경우보다, 제니포사이드(geniposide, GPS) 및 덱사메타손(dexamethasone, Dex)을 병용사용한 경우 ALP 활성도가 더 증가하는 것을 확인하였다. 상기 결과는 제니포사이드(geniposide, GPS) 및 덱사메타손(dexamethasone, Dex)의 병용사용에 의해 더 많은 석회화가 이루어짐을 확인할 수 있다.As a result, it was confirmed that ALP activity increased further when geniposide (GPS) and dexamethasone (Dex) were used in combination compared to the case of using geniposide (GPS) or dexamethasone (Dex) respectively. . The results can be seen that more calcification is achieved by the combined use of geniposide (GPS) and dexamethasone (Dex).
또한, 조골세포 (MC3T3E1)와 인간 유래 치수줄기세포 (human dental pulp stem cells; HDPSCs)에서 상아모세포 분화 유전자 마커인 DSPP(상아질 시알로포스포프로틴, dentin sialophosphoprotein)와 뼈모세포 및/또는 백악모세포 분화 유전자 마커인 BSP(뼈 시알로단백질, bone sialoprotein)의 유전자 발현 수준을 확인한 결과를 도 5C에 나타내었다.In addition, osteoblasts (MC3T3E1) and human dental pulp stem cells (HDPSCs) are differentiated from oblastoma cell differentiation gene marker DSPP (dentin sialophosphoprotein) and osteoblasts and/or cretaceous cells. The result of confirming the gene expression level of the genetic marker BSP (bone sialoprotein) is shown in FIG. 5C.
그 결과, 제니포사이드(geniposide, GPS) 또는 덱사메타손(dexamethasone, Dex)을 각각 사용한 경우보다, 제니포사이드(geniposide, GPS) 및 덱사메타손(dexamethasone, Dex)을 병용사용한 경우 DSPP 및 BSP 유전자 발현이 더 증가하는 것을 확인하였다.As a result, when geniposide (GPS) and dexamethasone (Dex) were used in combination, DSPP and BSP gene expression increased more than when geniposide (GPS) or dexamethasone (Dex) was used respectively. Confirmed.
그 결과, 제니포사이드(geniposide, GPS) 또는 덱사메타손(dexamethasone, Dex)을 각각 사용한 경우보다, 제니포사이드(geniposide, GPS) 및 덱사메타손(dexamethasone, Dex)을 병용사용한 경우 DSPP 및 BSP 유전자 발현이 더 증가하는 것을 확인하였다.As a result, when geniposide (GPS) and dexamethasone (Dex) were used in combination, DSPP and BSP gene expression increased more than when geniposide (GPS) or dexamethasone (Dex) was used respectively. Confirmed.
<통계분석><Statistical Analysis>
결과들은 평균 ± 표준오차로 표시하고, 변수의 분석들은 Duncan's test를 사용하였다. P 값이 0.05 미만인 경우에 통계적으로 유의하다는 판정을 하였으며, 모든 실험은 독립적으로 3회 이상 실시하여 통계 처리를 하였다.Results were expressed as mean ± standard error, and Duncan's test was used for analysis of variables. When the P value was less than 0.05, it was determined that it was statistically significant, and all experiments were independently conducted three or more times to perform statistical processing.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특히 청구범위에 나타나 있으며, 그와 동등한 범위내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is not shown in the foregoing description, but in particular in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.
Claims (12)
상기 조성물은 덱사메타손(dexamethasone)을 더 포함하는 것을 특징으로 하는 조성물.The method of claim 1,
The composition is a composition, characterized in that it further comprises dexamethasone (dexamethasone).
상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)은 두충(Eucommia ulmoides Oliv.) 추출물로부터 분리된 화합물인 것을 특징으로 하는 조성물.The method of claim 1,
The geniposide (geniposide) or geniposidic acid (geniposidic acid) is a composition characterized in that the compound isolated from the Eucommia ulmoides Oliv. extract.
를 포함하는 치수줄기세포의 상아 전구세포로의 분화 유도 방법.Treating the pulp stem cells with geniposide or geniposidic acid;
A method for inducing differentiation of pulp stem cells into dentinal progenitor cells comprising a.
상기 처리는 7일 내지 14일 동안 수행되는 것을 특징으로 하는 치수줄기세포의 상아 전구세포로의 분화 유도 방법.The method of claim 4,
The treatment is a method for inducing the differentiation of pulp stem cells into dentinal progenitor cells, characterized in that performed for 7 to 14 days.
상기 조성물은 덱사메타손(dexamethasone)을 더 포함하는 것을 특징으로 하는 조성물.The method according to any one of claims 7 to 9,
The composition is a composition, characterized in that it further comprises dexamethasone (dexamethasone).
상기 제니포사이드(geniposide) 또는 제니포시딕산(geniposidic acid)은 두충(Eucommia ulmoides Oliv.) 추출물로부터 분리된 화합물인 것을 특징으로 하는 조성물.The method according to any one of claims 7 to 9,
The geniposide (geniposide) or geniposidic acid (geniposidic acid) is a composition characterized in that the compound isolated from the Eucommia ulmoides Oliv. extract.
상기 상아질-치수질환은 상아질 지각과민증, 치수충혈, 치수염, 치수변성, 치수의 괴사 및 괴저증으로 이루어진 군으로부터 선택되는 것을 특징으로 하는 조성물.The method according to any one of claims 7 to 9,
The dentin-dimensional disease is a composition, characterized in that selected from the group consisting of dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis and gangrene.
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