KR20010044235A - The method for increasing saponin content and controlling the ratio of triol and diol during adventitus roots culture in ginseng - Google Patents
The method for increasing saponin content and controlling the ratio of triol and diol during adventitus roots culture in ginseng Download PDFInfo
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- 235000003140 Panax quinquefolius Nutrition 0.000 title claims abstract description 86
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 title claims abstract description 85
- 229930182490 saponin Natural products 0.000 title claims abstract description 62
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- 238000000034 method Methods 0.000 title claims abstract description 16
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- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 title description 13
- 150000002009 diols Chemical class 0.000 title description 13
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- UPPXMONNNGGKMB-UHFFFAOYSA-N 3-O-(alpha-L-rhamnopyranosyl-(1->2)-beta-D-glucopyranosyl-(1->2)-beta-D-glucopyranosyl)spergulagenic acid Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(OC(CO)C(O)C2O)OC2C(C3C(C4C(C5(CCC6(CCC(C)(CC6C5=CC4)C(O)=O)C(O)=O)C)(C)CC3)(C)CC2)(C)C)OC(CO)C(O)C1O UPPXMONNNGGKMB-UHFFFAOYSA-N 0.000 description 1
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- 229920001197 polyacetylene Polymers 0.000 description 1
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- 239000005017 polysaccharide Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- SWQINCWATANGKN-UHFFFAOYSA-N protopanaxadiol Natural products CC(CCC=C(C)C)C1CCC2(C)C1C(O)CC1C3(C)CCC(O)C(C)(C)C3CCC21C SWQINCWATANGKN-UHFFFAOYSA-N 0.000 description 1
- SHCBCKBYTHZQGZ-DLHMIPLTSA-N protopanaxatriol Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2[C@@H](O)C[C@@]3(C)[C@]4(C)CC[C@H]([C@](C)(O)CCC=C(C)C)[C@H]4[C@H](O)C[C@@H]3[C@]21C SHCBCKBYTHZQGZ-DLHMIPLTSA-N 0.000 description 1
- BBEUDPAEKGPXDG-UHFFFAOYSA-N protopanaxatriol Natural products CC(CCC=C(C)C)C1CCC2(C)C1C(O)CC3C4(C)CCC(O)C(C)(C)C4C(O)CC23C BBEUDPAEKGPXDG-UHFFFAOYSA-N 0.000 description 1
- 150000003216 pyrazines Chemical class 0.000 description 1
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- 238000004114 suspension culture Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/06—Roots
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
Description
인삼은 식물학적으로 오가과(Araliacea), 인삼속(Panax)에 속하는 식물로서 뿌리를 약용으로 이용한다.Ginseng is botanically belonging to the genus Araliacea and Panax ginseng, and its roots are used for medicinal purposes.
세계적으로 여기에 속하는 식물종은 6∼7종이 알려지고 있으나 경제적으로 재배되어 인삼 시장에서 상품으로 유통되고 있는 인삼종은 크게 3가지 종류가 있다.There are six to seven species of plants belonging to the world, but three kinds of ginseng are economically cultivated and distributed as commodities in the ginseng market.
지리적으로 아시아 극동지역에 분포, 재배되고 있는 "Panax ginseng C.A.Meyer"의 식물명을 가진 인삼은 전통적으로 중국의 한방 생약 중 가장 중요한 강장약으로 이용되어왔다.Ginseng with the botanical name of "Panax ginseng C.A.Meyer", which is geographically distributed and cultivated in the Far East of Asia, has traditionally been used as the most important tonic of Chinese herbal medicines.
인삼은 예로부터 여러 가지 질병의 치료와 병의 회복 촉진에 놀라운 효험을 발휘하는 효능을 발휘해왔고 이러한 인삼의 효능에 대하여 인삼의 약효 성분과 약리적 효능을 탐구하기 위하여 광범위한 연구를 계속하고 있으며 지금까지 과학적으로 밝혀진 대표적 효능으로는 신체 조절 기능의 항상성 유지 작용이라 할 수 있으며 이러한 작용에 근거하여 항피로 및 항스트레스 작용, 항당뇨 작용, 혈압 조절 작용, 항암작용, 동맥 경화 및 고혈압의 예방, 두뇌 기능 강화, 위장 기능 강화, 면역 기능 강화, 항바이러스 작용 등이 보고되고 있다.Ginseng has long been effective in treating various diseases and facilitating the recovery of diseases, and has been extensively studied to explore the efficacy and pharmacological effects of ginseng. Representative efficacy of this study is the homeostasis action of body control function, and based on this action, anti-fatigue and anti-stress action, anti-diabetic action, blood pressure control action, anti-cancer action, prevention of atherosclerosis and hypertension, strengthening of brain function , Gastrointestinal function, immune function, antiviral action, etc. have been reported.
