KR102580993B1 - Method of culturing Ixeris dentata NAKAI embryogenic callus for mass production of chrysoeriol-7-0-glucoside - Google Patents
Method of culturing Ixeris dentata NAKAI embryogenic callus for mass production of chrysoeriol-7-0-glucoside Download PDFInfo
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- KR102580993B1 KR102580993B1 KR1020200135450A KR20200135450A KR102580993B1 KR 102580993 B1 KR102580993 B1 KR 102580993B1 KR 1020200135450 A KR1020200135450 A KR 1020200135450A KR 20200135450 A KR20200135450 A KR 20200135450A KR 102580993 B1 KR102580993 B1 KR 102580993B1
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- embryogenic callus
- glucoside
- chrysoeriol
- callus
- mass production
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- A23V2200/00—Function of food ingredients
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Abstract
본 발명은 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법 및 상기 배양 방법으로 배양된 씀바귀 배발생 캘러스에 관한 것이다. 본 발명은 씀바귀 조직을 2,4-D 및 NAA(naphthaleneacetic acid)가 포함된 배지에 배양하여 배발생 캘러스(embryogenic callus, EC)로 유도하는 단계를 포함하는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법이다.The present invention relates to a method for cultivating embryogenic callus (EC) for mass production of chrysoeriol-7-0-glucoside and to embryogenic callus cultured by the culture method. will be. The present invention relates to chrysoeriol-7-0-glucoside, which includes the step of inducing embryogenic callus (EC) by culturing the soybean tissue in a medium containing 2,4-D and naphthaleneacetic acid (NAA). (chrysoeriol-7-0-glucoside) This is a method of cultivating embryogenic callus (EC) for mass production.
Description
본 발명은 크리소에리올-7-0-글루코사이드 대량생산용 씀바귀 배발생 캘러스 배양 방법에 관한 것이다.The present invention relates to a method of cultivating embryogenic callus of Chrysoeriol-7-0-glucoside for mass production.
약용 작물은 전통생약으로서 오랫동안 복용해오고 있으며, 안전성에 대한 우려가 적은 장점이 있으며, 현대 생명공학 기술을 바탕으로 새롭게 연구가 이어지고 있다. 특히 약용식물은 오랜 임상 경험을 통해 유효성 및 안전성이 입증되어 바이오소재 제품으로의 실패를 줄일 수 있고 빠른 개발이 가능하다.Medicinal crops have been used as traditional herbal medicines for a long time, have the advantage of less concerns about safety, and new research is continuing based on modern biotechnology. In particular, medicinal plants have proven effectiveness and safety through long-term clinical experience, which can reduce failures in biomaterial products and enable rapid development.
이러한 장점을 바탕으로 약용식물을 이용한 천연물 바이오 소재의 의약품 및 건강 기능식품으로 산업화가 가속화 되고 있으며, 국내외 의약품의 50%가 천연물에서 유래된 단일물질이다. 우리나라에서 약용자원으로 사용할 수 있는 식물은 약용 1,253종, 식용 826종으로 총 2,104종이 자생하고 있다. Based on these advantages, the industrialization of medicines and health functional foods made from natural biomaterials using medicinal plants is accelerating, and 50% of domestic and foreign medicines are single substances derived from natural products. In Korea, a total of 2,104 species of plants that can be used as medicinal resources grow naturally, including 1,253 medicinal species and 826 edible species.
그 중, 씀바귀(Ixeris dentata NAKAI)는 한국, 일본 및 중국 등지에서 주로 서식하는 약용 식물로서 간염, 소화불량, 당뇨병, 알러지 및 암의 치료 등 다양한 질병의 치료에 사용되고 있다. 씀바귀는 크리소에리올-7-0-글루코사이드를 함유하고 있으며, 크리소에리올-7-0-글루코사이드는 플라노보이드 계통의 이차 대사 산물로서, 기존 혈당강하 및 지질 강하 효과가 있다고 알려진 시나로사이드(cynaroside)보다 항균 및 살균 효과가 우수하고, 특히 메틸기가 존재하여 생물체로의 흡수가 더욱 용이할 것으로 추정된다.Among them, Ixeris dentata NAKAI is a medicinal plant that mainly grows in Korea, Japan, and China, and is used to treat various diseases such as hepatitis, indigestion, diabetes, allergies, and cancer. Cinnamon contains chrysoeriol-7-0-glucoside, which is a secondary metabolite of the flavonoid family and is known to have blood sugar-lowering and lipid-lowering effects. It is estimated that it has better antibacterial and sterilizing effects than cynaroside, and in particular, the presence of a methyl group makes it easier to absorb into living organisms.
그러나, 씀바귀는 시중 유통가격이 비싸고, 재배의 어려움으로 재배농가가 적어 경제성 있는 대량재배가 어려워 널리 보급하는데 결정적인 한계가 있으므로, 적은 비용으로 대량생산이 가능한 기술이 필요한 실정이다.However, the market price of bitter gourd is high, and due to the difficulty of cultivation, there are only a few farmers, making it difficult to cultivate economically in large quantities, so there is a critical limit to its widespread distribution. Therefore, technology that allows mass production at low cost is needed.
본 발명이 해결하고자 하는 과제는, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법 및 상기 배양 방법으로 배양된 씀바귀 배발생 캘러스에 관한 것이다.The problem to be solved by the present invention is a method for cultivating embryogenic callus (EC) for mass production of chrysoeriol-7-0-glucoside and a method for cultivating embryogenic callus (EC) cultured by the culture method. It is about the embryogenic callus of the snail.
또한, 본 발명은 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 이의 배양액을 포함하는 충치 예방 및 구취 억제용 약학적 조성물 또는 식품 조성물을 제공하고자 한다.In addition, the present invention seeks to provide a pharmaceutical composition or food composition for preventing tooth decay and suppressing bad breath, comprising embryogenic callus (EC), its extract, or its culture solution.
본 발명의 일실시예는 씀바귀 조직을 2,4-D 및 NAA(naphthaleneacetic acid)가 포함된 배지에 치상하고 배양하여 배발생 캘러스(embryogenic callus, EC)로 유도하는 단계를 포함하는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법이다.One embodiment of the present invention is chrysoeriol, which includes the step of inducing embryogenic callus (EC) by plating and culturing the tissue in a medium containing 2,4-D and NAA (naphthaleneacetic acid). This is a method of cultivating embryogenic callus (EC) for mass production of -7-0-glucoside (chrysoeriol-7-0-glucoside).
상기 씀바귀 조직은 잎, 엽병 또는 배축 조직일 수 있다.The shoot tissue may be a leaf, petiole, or hypocotyl tissue.
상기 2.4-D의 함량은 0.01~3.0mg/L이고, NAA의 함량은 0.005~0.2mg/L일 수 있다.The content of 2.4-D may be 0.01 to 3.0 mg/L, and the content of NAA may be 0.005 to 0.2 mg/L.
상기 배지는 수크로오스(Sucrose) 및 겔라이트(Gerlite)를 더 포함할 수 있다.The medium may further include sucrose and Gerlite.
상기 배양은 pH 5.5~6.5 및 20~30℃에서 암배양할 수 있다.The culture can be cultured in the dark at pH 5.5-6.5 and 20-30°C.
상기 배발생 캘러스(embryogenic callus, EC)는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 생합성량이 씀바귀 조직의 10~30배일 수 있다.The embryogenic callus (EC) may have a biosynthetic amount of chrysoeriol-7-0-glucoside that is 10 to 30 times that of callus tissue.