이러한 인삼의 주요 유효 성분으로는 사포닌, 사포게닌, 폴리아세틸렌, 피라진 유도체, 말톨 등이 알려져 있다.The main active ingredients of such ginseng are saponins, sapongenins, polyacetylenes, pyrazine derivatives, maltol, and the like.
인삼 사포닌은 화학구조의 특성에 따라 protopanaxadiol계, protopanaxatriol계 그리고 oleanonane계 사포닌으로 구분하는데, 현재까지 각각 19종, 10종, 1종의 화합물이 분리 정제되었으며, diol계와 triol계는 체내에서의 약리 작용이 서로 다른 것으로 알려져 있다.Ginseng saponins are divided into protopanaxadiol, protopanaxatriol, and oleanonane-based saponins according to the characteristics of the chemical structure.To date, 19, 10, and 1 compounds have been separately purified and diol and triol are pharmacological in the body. It is known that the action is different.
이와 같은 인삼 사포닌은 중추 신경 억제 작용, 단백질 합성 촉진 작용, 부신 피질 호르몬 분비 촉진 작용, 인슐린 유사 작용, 해독 작용, 항염증, 혈소판 응집 억제 등 이외에도 많은 효능이 보고되고 있다.Such ginseng saponins have been reported to have many effects in addition to central nerve suppression, protein synthesis promoting action, corticosteroid secretion promoting action, insulin-like action, detoxification action, anti-inflammatory, platelet aggregation inhibition and the like.
지금까지 식물 조직 배양 기술을 이용한 인삼의 캘러스 및 세포 현탁 배양 그리고 대량 배양하는 기술은 주로 고려인삼, 미국 인삼, 전칠삼을 중심으로 소규모 배양에서 출발하고 있으며 일부 학회지에 이미 세포 및 캘러스의 증식, 사포닌 및 다당류 함량에 관한 분석 비교가 보고되어 있다.Until now, the callus and cell suspension culture and mass cultivation of ginseng using plant tissue culture technology started mainly from small-scale cultivation mainly on Korean ginseng, American ginseng, and chile ginseng. An analytical comparison of polysaccharide content is reported.
또한 일본에서는 세포 배양을 통한 상품화가 이루어지고 있으며 특허화된 것도 있다(일본 공개특허 제 소58-43725호, 제 소58-43726호, 제 소58-43737호).In Japan, commercialization through cell culture is being carried out and there are also patented ones (Japanese Patent Laid-Open Nos. 58-43725, 58-43726 and 58-43737).
그러나 재배삼, 장뢰삼, 산삼의 조직으로부터 캘러스를 얻고 이들 조직에서 부정근을 유기시켜 대량 증식시키면 부정근은 대량 생산이 가능하지만 자연삼에 비해 사포닌 함량이 낮고 diol:triol의 비율이 자연삼에 비해 큰 차이가 나기 때문에 경제성의 측면에서 문제가 발생할 수 있었다.However, if callus is obtained from the tissues of cultivated ginseng, rapeseed and wild ginseng, and organically grown in the roots of these ginsengs, the roots can be mass-produced. Because of this, problems could arise in terms of economics.
또한 재배삼, 장뢰삼, 산삼의 부정근 배양시 증식 과정이나 수확 후 사포닌 함량을 현저히 증가시키고 diol계 사포닌과 triol계 사포닌의 비율을 자연삼과 비슷하게 조절할 수 있는 방법에 관한 연구는 전무한 실정이다.In addition, there are no studies on how to increase the saponin content significantly during the growth or harvesting of cultivated ginseng, rapeseed and wild ginseng, and to control the ratio of diol-based saponins and triol-based saponins similar to natural ginseng.