본 발명의 다른 실시예는 상기 배양 방법으로 배양된 크리소에리올-3-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC)이다.Another embodiment of the present invention is embryogenic callus (EC) for mass production of chrysoeriol-3-0-glucoside (chrysoeriol-7-0-glucoside) cultured using the above culture method.
본 발명의 또 다른 실시예는 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC)의 배양액을 포함하는 충치 예방 및 구취 억제용 약학적 조성물이다.Another embodiment of the present invention is a pharmaceutical composition for preventing tooth decay and suppressing bad breath, comprising embryogenic callus (EC), an extract thereof, or a culture medium of embryogenic callus (EC).
상기 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액은 침샘세포의 아밀라아제 분비능을 향상시킬 수 있다.The embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium can improve the amylase secretion ability of salivary gland cells.
본 발명의 또 다른 실시예는 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액을 포함하는 충치 예방 및 구취 억제용 식품 조성물이다.Another embodiment of the present invention is a food composition for preventing tooth decay and suppressing bad breath, comprising embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium.
본 발명의 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법을 이용하여 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)를 대량생산 할 수 있으며, 이에 따라 배양된 배발생 캘러스로부터 고 함량의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)를 대량으로 생산하여 산업화 할 수 있다.Chrysoeriol-7-0-glucoside can be mass-produced using the embryogenic callus (EC) culture method of the present invention, and thus cultured embryogenesis High-content chrysoeriol-7-0-glucoside can be produced and industrialized in large quantities from callus.
또한, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)을 포함하는 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC)의 배양액을 포함하는 충치 예방 및 구취 억제용 약학적 조성물 또는 식품 조성물을 제공할 수 있다.In addition, embryogenic callus (EC) containing chrysoeriol-7-0-glucoside, its extract, or culture medium of embryogenic callus (EC) A pharmaceutical composition or food composition for preventing cavities and suppressing bad breath containing a can be provided.
도 1은 본 발명의 일 실시예에 따라 씀바귀로부터 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)의 사진이다.
도 2는 본 발명에 따른 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 생합성 모식도이다.
도 3은 본 발명에 따라 배양 조건을 달리하여 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)의 사진이다.
도 4는 본 발명에 따라 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)의 과산화수소수 반응을 나타낸 사진이다.
도 5는 본 발명에 따라 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)를 주사전자현미경(scanning electron microscope, SEM)으로 촬영한 사진이다.
도 6은 본 발명에 따라 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)를 투과전자현미경(transmission electron microscope, TEM)으로 촬영한 사진이다.
도 7은 씀바귀 조직, 씀바귀 배발생 캘러스(embryogenic callus, EC) 및 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 생합성량을 비교한 그래프이다.
도 8은 씀바귀 배발생 캘러스(embryogenic callus, EC)의 추출물의 농도별 세포 생존도를 나타낸 도면이다.
도 9는 씀바귀 배발생 캘러스(embryogenic callus, EC)의 추출물의 농도별 침샘세포의 아밀라이제 분비능을 나타낸 도면이다.Figure 1 is a photograph of embryogenic callus (EC) derived from E. vulgaris according to an embodiment of the present invention.
Figure 2 is a schematic diagram of the biosynthesis of chrysoeriol-7-0-glucoside according to the present invention.
Figure 3 is a photograph of embryogenic callus (EC) induced under different culture conditions according to the present invention.
Figure 4 is a photograph showing the reaction of embryogenic callus (EC) induced according to the present invention to hydrogen peroxide.
Figure 5 is a photograph taken with a scanning electron microscope (SEM) of embryogenic callus (EC) induced according to the present invention.
Figure 6 is a photograph taken using a transmission electron microscope (TEM) of embryogenic callus (EC) induced according to the present invention.
Figure 7 shows the biosynthesis of chrysoeriol-7-0-glucoside in Erythrophyllosa tissue, embryogenic callus (EC), and embryogenic callus (EC) culture medium. This is a graph comparing quantities.
Figure 8 is a diagram showing cell viability according to concentration of extract of embryogenic callus (EC).
Figure 9 is a diagram showing the amylase secretion ability of salivary gland cells according to the concentration of extract of embryogenic callus (EC).
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 구체적인 실시예를 통하여 보다 상세하게 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다.Hereinafter, the present invention will be described in more detail through specific embodiments so that those skilled in the art can easily implement it. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.
본 발명자들은 씀바귀의 구강건조증 치료를 위한 구강 분무제에 대한 기술에 대하여 출원한 바 있으며, 이에 따라 특허출원 제10-2017-0023894호에 개시된 내용은 모두 본 발명에 대한 참고가 될 수 있다.The present inventors have applied for a technology for an oral spray for the treatment of dry mouth, and accordingly, all of the content disclosed in Patent Application No. 10-2017-0023894 can be used as a reference for the present invention.
본 발명의 일실시예는 씀바귀 조직을 2,4-D 및 NAA(naphthaleneacetic acid)가 포함된 배지에 배양하여 배발생 캘러스(embryogenic callus, EC)로 유도하는 단계를 포함하는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법이다.One embodiment of the present invention is chrysoeriol-7, which includes the step of inducing embryogenic callus (EC) by culturing the snail tissue in a medium containing 2,4-D and NAA (naphthaleneacetic acid). This is a method of cultivating embryogenic callus (EC) for mass production of -0-glucoside (chrysoeriol-7-0-glucoside).
도 1의 A는 본 발명의 일 실시예에 따라 씀바귀로부터 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)의 사진이고, 도 1의 B는 비배발생 캘러스의 사진이다. 도 2는 본 발명에 따른 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 생합성 모식도이다.A in FIG. 1 is a photograph of embryogenic callus (EC) derived from E. chinensis according to an embodiment of the present invention, and B in FIG. 1 is a photograph of non-embryogenic callus. Figure 2 is a schematic diagram of the biosynthesis of chrysoeriol-7-0-glucoside according to the present invention.
도 1에 따른 배발생 캘러스(embryogenic callus, EC)는 식물체의 조직배양 시 외식편(外植片)을 배지 위에 치상하고 적정 배양조건에서 배양하면 일정한 체제를 이루고 있는 세포괴를 형성하며 이 세포괴를 캘러스라 하고 배형성 능력을 가진 캘러스를 의미한다.Embryogenic callus (EC) according to Figure 1 forms a cell mass with a certain structure when an explant is placed on a medium and cultured under appropriate culture conditions during tissue culture of a plant, and this cell mass is called callus. It refers to callus that has the ability to form embryos.
상기 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)는 플라노보이드 계통인 이차 대사 산물로서, 도 2에 다른 모식도를 거쳐 생합성될 수 있다.The chrysoeriol-7-0-glucoside (chrysoeriol-7-0-glucoside) is a secondary metabolite of the flavonoid family and can be biosynthesized through another schematic diagram in FIG. 2.
본 발명에서 상기 씀바귀 조직은 잎, 엽병 또는 배축일 수 있으며, 바람직하게는 배축 또는 엽병일 수 있으며, 더욱 바람직하게는 엽병일 수 있다.In the present invention, the shoot tissue may be a leaf, petiole, or hypocotyl, preferably the hypocotyl or petiole, and more preferably the petiole.
상기 배축은 고등식물의 배에 속한 기관의 일부로서 자엽 아래 부위에 최초로 형성되는 줄기 부분을 말하며, 상기 엽병은 잎몸을 줄기나 가지에 붙게 하는 꼭지 부분을 말한다.The hypocotyl is a part of the organ belonging to the embryo of higher plants and refers to the stem part that is first formed in the area below the cotyledons, and the petiole refers to the apex part that attaches the leaf body to the stem or branch.