본 발명은 상기의 사항들을 고려하여 안출한 것으로서, 본 발명의 목적은 재배삼, 장뢰삼, 산삼으로부터 채취한 뿌리, 잎, 줄기 조직을 조직 배양하여 형성된 캘러스에서 부정근을 유도한 다음, 이와 같이 유도된 부정근을 진탕 배양 또는 생물반응기 배양을 통하여 대량 증식시키는 과정에 있어서 사포닌 함량을 재배삼이나 산삼등의 자연삼보다 증가시키고 사포닌의 조성 비율을 자연삼과 유사한 수준으로 유도할 수 있는 최적의 배양 조건을 규명하여 상품성 및 기능성이 우수한 기내 부정근을 생산할 수 있는 조직 배양에 의한 인삼·장뢰삼·산삼 부정근의 사포닌 함량 개선 방법을 제공하는데 있다.The present invention has been made in view of the above matters, an object of the present invention is to induce the root muscle in the callus formed by the tissue culture of root, leaf, stem tissue collected from cultivated ginseng, jangsam ginseng, wild ginseng, and then In the process of mass growth of inferior roots through shaking culture or bioreactor culture, the optimum culture conditions to increase the saponin content than natural ginseng such as cultivated ginseng or wild ginseng and to induce the composition ratio of saponin to the level similar to natural ginseng were investigated. The present invention provides a method for improving the saponin content of ginseng, chungsam ginseng and wild ginseng root muscle by tissue culture capable of producing in-vehicular root muscle with excellent commerciality and functionality.
이와 같은 목적을 달성하기 위해서 본 발명에 따른 조직 배양에 의한 인삼·장뢰삼·산삼 부정근의 사포닌 함량 개선 방법은 인삼, 장뢰삼, 산삼 중의 어느 하나를 조직 배양하여 얻은 부정근을 생장조절제로 전처리하는 단계와,In order to achieve the above object, the method for improving the saponin content of ginseng, jangsam ginseng and wild ginseng ginseng by tissue culture according to the present invention is a step of pretreatment of the ginseng root obtained by tissue culture of any of ginseng, jangsam ginseng and wild ginseng with a growth regulator. Wow,
상기 전처리한 부정근을 자스모닉산이나 메틸 자스몬산을 처리한 배지에 접종하는 단계와,Inoculating the pretreated labia root in a medium treated with jasmonic acid or methyl jasmonic acid,
상기 접종한 부정근을 풍선형 또는 원추형 생물반응기내에서 광처리 하에서 배양하는 단계와,Culturing the inoculated adductor under light treatment in a balloon or conical bioreactor,
상기 부정근을 배양하는 배지를 수확 5∼10일전 질소무첨가 배지로 교환하는 단계로 구성되는 것을 특징으로 하는 것으로서, 사포닌 함량을 증가시키고 diol계 사포닌과 triol계 사포닌의 비율을 자연삼과 유사한 수준으로 조절한 부정근의 생산이 가능해짐으로써 상품성 및 기능적 이용 가치가 증가된 부정근을 제공할 수 있다.It characterized in that it comprises the step of replacing the medium for culturing the root muscle with nitrogen-free medium 5 to 10 days before harvesting, by increasing the saponin content and adjusting the ratio of diol-based saponins and triol-based saponins to a level similar to natural ginseng By enabling the production of intestinal roots, it is possible to provide the roots with increased merchandise and functional value.
이하 본 발명을 상세하게 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
조직 배양하여 얻은 재배삼, 장뢰삼, 산삼의 부정근을 MS(Murashige-Skoog) 배지, SH(Schenk and Hildebrandt) 배지, B5(Gamborg) 배지에 배양하면 부정근의 증식은 잘 이루어지지만 사포닌 함량이 매우 낮아 상품성이 결여되며 또한 triol계 사포닌과 diol계 사포닌의 비율이 0.5∼2.5로 나타나 목표치 1:1 내지 1:2에 못 미치는 경우가 있다.Cultured cultivated ginseng, rapeseed, and wild ginseng obtained from tissue culture in Murashige-Skoog (MS) medium, Schenk and Hildebrandt (SH) medium, and B5 (Gamborg) medium are used for the growth of intestinal root, but the saponin content is very low. In addition, the ratio of triol-based saponins and diol-based saponins is 0.5 to 2.5, which is sometimes below the target 1: 1 to 1: 2.
일반적으로 배지에 아무런 처리를 하지 않을 경우 기내에서 생산된 부정근에서의 사포닌 함량은 2% 미만으로 나타나 자연삼에 비해 함량이 부족한 편이다.In general, if the medium is not treated, saponin content in the intestinal root produced in the aircraft is less than 2%, which is less than that of natural ginseng.
그러므로 본 발명에서는 기내 부정근 배양 과정 중 또는 수확한 부정근에 대해 사포닌 함량을 현저히 증가시키는 동시에 triol/diol의 비율을 자연삼과 비슷한 수준으로 조절하는 방법을 제시하고자 하였다.Therefore, the present invention aims to provide a method for significantly increasing the saponin content in the incubation process of intestinal root muscle or at the same time to control the triol / diol ratio to a level similar to that of natural ginseng.
따라서 본 발명의 구체적인 기술은 다음의 내용으로 구성된다.Therefore, the specific technology of this invention consists of the following content.
인삼, 장뢰삼, 산삼 중 어느 하나를 조직 배양하여 얻은 부정근을 조직 배양용 배지에 배양하기 전 생장조절제로서 사이토키닌류인 BA(benzyl adenine), 2iP, Zeatin, 메틸자스모네이트, TDZ, Kinetin중의 1종 또는 자스모닉산중 어느 하나를 1.0∼100㎎/L의 양으로 첨가하여 1∼10시간 동안 처리한 다음 당 3%, IBA와 NAA중 어느 하나를 0.5∼5.0㎎/L 양으로 첨가한 MS 배지에 접종하여 pH를 6.0으로 조절한 풍선형 생물반응기에서 배양하였다.As a growth regulator before incubating any of the ginseng root, ginseng, wild ginseng and wild ginseng in the tissue culture medium, cytokinins such as BA (benzyl adenine), 2iP, Zeatin, methyljasmonate, TDZ, and Kinetin One or one of jasmonic acid was added in an amount of 1.0 to 100 mg / L, and then treated for 1 to 10 hours, followed by 3% sugar, and one of IBA and NAA in an amount of 0.5 to 5.0 mg / L. Inoculation in MS medium was incubated in a balloon bioreactor adjusted to pH 6.0.
상기 생물반응기는 풍선형 외에 원추형 생물반응기를 사용하였을 때에도 사포닌 함량이 높은 결과가 나타나 원추형 생물반응기를 사용하는 것도 바람직할 것으로 판단되었다.The bioreactor showed a high saponin content even when a conical bioreactor was used in addition to the balloon type, and thus, it was determined that a conical bioreactor was also used.
상기 생물반응기 내의 부정근 배양 조건은 온도 22∼25℃ 그리고 형광등, 청색광, 적색광 중의 1종을 선택하여 생물반응기내에서 처리해 주는 것이 바람직하다.It is preferable to select one of the fluorescent lamp, blue light, and red light in the bioreactor for treating the root muscle culture in the bioreactor with a temperature of 22 to 25 ° C.
상기와 같은 조건에서 부정근을 배양한 후 수확 5∼10일 전 배지를 질소가 첨가되지 않은 배지로 교환해 주며 또한 자스모닉산과 메틸 자스몬산 중 1종을 1.0∼10.0㎎/L의 양으로 수확 10일 전 생물반응기 내에 7일간 처리해준 후 수확하였다.After culturing the root muscle under the above conditions, the medium was exchanged for 5 to 10 days before harvesting with a medium without nitrogen, and one of jasmonic acid and methyl jasmonic acid was harvested in an amount of 1.0 to 10.0 mg / L. 10 days before the harvest was treated for 7 days in the bioreactor.
자스모닉산 또는 메틸 자스몬산을 증식된 부정근을 수확한 후 물로 씻은 다음 1.0∼10.0㎎/L의 양으로 7일간 처리했을 경우에도 사포닌 함량이 현저히 증가되므로 수확 후의 처리도 바람직하다.When harvesting the grown root of jasmonic acid or methyl jasmonic acid, washed with water, and then treated with an amount of 1.0 to 10.0 mg / L for 7 days, saponin content is markedly increased, and thus treatment after harvesting is also preferable.
이하 실시예를 통하여 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail with reference to the following examples.