상기 잎, 엽병 또는 배축의 크기는 1~3mm일 수 있다.The size of the leaf, petiole, or hypocotyl may be 1 to 3 mm.
또한, 본 발명에서 상기 씀바귀 조직을 배지에 치상하기 전에, 씀바귀 조직을 소독하는 살균 과정이 진행될 수 있다.Additionally, in the present invention, before placing the tissue on a medium, a sterilization process may be performed to sterilize the tissue.
상기 살균은 다음과 같은 순서로 진행될 수 있으며, 살균 처리시간에 따라 시간이 길어질 경우 조직이 파괴되고, 시간이 부족할 경우 표면조직의 각종 곰팡이 및 세균이 완전히 살균되지 않아 오염이 심하기 때문에 적당한 처리시간으로 살균을 실시하는 것이 중요하다.The sterilization may be carried out in the following order. Depending on the sterilization treatment time, if the time is prolonged, the tissue is destroyed, and if the time is insufficient, various molds and bacteria on the surface tissue are not completely sterilized, resulting in severe contamination. Therefore, an appropriate treatment time is required. It is important to perform sterilization.
먼저, 50~90% 에탄올로 10~60초간 살균한다. 다음으로 살균된 조직을 멸균 증류수로 1~5회 세척한 후, 두 번에 걸쳐 차아염소산나트륨(Sodium Hypochlorite) 수용액에 소독한다. 첫번째 소독은 1% 차아염소산나트륨(Sodium Hypochlorite)에 3~5분간 침지한 후, 표피에 남아있는 소독약제가 내피세포로 침투하는 것을 방지하기 위하여 멸균된 증류수로 1~5회 세척한다. 이후, 다시 한 번 0.5% 차아염소산나트륨에 3~10분간 침지한 후, 멸균된 증류수로 3~5회 세척한다.First, sterilize with 50-90% ethanol for 10-60 seconds. Next, the sterilized tissue is washed 1 to 5 times with sterile distilled water and then disinfected twice with an aqueous solution of sodium hypochlorite. The first disinfection is immersed in 1% sodium hypochlorite for 3 to 5 minutes, then washed 1 to 5 times with sterilized distilled water to prevent the disinfectant remaining on the epidermis from penetrating into the endothelial cells. Afterwards, it is once again immersed in 0.5% sodium hypochlorite for 3 to 10 minutes and then washed 3 to 5 times with sterilized distilled water.
멸균된 씀바귀 조직은 2,4-D 및 NAA(naphthaleneacetic acid)가 포함된 배지에 치상할 수 있다.Sterilized tissue can be placed in a medium containing 2,4-D and NAA (naphthaleneacetic acid).
상기 2,4-D(2-4-dichlorophenoxy acetic acid)는 생장조절물질인 옥신의 한 종류이다. 상기 2,4-D의 함량은 0.01~3.0mg/L일 수 있으며, 바람직하게는 0.05~0.1mg/L일 수 있다. 상기 2,4-D의 함량이 0.01mg/L 미만이거나, 3.0mg/L을 초과하면 배발생 캘러스의 형성이 용이하지 않을 수 있다.The 2,4-D (2-4-dichlorophenoxy acetic acid) is a type of auxin, a growth regulator. The content of 2,4-D may be 0.01 to 3.0 mg/L, preferably 0.05 to 0.1 mg/L. If the content of 2,4-D is less than 0.01 mg/L or exceeds 3.0 mg/L, the formation of embryogenic callus may not be easy.
상기 NAA(naphthaleneacetic acid)는 나프탈렌 아세트산으로 생장조절물질인 옥신의 한 종류이다. 인돌아세트산과 거의 같지만 안정성이 높아서 발근을 촉진하거나 단위 결실을 유도하는 데 사용된다. 상기 NAA의 함량은 0.005~0.2mg/L일 수 있고, 바람직하게는 0.01~0.1mg/L일 수 있다. 상기 NAA의 함량이 0.005mg/L 미만이거나, 0.2mg/L를 초과하면 배발생 캘러스의 형성이 용이하지 않을 수 있다.The NAA (naphthaleneacetic acid) is naphthalene acetic acid and is a type of auxin, a growth regulator. It is almost the same as indole acetic acid, but has high stability, so it is used to promote rooting or induce parthenocarpy. The NAA content may be 0.005 to 0.2 mg/L, and preferably 0.01 to 0.1 mg/L. If the NAA content is less than 0.005 mg/L or more than 0.2 mg/L, it may not be easy to form embryogenic callus.
본 발명에서 2,4-D 및 NAA를 함께 첨가함으로써 시너지 효과가 발생하여 단독으로 첨가하는 경우보다 씀바귀 배발생 캘러스(embryogenic callus, EC)의 배양 효과를 극대할 수 있다.In the present invention, a synergistic effect occurs by adding 2,4-D and NAA together, which can maximize the culture effect of embryogenic callus (EC) compared to adding them alone.
상기 배지는 고체 MS 배지일 수 있고, MS 염류가 20~60중량% 포함된 액체 배지일 수 있으며, MS 배지는 하기 표 1과 같은 조성일 수 있으며, 수크로오스(Sucrose) 및 겔라이트(Gerlite)를 더 포함할 수 있다.The medium may be a solid MS medium or a liquid medium containing 20 to 60% by weight of MS salts. The MS medium may have a composition as shown in Table 1 below, and may further contain sucrose and Gerlite. It can be included.
본 발명에서 상기 배양은 pH 5.5~6.5 및 20~30℃에서 암배양될 수 있다. In the present invention, the culture can be cultured in the dark at pH 5.5-6.5 and 20-30°C.
또한, 본 발명에 따라 배양된 상기 배발생 캘러스(embryogenic callus, EC)는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 생합성량이 일반 씀바귀 조직의 10~30배일 수 있다.In addition, the embryogenic callus (EC) cultured according to the present invention may have a biosynthetic amount of chrysoeriol-7-0-glucoside that is 10 to 30 times that of general snail tissue. there is.
이와 같이, 본 발명에 따른 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법을 이용하면 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스를 배양할 수 있다.In this way, by using the embryogenic callus (EC) culture method according to the present invention, embryogenic callus of the embryonic callus for mass production of chrysoeriol-7-0-glucoside can be cultured. can do.
본 발명의 다른 실시예는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC)이며, 상기 배발생 캘러스(embryogenic callus, EC)는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 생합성량이 일반 씀바귀 조직의 10~30배일 수 있다.Another embodiment of the present invention is embryogenic callus (EC) for mass production of chrysoeriol-7-0-glucoside, and the embryogenic callus (EC) ) may have a biosynthetic amount of chrysoeriol-7-0-glucoside that is 10 to 30 times that of general snail tissue.
본 발명의 또 다른 실시예는 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액을 포함하는 충치 예방 및 구취 억제용 약학적 조성물이다.Another embodiment of the present invention is a pharmaceutical composition for preventing tooth decay and suppressing bad breath, comprising embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium.
본 발명에서 "추출물"이란 배발생 캘러스(embryogenic callus, EC)를 용매에 녹여 분리한 물질로, 증류 또는 증발을 이용하여 농축될 수 있다.In the present invention, “extract” is a material separated by dissolving embryogenic callus (EC) in a solvent, and can be concentrated using distillation or evaporation.