[실시예 1] 생물반응기의 형태가 사포닌 함량에 미치는 효과Example 1 Effect of Form of Bioreactor on Saponin Content
당 3%, IBA 2.0㎎/L를 첨가한 MS 배지 또는 SH 배지에 부정근을 1∼2㎝로 잘라서 접종한 다음 풍선형, 원추형, bulb형, 드럼형 배양 용기와 삼각플라스크를 사용하여 부정근을 배양한 4주 후에 사포닌 함량을 측정하여 그 결과를 표 1에 나타내었다.Inoculated by cutting 1 to 2 cm of negative root into MS medium or SH medium containing 3% sugar and 2.0 mg / L of IBA, and incubating the root muscle using a balloon, cone, bulb and drum culture vessel and a Erlenmeyer flask. After four weeks, the saponin content was measured and the results are shown in Table 1.
사포닌 함량 측정은 한국 인삼 연초 연구소의 인삼 성분 분석 표준법에 따라 실시하였다.Saponin content was measured according to the ginseng component analysis standard method of Korea Ginseng and Tobacco Research Institute.
본 실험 결과 사포닌 함량은 풍선형 생물반응기나 원추형 생물반응기에서 배양하였을 때 사포닌 함량이 높았고 삼각플라스크 드럼형은 함량이 낮았다. 따라서 인삼, 장뢰삼, 산삼의 부정근 배양시 풍선형이나 원추형 생물반응기를 이용하는 것이 바람직한 것으로 판단되었다.As a result, saponin content was high in saponin or conical bioreactor and low in triangular flask drum type. Therefore, it was judged that it is preferable to use a balloon-type or cone-shaped bioreactor for culturing ginseng root, ginseng root and wild ginseng.
[실시예 2] 자스모닉산 및 메칠 자스몬산 처리가 사포닌 함량에 미치는 효과Example 2 Effect of Jasmonic Acid and Methyl Jasmonic Acid Treatment on Saponin Content
재배삼, 장뢰삼, 산삼의 부정근을 풍선형 생물반응기에 배양하면서 배양 초기, 수확 10일전, 수확 후 두 물질을 농도별로 처리하였을 때의 사포닌 함량을 비교하였다.Saponin contents were compared when the two roots of cultivated ginseng, rapeseed ginseng and wild ginseng were cultured in a balloon-type bioreactor when the two materials were treated at different concentrations at the beginning of culture, 10 days before harvest, and after harvest.
MS 배지에 당 3%, IBA 2.0㎎/L을 첨가하고 pH를 6.0으로 조절한 후 실험을 실시하였다. 자스모닉산 또는 메틸자스모네이트 농도를 0, 1, 2, 5, 10㎎/L로 달리하여 수확 10일 전에 처리하는 방법과 수확 후 뿌리를 물로 씻은 다음 설탕을 첨가하지 않은 MS 배지나 수돗물에 자스모닉산 또는 메틸자스모네이트를 넣고 1주일간 처리한 후 사포닌 함량을 측정하여 그 결과를 표 2에 나타내었다.The experiment was performed after adding 3% sugar, 2.0 mg / L of IBA to the MS medium and adjusting the pH to 6.0. 10 days before harvesting by varying the concentration of jasmonic acid or methyljasmonate to 0, 1, 2, 5, 10mg / L and after harvesting, the roots were washed with water and added to MS-free or tap water without sugar. Jasmonic acid or methyl jasmonate was added and treated for 1 week, and then the saponin content was measured and the results are shown in Table 2.
배양 초기의 처리는 생장량을 억제시켰으며 수확 10일 전 생물반응기에 처리하거나 수확 후 증식된 부정근을 물로 씻은 다음 자스모닉산이나 메틸 자스몬산을 처리한 후 7일이 경과하였을 때의 사포닌 함량은 7∼8%에 달하여 재배삼의 2∼3%에 비하여 사포닌 함량이 현저히 증가되었음을 관찰할 수 있었다. 그러나 diol:triol의 비율이 재배삼에서 관찰되는 것보다 처리구에서 diol계 사포닌의 비율이 현저히 증가하는 경향을 나타내었다.Treatment at the beginning of the culture inhibited the growth and the saponin content after 7 days after treatment with the bioreactor 10 days before harvesting or washing the grown roots with water and then treated with jasmonic acid or methyl jasmonic acid was 7 It was observed that the saponin content was significantly increased compared to 2-3% of the cultivated ginseng, reaching ˜8%. However, the ratio of diol-triol was significantly increased in the treatment group than that of the cultivated ginseng.