또한, "배양액"이란 세포를 배양시킨 다음, 세포를 제외하고 남은 세포 배양용액을 의미한다. Additionally, “culture medium” refers to the cell culture solution remaining after culturing cells, excluding the cells.
본 발명에서 "캘러스"란, 탈분화 과정을 통하여 분화하지 않은 상태로 된 세포 또는 세포덩어리를 말하며, 줄기세포를 포함할 수 있다.In the present invention, “callus” refers to cells or cell masses that have become undifferentiated through a dedifferentiation process and may include stem cells.
상기 추출물은 1) 추출시료에 추출용매를 가하여 추출하는 단계; 2) 단계 1)의 추출물을 여과하는 단계; 및 3) 단계 2)의 여과한 추출물을 감압 농축한 후 건조하는 단계를 거쳐 제조될 수 있다.The extract is extracted by adding an extraction solvent to the extraction sample; 2) filtering the extract of step 1); and 3) concentrating the filtered extract of step 2) under reduced pressure and drying it.
상기 추출 용매는 물, 탄소수 1 내지 6인 알코올, 헥산, 에틸아세테이트 및 이들의 혼합용매로 이루어진 군에서 선택된 어느 하나의 용매로 추출한 것일 수 있다. 본 발명의 추출물은 바람직하게는 물, 탄소수 1 내지 6인 알코올 및 이들의 혼합용매에서 선택된 용매로 추출한 것일 수 있으며, 또는 탄소수 1 내지 6의 저급 알코올 수용액일 수 있다. 상기 알코올 수용액의 농도는 10 내지 100%(v/v)일 수 있으며, 바람직하게는 70 내지 100%(v/v)일 수 있다. 또한, 상기 추출용매는 건조된 씀바귀 분량의 2 내지 20 배 첨가하여 추출하는 것이 바람직하다. 추출온도는 20 내지 80℃인 것이 바람직하나 이에 한정하지 않는다. 또한, 추출시간은 10 내지 100 시간인 것이 바람직하며, 구체적으로 24 내지 96 시간이 더욱 바람직하다.The extraction solvent may be one extracted with any one solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, hexane, ethyl acetate, and mixed solvents thereof. The extract of the present invention may preferably be extracted with a solvent selected from water, alcohols having 1 to 6 carbon atoms, and mixed solvents thereof, or may be an aqueous solution of lower alcohols having 1 to 6 carbon atoms. The concentration of the alcohol aqueous solution may be 10 to 100% (v/v), preferably 70 to 100% (v/v). In addition, it is preferable to extract the extract by adding 2 to 20 times the amount of dried waste. The extraction temperature is preferably 20 to 80°C, but is not limited thereto. Additionally, the extraction time is preferably 10 to 100 hours, and more specifically, 24 to 96 hours.
본 발명에서의 추출 방법은 당업계에 공지된 것이면 제한 없이 사용할 수 있으며, 그 예로 냉침, 열수 추출, 초음파 추출, 환류냉각 추출 등의 방법이 있으나 여기에 국한되는 것은 아니다.The extraction method in the present invention can be used without limitation as long as it is known in the art, and examples include cold immersion, hot water extraction, ultrasonic extraction, and reflux cooling extraction, but are not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the vacuum concentration in step 3) is preferably performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.
본 발명의 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액은 1 ㎍ /㎖ 내지 100 ㎎/㎖의 농도로 충치 예방 및 구취 억제용 약학적 조성물에 포함될 수 있다. 구체적으로, 상기 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액은 50 ㎍ /㎖ 내지 100 ㎎/㎖의 농도로 포함될 수 있으며, 보다 구체적으로 1 ㎎ /㎖ 내지 80㎎/㎖의 농도로 포함되는 것이 가장 바람직하다.Embryogenic callus (EC) of the present invention, its extract, or embryogenic callus (EC) culture medium is a pharmaceutical composition for preventing tooth decay and suppressing bad breath at a concentration of 1 μg / ml to 100 mg / ml. may be included in Specifically, the embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium may be included at a concentration of 50 ㎍ / ㎖ to 100 mg / ㎖, more specifically 1 It is most preferable that it is contained at a concentration of ㎎ / ㎖ to 80㎎ / ㎖.
또한, 상기 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액은 침샘세포의 아밀라이제 분비능을 향상시킬 수 있다.In addition, the embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium can improve the amylase secretion ability of salivary gland cells.
상기 조성물이 약학적 조성물의 형태로 제공되는 경우에는, 본 발명의 상기 조성물 이외에 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 함유할 수 있다. 상기에서 "약학적으로 허용되는" 이란 생리학적으로 허용되고 인간에게 투여될 때, 활성성분의 작용을 저해하지 않으며 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 비독성의 조성물을 말한다.When the composition is provided in the form of a pharmaceutical composition, it may further contain one or more pharmaceutically acceptable carriers, excipients, or diluents in addition to the composition of the present invention. As used above, “pharmaceutically acceptable” means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions. says
약학적으로 허용되는 담체로는 예컨대, 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 아울러, 펩티드 제제에 대한 경구투여용으로 사용되는 다양한 약물전달물질을 포함할 수 있다. 또한, 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코오스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현택제 등을 추가로 포함할 수 있다. 그 밖의 약학적으로 허용되는 담체 및 제제는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, it may contain various drug delivery substances used for oral administration of peptide preparations. Additionally, the carrier for parenteral administration may include water, suitable oil, saline solution, aqueous glucose, glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. in addition to the above components. Other pharmaceutically acceptable carriers and agents may be referred to as described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
본 발명의 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들면, 경구 또는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다.The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. It can be.
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있으며, 분무제의 형태로 제형화될 수 있다.The pharmaceutical composition of the present invention can be formulated as a preparation for oral or parenteral administration according to the route of administration as described above, and may be formulated in the form of a spray.
경구 투여용 제제의 경우에 본 발명의 조성물은 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 등으로 당업계에 공지된 방법을 이용하여 제형화될 수 있다. 예를 들어, 경구용 제제는 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈,메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In the case of a formulation for oral administration, the composition of the present invention can be formulated into powder, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, slurries, suspensions, etc. using methods known in the art. You can. For example, oral preparations can be prepared by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose, gelatin, polyvinylpyrrolidone, etc. may be included. Additionally, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.
비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌(Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA,1995)에 기재되어 있다.In the case of preparations for parenteral administration, they can be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a commonly known text in all pharmaceutical chemistry.
본 발명의 조성물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 바람직하게 본 발명의 약학적 조성물의 바람직한 전체 용량은 1일당 환자 체중 1㎏ 당 약 0.01㎍ 내지 10,000mg, 가장 바람직하게는 0.1㎍ 내지 1,000mg일 수 있다. 그러나 상기 약학적 조성물의 용량은 제제화 방법, 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율등 다양한 요인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 본 발명의 조성물의 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다. The total effective amount of the composition of the present invention can be administered to a patient in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 1,000 mg per kg of patient body weight per day. However, the effective dose for the patient is determined by considering various factors such as the formulation method, administration route, and number of treatments as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate. Therefore, taking this into account, anyone skilled in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.
본 발명의 또 다른 실시예는 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액을 포함하는 충치 예방 및 구취 억제용 식품 조성물이다.Another embodiment of the present invention is a food composition for preventing tooth decay and suppressing bad breath, comprising embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium.
본 발명의 상기 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food)및 식품 첨가제(food additives)등의 모든 형태를 포함한다. 상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.The food composition of the present invention includes all types of functional foods, nutritional supplements, health foods, and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.
예를 들면, 건강 식품으로는 상기 조성물 자체를 차, 주스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마말레이드 등), 어류, 육류, 및 그 가공식품(예: 햄, 소시지 콘비프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등)등에 추출물을 첨가하여 제조할 수 있다. 또한, 본 발명의 조성물을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다. For example, as a health food, the composition itself can be prepared and consumed in the form of tea, juice, and drinks, or can be consumed by granulating, encapsulating, and powdering. In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, marmalades, etc.), fish, meat, and their processed foods (e.g. ham, sausages, corned beef, etc.), Bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, It can be manufactured by adding extracts to frozen foods and various seasonings (e.g. soybean paste, soy sauce, sauce, etc.). Additionally, in order to use the composition of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.
본 발명의 식품 조성물 중 상기 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액의 바람직한 함량은 식품용 조성물 총 중량에 대하여 0.001 내지 50% 일 수 있으며, 바람직하게는 0.1 내지 50% 범위로 함유될 수 있다.In the food composition of the present invention, the preferred content of embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium may be 0.001 to 50% based on the total weight of the food composition. , preferably in the range of 0.1 to 50%.
이하, 본 발명에 따른 구체적인 실시예를 들어 설명한다.Hereinafter, specific embodiments according to the present invention will be described.
실시예 1 : 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양Example 1: Embryogenic callus (EC) culture
본 발명에서 사용한 씀바귀의 배양용 재료는 농업과학원 생물자원부 온실 내 화분에서 재배 중인 식물체에서 잎의 크기 2.5cm, 전체 크기 7~8cm 정도의 씀바귀를 선별하여 사용하였다.The material used in the present invention for cultivating the sagebrush was selected from plants growing in pots in the greenhouse of the Department of Biological Resources of the National Institute of Agricultural Sciences, with a leaf size of 2.5 cm and an overall size of about 7-8 cm.
재료 살균Material sterilization
씀바귀의 내피조직은 보존하고 표피조직의 완벽한 살균을 위하여, 70% 에탄올에 30초 침지한 후, 멸균증류수로 3회 세척하고, 두 번에 걸쳐 차아염소산나트륨(Sodium Hypochlorite) 수용액에 소독하는 데 첫번째 소독은 1% 차아염소산나트륨(Sodium Hypochlorite) 3~5분간 침지한 후, 표피에 남아있는 소독약제가 내피세포로 침투하는 것을 방지하기 위하여 멸균된 증류수로 1~5회 세척한다. 그리고 다시 한 번 0.5% 차아염소산나트륨에 3~10분간 침지한 후 멸균수로 3~5회 세척한다.To preserve the endothelial tissue and completely sterilize the epidermal tissue, the first step is to immerse it in 70% ethanol for 30 seconds, wash it three times with sterilized distilled water, and disinfect it twice with an aqueous solution of sodium hypochlorite. For disinfection, immerse in 1% sodium hypochlorite for 3 to 5 minutes, then wash with sterilized distilled water 1 to 5 times to prevent the disinfectant remaining on the epidermis from penetrating into the endothelial cells. Then, soak it again in 0.5% sodium hypochlorite for 3 to 10 minutes and wash it 3 to 5 times with sterilized water.
배양culture
씀바귀의 잎은 2~3mm로 준비하고, 엽병은 잎과 배축을 이여주는 부분을 2mm로 준비하고, 배축은 2mm로 준비하고, 뿌리는 MS 기본 배지에 생장점 배양을 하여 식물체로 발달된 씀바귀를 뿌리를 8주 배양하여 2mm로 잘라 준비하였다.The leaves of the plant are prepared to 2-3 mm, the petiole connecting the leaf and the hypocotyl is prepared to be 2 mm, the hypocotyl is prepared to be 2 mm, and the roots are grown at the growing point on MS basic medium and the developed plant is rooted. were cultured for 8 weeks and cut into 2mm pieces.
상기 잎, 엽병, 배축 및 뿌리를 배양 배지에 치상하였으며, 25±1℃에서 암배양하여 배발생 캘러스(embryogenic callus) 유도 배양을 실시하였으며, 식물을 부위별로 배양한 지 10일 정도부터 callus 형성을 관찰하기 시작하였다.The leaves, petioles, hypocotyls, and roots were placed on a culture medium, and cultured in the dark at 25 ± 1°C to induce embryogenic callus. Callus formation was observed from about 10 days after culturing the plants for each part. I started observing.
상기 배양배지는 표 1의 기본 MS 배지에 30g의 수크로오스 및 4g의 겔라이트를 첨가하였으며, 2,4D, NAA 및 사이토카인의 한 종류인 BAP(벤조피렌)를 하기 표 2와 같이 첨가하였으며, 유도된 캘러스를 도 3에 나타내었다.The culture medium was prepared by adding 30 g of sucrose and 4 g of gelite to the basic MS medium in Table 1, and 2,4D, NAA, and BAP (benzopyrene), a type of cytokine, were added as shown in Table 2 below, and the induced The callus is shown in Figure 3.
도 3은 본 발명에 따라 배양 조건을 달리하여 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)의 사진으로서, 도 3a는 잎, 엽병 및 배축의 배양 조건을 달리하여 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)의 사진이고, 도 3b는 뿌리의 배양 조건을 달리하여 유도된 씀바귀 배발생 캘러스(embryogenic callus, EC)의 사진이다. 도 3a에서 A는 잎, B는 엽병, C는 배축이고, 도 3a 및 도 3b에서 D는 MS 기본 배지에 첨가된 2,4-D의 함량(mg/L)이고, N은 MS 기본 배지에 첨가된 NAA의 함량(mg/L)이며, B는 MS 기본 배지에 첨가된 BAP의 함량이다.Figure 3 is a photograph of embryogenic callus (EC) induced by varying the culture conditions according to the present invention, and Figure 3a is a photograph of embryogenic callus (EC) induced by varying the culture conditions of leaves, petioles, and hypocotyls. callus (EC), and Figure 3b is a photograph of embryogenic callus (EC) induced by different root culture conditions. In Figure 3a, A is the leaf, B is the petiole, and C is the hypocotyl. In Figures 3a and 3b, D is the content (mg/L) of 2,4-D added to the MS basic medium, and N is the content of 2,4-D added to the MS basic medium. This is the content of added NAA (mg/L), and B is the content of BAP added to MS basic medium.
도 3을 참조하면, 호르몬 무첨가 배지에서는 완전한 식물체로 분화하거나 본연의 색인 초록색을 띠며 유지해 나갔다. 배발생 캘러스 유도에 있어서 2,4-D를 다양한 농도(0.05~3mg/L)로 처리한 결과(배양배지 1 내지 7) 낮은 농도인 0.05~0.1mg/L(배양배지 1 및 2)에서 배발생 캘러스 유도가 가장 잘 되었으며, 배발생 캘러스의 특징인 하얗고 연한 노란색이면서 부슬거리며 구상배 전단계의 배발생 캘러스를 육안으로 확인할 수 있었다. Referring to Figure 3, in the hormone-free medium, plants differentiated into complete plants or maintained their natural green color. As a result of treating 2,4-D at various concentrations (0.05 to 3 mg/L) in inducing embryogenic callus (culture media 1 to 7), embryo growth occurred at low concentrations of 0.05 to 0.1 mg/L (culture media 1 and 2). The induction of callus development was the best, and the white, light yellow, and crumbly embryonic callus in the pre-globular embryo stage, which are the characteristics of embryogenic callus, could be confirmed with the naked eye.