따라서 전체 사포닌 함량은 자스모닉산 처리구가 무처리구에 비하여 사포닌의 함량이 6배 이상 증가된 것으로 나타나 자스모닉산 또는 메틸 자스몬산을 처리하는 것이 바람직하다.Therefore, the total saponin content of the jasmonic acid treated group was found to increase the content of saponin 6 times or more compared to the untreated group, it is preferable to treat jasmonic acid or methyl jasmonic acid.
[실시예 3] 배지내의 질소 제거가 사포닌 함량에 미치는 효과Example 3 Effect of Nitrogen Removal in Medium on Saponin Content
인삼 부정근 배양시 사포닌의 함량을 증가시키기 위하여 5ℓ 원추형 생물반응기에서 당 3%, IBA 2.0㎎/L를 첨가한 MS 배지를 사용하였고 pH는 6.0으로 조절하여 20∼30일 동안 최대 부정근 증식이 이루어지게 한 다음 수확 5∼10일전 질소가 첨가되지 않은 배지로 교환해 주었을 때의 사포닌 함량을 측정하여 그 결과를 표 3에 나타내었다.In order to increase the saponin content in ginseng root muscle culture, MS medium with 3% sugar and 2.0 mg / L of IBA was used in a 5L conical bioreactor.The pH was adjusted to 6.0 to allow maximum root muscle growth for 20 to 30 days. Next, the saponin content was measured when exchanged with a medium without nitrogen 5-10 days before harvesting, and the results are shown in Table 3.
표 3의 결과에서 알 수 있듯이 배양용 배지에 적정의 질소를 첨가시켜 부정근이 최대한 증식되도록 한 다음 수확 5∼10일 전 배지를 교환하여 주었을 때 사포닌 함량이 증가되었다.As can be seen from the results of Table 3, saponin content was increased when the medium was added to the culture medium to maximize the growth of the adventitious root, and then the medium was exchanged 5-10 days before harvest.
배지 교환시에는 질산태 및 암모니아태 질소가 전혀 첨가되지 않아야 하며 이 시기에 상기 실시예 2와 같이 자스모닉산을 첨가해주면 사포닌 함량 증가에 더욱 효과적인 결과를 가져올 수 있는 것으로 판단되었다.At the time of medium exchange, nitrate and ammonia nitrogen should not be added at all, and it was judged that addition of jasmonic acid as in Example 2 at this time could result in more effective increase in saponin content.
[실시예 4] 광원이 사포닌 함량에 미치는 영향Example 4 Effect of Light Source on Saponin Content
재배삼, 장뢰삼, 산삼 부정근 배양시 여러 가지 광원을 달리했을 때 사포닌 함량과 diol 및 triol계 사포닌의 비율에 미치는 효과를 조사하기 위해 100㎖ 삼각플라스크에 상기 실시예 1과 동일한 조건의 MS 배지 30㎖를 분주한 후 인삼 부정근을 1∼2㎝ 간격으로 절단하여 접종한 다음 암상태, 형광등, 메틸할라이트, 청색광(430㎚), 적색광(650㎚)을 40μ㏖·m-2·s-1의 광도로 16시간 내지 24시간 조명하에서 배양하였다. 배양실 온도는 22∼25℃로 조절하였고 4주 후 사포닌 함량을 측정하여 그 결과를 표 4에 나타내었다.In order to investigate the effect on the saponin content and the ratio of diol and triol saponins when different light sources were used in cultivation of cultivated ginseng, rapeseed ginseng and wild ginseng, 30 ml of MS medium under the same conditions as in Example 1 in a 100 ml Erlenmeyer flask And then inoculated by cutting the ginseng roots at intervals of 1 to 2 cm and inoculating dark, fluorescent, methylhalite, blue light (430 nm) and red light (650 nm) at 40 μmol · m −2 · s −1 . Incubation was carried out under luminous intensity for 16 to 24 hours. The culture room temperature was adjusted to 22-25 ° C. and after 4 weeks the saponin content was measured and the results are shown in Table 4.
본 실험 결과 암상태에서보다 형광등 하에서 배양하는 것이 생장율은 다소 저하되지만 사포닌 함량이 증가하였으며 특히 triol계 사포닌의 함량을 높일 수 있는 효과적인 방법이었다.As a result of this experiment, growth under fluorescent lamps was slightly decreased, but saponin content was increased, and triol saponin content was especially effective.