또한, 0.2mg/L 처리구(배양배지 6)부터 농도가 올라갈수록 배발생 캘러스의 색이 노란색을 점점 띠며 형성량은 적어짐을 관찰할 수 있었다. 씀바귀는 부위별로 배양했을 때 잎, 엽병 및 배축 부위에서 배발생 캘러스가 잘 관찰 되었으며, 특히 엽병에서 가장 좋은 배발생 캘러스를 얻을 수 있었으나, 뿌리에서는 배발생 캘러스 형성률이 낮았다. In addition, it was observed that as the concentration increased from the 0.2 mg/L treatment group (culture medium 6), the color of embryonic callus gradually became yellow and the amount formed decreased. When Siberia was cultured by region, embryogenic callus was well observed in the leaves, petioles, and hypocotyl regions. In particular, the best embryogenic callus was obtained from the petioles, but the rate of embryogenic callus formation was low in the roots.
또한, NAA를 혼합처리한 결과, 2,4-D는 0.1mg/L로 고정하고, NAA를 0.01~0.1mg/L 혼합처리한구(배양배지 8 내지 10)에서 2,4-D 단독처리구(배양배지 1 내지 7)보다 더 많은 배발생 캘러스를 얻을 수 있었다. 반면 2,4-D를 2.0mg/L로 고정하고, NAA를 0.1~0.2mg/L로 혼합처리한구(배양배지 11 및 12)에서는 배발생 캘러스 형성률이 낮았고 노란색을 띠며 배발생 캘러스는 관찰할 수 없었다. 이로부터 식물의 종류나 호르몬의 종류 및 농도에 따라 상승적 효과가 나타나기도 하고 억제하기도 한다는 것을 확인할 수 있다.In addition, as a result of the NAA mixed treatment, 2,4-D was fixed at 0.1 mg/L, and the 2,4-D alone treatment group (culture medium 8 to 10) was different from the group treated with the mixture of NAA at 0.01 to 0.1 mg/L (culture medium 8 to 10). More embryogenic callus could be obtained than in culture media 1 to 7). On the other hand, in the group where 2,4-D was fixed at 2.0 mg/L and NAA was mixed at 0.1~0.2 mg/L (culture medium 11 and 12), the rate of embryogenic callus formation was low, yellow color, and embryogenic callus was not observable. I couldn't. From this, it can be seen that depending on the type of plant or the type and concentration of hormone, a synergistic effect can occur or it can be suppressed.
반면, 2,4-D와 cytokinin계통의 대표적인 호르몬인 BAP를 혼한처리한구(배양배지 13 내지 15, 미도시)에서는 어떠한 처리구에서도 반응이 나타나지 않았고 오히려 배양 기간이 길어질수록 갈변되며 죽었다.On the other hand, in the group treated with a mixture of 2,4-D and BAP, a representative hormone of the cytokinin system (culture medium 13 to 15, not shown), no reaction was observed in any of the treatments, and rather, the longer the culture period, the more browning and death occurred.
실시예 2 : 씀바귀 배발생 캘러스(embryogenic callus, EC)의 과산화수소수 반응Example 2: Hydrogen peroxide reaction of embryogenic callus (EC)
실시예 1에서 배양된 씀바귀 배발생 캘러스가 배아줄기세포임을 확인하기 위하여 배양배지 9에서 배양된 씀바귀 배발생 캘러스(embryogenic callus, EC)에 과산화수소수를 처리하였으며, 이를 도 4에 나타내었다.To confirm that the embryogenic callus (EC) cultured in Example 1 was an embryonic stem cell, the embryogenic callus (EC) cultured in culture medium 9 was treated with hydrogen peroxide, which is shown in Figure 4.
형성층 유래의 어린 줄기세포에 다량 존재하는 것으로 알려진 peroxidase 및 catalase에 의해 과산화수소수는 물과 산소로 분해된다. 도 4를 참조하면, 본 발명에서 분리 배양한 씀바귀 배발생 캘러스에 과산화수소수를 처리한 결과, 물과 다량의 산소가 발생되어 기포로 뒤덮이는 결과를 보였으므로, 씀바귀 배발생 캘러스가 형성층 유래의 씀바귀 배아 줄기세포임을 알 수 있다.Hydrogen peroxide is decomposed into water and oxygen by peroxidase and catalase, which are known to exist in large quantities in cambium-derived young stem cells. Referring to Figure 4, as a result of treating hydrogen peroxide solution to the embryonic callus of the embryonic callus isolated and cultured in the present invention, a large amount of water and oxygen were generated and the result was covered with air bubbles, so the embryonic callus of the embryonic callus was derived from the cambium. It can be seen that these are embryonic stem cells.
실시예 3 : 씀바귀 배발생 캘러스(embryogenic callus, EC)의 SEM 및 TEM 촬영Example 3: SEM and TEM imaging of embryogenic callus (EC)
실시예 1에서 배양된 씀바귀 배발생 캘러스가 체세포배임을 확인하기 위하여 SEM 및 TEM으로 촬영하여 이를 도 5 및 도 6에 나타내었다.In order to confirm that the embryogenic callus of the embryonic callus cultured in Example 1 was a somatic embryo, it was photographed using SEM and TEM, and these are shown in Figures 5 and 6.
도 5를 참조하면, 배발생 캘러스의 특징적인 여러 과정의 발달단계를 확인할 수 있으며, 도 6을 참조하면, 배발생 캘러스의 특징인 핵이 뚜렷하게 존재하며 작은 액포가 다량으로 존재하고, 에너지 저장소인 미토콘드리아가 다량으로 존재함을 확인할 수 있었다. 또한, 세포질이 응집되어 있으며, 백색체 내부에 전분립 등 여러 소기관이 발달됨을 관찰할 수 있었다. 이에 따라 실시예 1에서 배양된 씀바귀 배발생 캘러스가 체세포배 발달 과정 중에 있음을 확인할 수 있었다.Referring to Figure 5, the development stages of various processes characteristic of embryogenic callus can be confirmed. Referring to Figure 6, the nucleus, which is a characteristic of embryogenic callus, is clearly present, a large number of small vacuoles are present, and the energy storage It was confirmed that mitochondria were present in large quantities. In addition, it was observed that the cytoplasm was aggregated and that various organelles, such as starch granules, were developed inside the white body. Accordingly, it was confirmed that the embryogenic callus of Siberian snail cultured in Example 1 was in the process of somatic embryo development.
실시예 4 : 씀바귀 배발생 캘러스, 씀바귀 조직 및 배양액 내 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 함량 비교Example 4: Comparison of chrysoeriol-7-0-glucoside content in callus embryogenic callus, tissue and culture medium
크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 함량을 분석하기 위하여, 액체 크로마토그래피-질량분석법(LC-MS/MS)를 이용하였다.To analyze the content of chrysoeriol-7-0-glucoside, liquid chromatography-mass spectrometry (LC-MS/MS) was used.
구체적으로, 실시예 1에서 배양한 씀바귀 배발생 캘러스, 씀바귀 식물체(배축) 및 씀바귀 배발생 캘러스가 제외된 배양액을 건조한 후, 각각의 50mg 건조시료를 5, 25, 25㎖ 메탄올로 3회 추출한 후 추출물에 물 25㎖을 첨가한 다음 헥산 10㎖로 2회 상분배하여 메탄올/물 층을 수거한 다음 농축하고 500μM 메탄올로 재용해하여 분석 시료로 사용하였고, 추출물의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 함량을 분석하기 위하여, 액체 크로마토그래피-질량분석법(LC-MS/MS)를 이용하였다. 본 발명의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 함량 분석에 사용한 LC-MS/MS의 조건은 하기 표 3과 같다. Specifically, after drying the culture medium excluding the embryonic callus, the embryonic callus, the embryonic callus, and the embryonic callus, the 50 mg dried samples were extracted three times with 5, 25, and 25 mL of methanol. 25 ml of water was added to the extract, and the methanol/water layer was collected by phase distribution with 10 ml of hexane twice, concentrated, and re-dissolved in 500 μM methanol to be used as an analysis sample. Chrysoeriol-7-0 in the extract -To analyze the content of glucoside (chrysoeriol-7-0-glucoside), liquid chromatography-mass spectrometry (LC-MS/MS) was used. The LC-MS/MS conditions used to analyze the chrysoeriol-7-0-glucoside content of the present invention are shown in Table 3 below.
씀바귀 배발생 캘러스, 씀바귀 식물체(배축) 및 씀바귀 배발생 캘러스가 제외된 배양액의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 함량은 도 7에 나타내었다.The content of chrysoeriol-7-0-glucoside in the culture medium excluding the embryonic callus, the embryonic callus plant (hypocotyl), and the embryonic callus of the embryonic callus are shown in Figure 7.
도 7a는 씀바귀 식물체(배축), 도 7b는 씀바귀 배발생 캘러스, 도 7c는 배양액의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 생합성량을 나타낸 도면이다. 도 7a 내지 7c를 참조하면, 씀바귀 배발생 캘러스의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 생합성량은 12.30 ± 1.88 mg/L로 씀바귀 식물체(배축)의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 생합성량인 0.61 ± 0.26 mg/L의 약 20배인 것을 확인할 수 있으며, 씀바귀 배발생 캘러스에서 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 생합성량이 현저하게 증가한 것을 확인할 수 있다.Figure 7a is a diagram showing a plant (hypocotyl), Figure 7b is a view showing a callus developing embryo, and Figure 7c is a diagram showing the amount of chrysoeriol-7-0-glucoside (chrysoeriol-7-0-glucoside) biosynthesis in the culture medium. Referring to Figures 7a to 7c, the biosynthetic amount of chrysoeriol-7-0-glucoside in callus embryogenesis of Chrysoeriol-7-0-glucoside is 12.30 ± 1.88 mg/L, which is equivalent to the amount of chrysoeriol-7-0-glucoside in the embryonic callus of Chrysoeriol-7-0-glucoside. It can be confirmed that the amount of chrysoeriol-7-0-glucoside biosynthesis is about 20 times that of 0.61 ± 0.26 mg/L. It can be seen that the amount of biosynthesis (chrysoeriol-7-0-glucoside) has increased significantly.
또한, 배발생 캘러스의 특성을 고려할 때, 지속적인 대량 계대배양을 추진할 경우에도, 이러한 고농도의 기능성물질의 축적을 기대할수 있다.Additionally, considering the characteristics of embryogenic callus, accumulation of such high concentrations of functional substances can be expected even when continuous mass subculture is pursued.
한편, 배발생 캘러스의 배양액의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 생합성량은 2.63±0.96mg/L로 씀바귀식물체의 4.3배임을 알 수 있고, 이에 따라 대량생산을 통한 실용화 및 산업화에 결정적인 가능성을 확인할 수 있다.Meanwhile, the biosynthetic amount of chrysoeriol-7-0-glucoside in the culture medium of embryogenic callus is 2.63 ± 0.96 mg/L, which is 4.3 times that of the plant. Accordingly, the mass Critical possibilities for commercialization and industrialization through production can be confirmed.
실시예 5 : 씀바귀 배발생 캘러스 추출물의 세포 생존도 및 침샘 세포의 아밀라아제 분비능 측정Example 5: Measurement of cell viability and amylase secretion ability of salivary gland cells of embryonic callus extract
실시예 1에서 배양한 씀바귀 배발생 캘러스에서 배양액을 제거시킨 다음 동결건조한다. 이후, 동결건조한 씀바귀 배발생 캘러스 10g에 100ml의 80% 에탄올에 침지하여, 35℃에서 72시간 동안 초음파 추출하고, 추출액을 여과하였다. 여과하여 수득한 용매는 진공 상태에서 감압 농축하여 5g의 씀바귀 배발생 캘러스 에탄올 추출물을 수득하였다. 에탄올 추출물은 사용 전에 원하는 농도에 맞춰 물에 용해하여 사용하였다.The culture medium was removed from the embryonic callus cultured in Example 1, and then freeze-dried. Thereafter, 10 g of freeze-dried callus embryogenesis was immersed in 100 ml of 80% ethanol, subjected to ultrasonic extraction at 35°C for 72 hours, and the extract was filtered. The solvent obtained by filtration was concentrated under reduced pressure in a vacuum to obtain 5 g of ethanol extract of callus embryogenesis. The ethanol extract was dissolved in water to the desired concentration before use.
또한, 세포 배양은 다음과 같이 진행되었다.Additionally, cell culture was carried out as follows.
Human salivary gland (HSG) cells를 RPMI 1640(sigma) 배지에 항생제 (antibiotics, 15140, gibco)와 10% fetal bovine serum (FBS, gibco, 10437028)을 첨가하여 배양 하였다. 세포는 일정한 습도를 유지하는 37℃ 항온기에서 공기(95%)와 CO2 (5%)의 혼합기체를 공급하면서 배양하고, 2~3일 마다 계대 배양하였다.Human salivary gland (HSG) cells were cultured in RPMI 1640 (sigma) medium with antibiotics (antibiotics, 15140, gibco) and 10% fetal bovine serum (FBS, gibco, 10437028). Cells were cultured in a thermostat at 37°C maintaining a constant humidity while supplying a mixed gas of air (95%) and CO2 (5%), and subcultured every 2 to 3 days.
세포 생존도 측정Cell viability measurement
MTT((3-(4,5-디메틸티아졸-2-일)-2,5-디페닐테트라졸륨)브로마이드((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide; MTT))의 노란색 테트라졸륨 염이 살아있는 세포 내에서 보라색 포르마잔으로 환원되는 것을 기초로 하는 표준 비색 MTT 분석법을 사용함으로써 평가하였다.MTT((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium)bromide((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) ) bromide; MTT)) was evaluated by using a standard colorimetric MTT assay based on the reduction of the yellow tetrazolium salt to purple formazan in living cells.
Human salivary gland (HSG) cells을 1×105 세포수/mL의 밀도로 96-웰 플레이트에 씨딩하였고, 16 시간 동안 배양시켰으며, 본 발명의 씀바귀 배발생 캘러스 추출물(IXD)을 25, 50, 100 및 200 μg/mL의 농도로 처리하였다. 추가적으로 24 시간 동안 더 배양시킨 후, MTT 저장 용액(50 μL, 2 mg/mL)을 각 웰에 전체 반응 부피가 250 μL가 되도록 첨가하였다. 4시간 후, 상청액을 흡입하여 버리고, 디메틸 설폭사이드(DMSO, 150 μL)를 각 웰에 첨가하여 포르마잔 결정을 용해시켰다. Carmichael et al 1987에 개시된 바와 같이, 용액의 흡광도를 스캐닝 멀티-웰 분광 광도계(scanning multiwell spectrophotometer)로 540 nm에서 측정하였으며, 이에 대한 결과를 도 8에 나타내었다.Human salivary gland (HSG) cells were seeded in a 96-well plate at a density of 1×10 5 cells/mL, cultured for 16 hours, and the callus extract (IXD) of the present invention was added at 25, 50, Treated at concentrations of 100 and 200 μg/mL. After further incubation for 24 hours, MTT stock solution (50 μL, 2 mg/mL) was added to each well to make a total reaction volume of 250 μL. After 4 hours, the supernatant was aspirated and discarded, and dimethyl sulfoxide (DMSO, 150 μL) was added to each well to dissolve the formazan crystals. As disclosed in Carmichael et al 1987, the absorbance of the solution was measured at 540 nm using a scanning multiwell spectrophotometer, and the results are shown in FIG. 8.