또한 청색광에서는 자연 상태에서 재배한 인삼의 diol과 triol 비율 1:1에 가장 근접한 1.03의 비율을 보였으며 적색광도 유사한 경향을 나타내었다.In the blue light, the ratio of 1.03, which is the closest to the diol and triol ratio 1: 1 of ginseng grown in the natural state, was similar, and the red light also showed a similar tendency.
따라서 인삼류의 부정근 배양시 문제점 중의 하나인 전체 사포닌 함량 중에서 triol계 사포닌 함량을 증가시키는데는 효과적이었으며, 인삼의 부정근 배양시 형광등, 적색광 및 청색광의 처리는 자연 상태에서 재배한 인삼과 비슷한 수준의 사포닌 조성을 유지시키는데 매우 중요한 요인인 것으로 판단되었다.Therefore, it was effective to increase triol-based saponin content among total saponins, one of the problems in ginseng root culture of ginseng. Fluorescent lamp, red light and blue light treatment of ginseng root muscle were similar to ginseng grown in nature. It was judged to be a very important factor to maintain.
[실시예 5] 생장조절제 처리가 사포닌 함량 빛 비율에 미치는 영향Example 5 Effect of Growth Regulator Treatment on Saponin Content Light Ratio
생장조절제 중 사이토키닌류인 BA(Benzyl adenine), 2iP, Zeatin, 메틸자스모네이트, TDZ, Kinetin을 재배삼, 장뢰삼, 산삼의 뿌리조직을 배양하기 전 1∼100mg/L 농도로 1시간에서 10시간 전처리한 다음 배양하였다. 전처리 직후 사포닌 함량과 diol:triol의 함량을 비교 분석하여 그 결과를 표 5에 나타내었다.Among the growth regulators, cytokinins BA (Benzyl adenine), 2iP, Zeatin, methyljasmonate, TDZ, and Kinetin were grown at 1 to 100mg / L for 1 hour to 10 hours before incubating the root tissues of cultivated ginseng, rapeseed and wild ginseng. Time pretreatment followed by incubation. Immediately after the pretreatment, saponin content and diol: triol content were analyzed and the results are shown in Table 5.
표 5의 결과에서 알 수 있듯이 전처리한 다음 부정근을 배양하였을 경우에는 사포닌 함량이 5배 정도 증가하면서 triol:diol의 비율은 2.4 정도로 자연 재배삼과 비슷한 수준이었다. 특히 사이토키닌류를 처리했을 경우 사포닌 함량은 낮아지지만 triol:diol의 비율이 자연 재배삼과 매우 유사하게 나타났다.As can be seen from the results of Table 5, the pretreatment and incubation of the root muscle increased the saponin content by 5 times and the triol: diol ratio was about 2.4, similar to that of natural cultivated ginseng. In particular, the cytokine treatment lowered the saponin content, but the triol: diol ratio was very similar to that of natural cultivated ginseng.
따라서 배양 부정근의 triol:diol의 비율을 1:1∼1:2로 유지시키고자 할 경우에 배양 전 사이토키닌류나 자스모닉산의 처리는 부정근의 품질을 높이는데 매우 효과적인 결과를 가져올 수 있을 것으로 판단되었다.Therefore, in the case of maintaining the ratio of triol: diol in the intestinal root muscle at 1: 1 to 1: 2, the treatment of cytokinin or jasmonic acid before the incubation can have a very effective result in improving the quality of the root muscle. Judging.
이상에서 본 바와 같이 본 발명에 따른 조직 배양에 의한 인삼·장뢰삼·산삼 부정근의 사포닌 함량 개선 방법은 생물반응기의 형태, 생장조절제의 종류 및 처리 시기, 배지의 질소 첨가 유무, 광원의 종류에 따른 최적 조건을 규명함으로써 사포닌 함량이 자연삼을 능가하며 그 구성 비율도 자연삼과 유사한 부정근을 배양할 수 있는 우수한 방법임을 알 수 있었다.As described above, the method for improving the saponin content of ginseng, chungsam ginseng and wild ginseng root of ginseng by tissue culture according to the present invention depends on the type of bioreactor, the type and growth time of the growth regulator, the presence of nitrogen in the medium, and the type of light source. By identifying the optimum conditions, saponin content was higher than natural ginseng and its composition ratio was found to be an excellent method for cultivating adventitious roots similar to natural ginseng.