도 8을 참조하면, 본 발명에 따른 씀바귀 배발생 캘러스 추출물(IXD)은 Human salivary gland (HSG) cells에 대하여 독성이 없음을 확인할 수 있다.Referring to Figure 8, it can be confirmed that the callus extract (IXD) according to the present invention is not toxic to human salivary gland (HSG) cells.
아밀라아제 활성 측정Amylase activity measurement
Human salivary gland (HSG) cells를 5mM 및 40mM의 글루코오스를 함유한 배양액에서 배양한 후, 40mM의 글루코오스를 함유한 배양액에 24시간 동안 씀바귀 배발생 캘러스 추출물(IXD) 25, 50 및 100 μg/mL를 처리하였고, 아밀라아제 활성은 amylase assay kit (BioVision Co, K711-100)를 사용하여 405nm 파장에서 25℃로 설정한 후 ELISA를 이용하여 측정하였다. 이에 대한 결과를 도 9에 나타내었다.Human salivary gland (HSG) cells were cultured in a culture medium containing 5mM and 40mM glucose, and then 25, 50, and 100 μg/mL of callus extract (IXD) was added to the culture medium containing 40mM glucose for 24 hours. The amylase activity was measured using an amylase assay kit (BioVision Co, K711-100) at 25°C at a wavelength of 405 nm and then using ELISA. The results are shown in Figure 9.
도 9를 참조하면, 40mM 고농도의 글루코오스를 처리하는 경우, 아밀라아제의 분비가 감소하는 것을 알 수 있으나, 40mM 고농도 글루코오스에 씀바귀 배발생 캘러스 추출물(IXD)을 농도 의존적으로 처리 시 아밀라아제의 분비가 증가함을 확인하였다. Referring to FIG. 9, it can be seen that the secretion of amylase decreases when treated with 40mM high concentration glucose, but when the callus extract (IXD) is treated with 40mM high concentration glucose, the secretion of amylase increases in a concentration-dependent manner. was confirmed.
이로부터 다양한 혐기성 미생물들이 상존하는 구강 내 환경에서 씀바귀 배발생 캘러스 추출물(IXD)이 효소를 보호하는 것을 확인할 수 있고 이에 따라 씀바귀 배발생 캘러스 추출물(IXD)이 구강 청결 및 구강건강에 기여할수 있음을 알 수 있다.From this, it can be confirmed that callus extract (IXD) protects enzymes in the oral environment where various anaerobic microorganisms exist, and thus, callus extract (IXD) can contribute to oral cleanliness and oral health. Able to know.
이와 같이, 본 발명의 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법을 이용하여 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 함량을 증가시킬 수 있으며, 이에 따라 배양된 배발생 캘러스로부터 고 함량의 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)를 대량으로 생산하여 산업화 할 수 있다.In this way, the content of chrysoeriol-7-0-glucoside can be increased using the embryogenic callus (EC) culture method of the present invention, thereby High-content chrysoeriol-7-0-glucoside can be produced and industrialized in large quantities from cultured embryogenic callus.
또한, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)을 포함하는 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC)의 배양액을 포함하는 충치 예방 및 구취 억제용 약학적 조성물을 제공할 수 있다.In addition, embryogenic callus (EC) containing chrysoeriol-7-0-glucoside, its extract, or culture medium of embryogenic callus (EC) It is possible to provide a pharmaceutical composition for preventing cavities and suppressing bad breath containing a.
이상으로 본 발명의 바람직한 실시예를 상세하게 설명하였다. 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다.Preferred embodiments of the present invention have been described in detail above. The description of the present invention is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing its technical spirit or essential features.
따라서, 본 발명의 범위는 상세한 설명보다는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미, 범위 및 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.Therefore, the scope of the present invention is indicated by the claims described later rather than the detailed description, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts are interpreted to be included in the scope of the present invention. It has to be.
Claims (10)
상기 2,4-D의 함량은 0.05~0.1mg/L이고, NAA의 함량은 0.01~0.1mg/L인 것인, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법.Chrysoeriol-7-0-glucoside (chrysoeriol-7-0-glucoside), which includes the step of inducing embryogenic callus (EC) by culturing the soybean tissue in a medium containing 2,4-D and naphthaleneacetic acid (NAA). 7-0-glucoside) A method of cultivating embryogenic callus (EC) for mass production,
Chrysoeriol-7-0-glucoside, wherein the 2,4-D content is 0.05 to 0.1 mg/L and the NAA content is 0.01 to 0.1 mg/L. Method for cultivating embryogenic callus (EC) for mass production.
상기 씀바귀 조직은 잎, 엽병 또는 배축 조직인 것을 특징으로 하는, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법.According to paragraph 1,
A method of cultivating embryogenic callus (EC) for mass production of chrysoeriol-7-0-glucoside, wherein the tissue is a leaf, petiole, or hypocotyl tissue.
상기 배지는 수크로오스(Sucrose) 및 겔라이트(Gerlite)를 더 포함하는 것을 특징으로 하는, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법.According to paragraph 1,
The medium is an embryogenic callus for mass production of chrysoeriol-7-0-glucoside, characterized in that it further contains sucrose and Gerlite. callus, EC) culture method.
상기 배양은 pH 5.5~6.5 및 20~30℃에서 암배양하는 것을 특징으로 하는, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법.According to paragraph 1,
The culture is an embryogenic callus for mass production of chrysoeriol-7-0-glucoside, characterized in that it is cultured in the dark at pH 5.5-6.5 and 20-30°C. , EC) Culture method.
상기 배발생 캘러스(embryogenic callus, EC)는 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside)의 생합성량이 씀바귀 조직의 10~30배인 것을 특징으로 하는, 크리소에리올-7-0-글루코사이드(chrysoeriol-7-0-glucoside) 대량생산용 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양 방법.According to paragraph 1,
The embryogenic callus (EC) is characterized in that the biosynthetic amount of chrysoeriol-7-0-glucoside is 10 to 30 times that of the chrysoeriol-7-0-glucoside. Method for cultivating embryogenic callus (EC) for mass production of 7-0-glucoside (chrysoeriol-7-0-glucoside).
상기 씀바귀 배발생 캘러스(embryogenic callus, EC), 이의 추출물 또는 씀바귀 배발생 캘러스(embryogenic callus, EC) 배양액은 침샘세포의 아밀라아제 분비능을 향상시키는 것을 특징으로 하는, 충치 예방 및 구취 억제용 약학적 조성물.According to clause 8,
A pharmaceutical composition for preventing tooth decay and suppressing bad breath, wherein the embryogenic callus (EC), its extract, or embryogenic callus (EC) culture medium improves the amylase secretion ability of salivary gland cells.
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