상기와 같이 이루어지는 본 발명에 의하면, 조직 배양에 의해 인삼, 장뢰삼, 산삼 부정근을 생물반응기내에서 배양하는 경우 생장조절제, 생물반응기의 형태, 부정근의 최대 생장 이후 질소 공급 중단, 광처리 등 사포닌 함량 및 조성을 개선시킬 수 있는 최적 조건들을 규명함으로써 자연삼보다 사포닌 함량이 2∼3배 높으며, diol계 사포닌과 triol계 사포닌의 구성 비율도 자연삼과 유사한 기내 부정근을 생산할 수 있으며, 고부가가치가 있는 기내 삼을 다양한 수요층에 공급할 수 있다.According to the present invention made as described above, in the case of culturing ginseng, chungsam ginseng, wild ginseng root muscle in the bioreactor by tissue culture, growth regulators, bioreactor type, nitrogen supply after the maximum growth of the root of the root muscle, saponin content such as light treatment and By identifying the optimal conditions to improve the composition, saponin content is 2-3 times higher than natural ginseng, and the ratio of diol-based saponins and triol-based saponins can produce in-situ roots similar to natural ginseng. It can be supplied to various demand groups.
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| US09/998,136 US6713303B2 (en) | 2001-01-19 | 2001-12-03 | Method for the mass propagation of adventitious roots of ginseng, camphor ginseng and wild ginseng by tissue culture and the improvement of their saponin content |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005020672A1 (en) * | 2003-09-02 | 2005-03-10 | Neo Bio Co., Ltd. | Method for mass production of wild ginseng roots by plant tissue culture |
| KR100478213B1 (en) * | 2002-01-23 | 2005-03-22 | 손성호 | Production method of Callus and Adventitious root from the leaves and stems of ginseng |
| KR100711954B1 (en) * | 2006-09-21 | 2007-05-02 | 주식회사 씨비엔바이오텍 | Sansam Cultured Root Extract with High Saponin Content |
| KR101294742B1 (en) * | 2011-06-28 | 2013-08-08 | 주식회사김정문알로에 | Method for producing aloe adventitious root with increased medical active material |
| KR20180007203A (en) * | 2016-07-12 | 2018-01-22 | (주)에이씨티 | Method for cultivating cultured wild ginseng roots having high content of biologically active substances |
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| CN115837041A (en) * | 2022-12-14 | 2023-03-24 | 通化本草生物科技有限公司 | Fermentation medium for ginseng tissue culture adventitious roots and application thereof |
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| KR101206707B1 (en) | 2010-04-15 | 2012-11-29 | 한국식품연구원 | Cultivation method of plant with increased contents of functional materials |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100478213B1 (en) * | 2002-01-23 | 2005-03-22 | 손성호 | Production method of Callus and Adventitious root from the leaves and stems of ginseng |
| WO2005020672A1 (en) * | 2003-09-02 | 2005-03-10 | Neo Bio Co., Ltd. | Method for mass production of wild ginseng roots by plant tissue culture |
| KR100711954B1 (en) * | 2006-09-21 | 2007-05-02 | 주식회사 씨비엔바이오텍 | Sansam Cultured Root Extract with High Saponin Content |
| KR101294742B1 (en) * | 2011-06-28 | 2013-08-08 | 주식회사김정문알로에 | Method for producing aloe adventitious root with increased medical active material |
| KR20180007203A (en) * | 2016-07-12 | 2018-01-22 | (주)에이씨티 | Method for cultivating cultured wild ginseng roots having high content of biologically active substances |
| CN112956415A (en) * | 2021-01-18 | 2021-06-15 | 山东安然纳米实业发展有限公司 | Liquid culture medium for culturing ginseng adventitious roots and culture method |
| CN112956415B (en) * | 2021-01-18 | 2021-09-24 | 山东安然纳米实业发展有限公司 | Liquid culture medium for culturing ginseng adventitious roots and culture method |
| CN115837041A (en) * | 2022-12-14 | 2023-03-24 | 通化本草生物科技有限公司 | Fermentation medium for ginseng tissue culture adventitious roots and application thereof |
| CN115837041B (en) * | 2022-12-14 | 2024-04-26 | 通化本草生物科技有限公司 | Fermentation medium for tissue culture adventitious roots of ginseng and application thereof |
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