Protective effects of Artemisia iwayomogi and Codonopsis lanceolata hot water extracts against ethanol-induced dyslipidemia and fatty liver in rats

김소진 Graduate School, Yonsei University 2006 국내석사

알코올성 지방간은 만성적인 알코올 섭취로 인해 간 세포내 지질이 이상적으로 축적하여 유발되는 것으로 알려져 있다. 장기간의 알코올 섭취는 cytochrome P450 2E1의 유도에 따른 산화적 스트레스와 자유 라디칼의 생성을 증가시키며, 지질과산화물 생성을 촉진한다. 국내외에서 다양한 종류의 숙취해소 및 알코올성 간 손상 예방 음료들이 개발되어 판매되어 왔으나, 이들 식품소재의 정확한 효능 및 작용기전, 그리고 장기 섭취 시 영양소균형에 미치는 영향에 관한 연구는 매우 드문 실정이다. 따라서, 본 연구는 만성알코올 섭취 흰쥐를 대상으로 지질대사 개선용 기능성 식품소재가 알코올대사, 알코올성 고지혈증 및 지방간에 미치는 효과를 확인하고, 기타 간손상에 대한 보호활성 규명, 더 나아가 알코올 및 콜레스테롤 대사관련 간조직 유전자발현 변화에 미치는 영향을 규명하고자 시도되었다.본 연구는 SD 수컷 흰쥐를 구입하여 난괴법 (randomized complete block design)에 의하여 6군으로 분류하여 각 실험식이로 8주간 사육하였다: 정상군 (normal diet, ND), 알코올대조군 (ethanol control diet, ED), 알코올+ tannic acid군 (0.5% tannic acid-supplemented ethanol diet, TC), 알코올+ betaine군 (0.5% betaine-supplemented ethanol diet, BE), 알코올+ 인진쑥열수 추출분말군 (0.2% Artemisia iwayomogi-supplemented ethanol diet, AE), 알코올+ 더덕열수추출분말군 (0.5% Codonopsis lanceolata-supplemented ethanol diet, CE).그 결과, 에탄올대조군의 누적 체중증가량은 정상군에 비해 유의적으로 더 낮았다 (p<0.05). 에탄올 식이에 betaine, 인진쑥 또는 더덕열수추출물을 첨가시킨 군의 누적 체중증가량은 EC군에 비해 유의한 증가가 관찰되었다 (p<0.05). 식이효율(FER)은 ND군에 비해 EC군이 유의적으로 낮았으며, AE군과 CE군에서 EC군에 비해 유의적으로 더 높았다 (p<0.05). 체중 당 간조직의 무게는, EC군이 ND군보다 더 무거웠고, 신장무게도 EC군에서 ND군에 비해 유의적으로 무거웠다 (p<0.05). 반면, 에탄올식이에 더덕열수추출물을 첨가시킨 군의 간조직 및 신장 무게는 EC군에 비해 유의적으로 더 낮았다 (p<0.05).한편, 에탄올식이에 인진쑥열수추출물를 첨가시킨 군은 혈장 중성지방 농도와 총콜레스테롤이 EC군에 비해 유의적으로 낮음이 관찰되었다 (p<0.05). 혈장 총콜레스테롤 및 HDL-콜레스테롤 농도는 EC군이 ND군에 비해 유의적으로 더 높았다. 에탄올과 함께 tannic acid, 인진쑥열수추출물, 또는 더덕열수추출물 섭취를 병행한 TE군, AE군, CE군의 혈장 HDL 콜레스테롤 농도는 EC군에 비해 유의적으로 더 낮았다 (p<0.05). 그러나 에탄올식이에 betaine을 첨가 시킨 결과 HDL 콜레스테롤 농도가 17% 증가하는 것으로 나타났다. 이는 만성알코올 섭취에 대한 체내 적응 현상으로 심장보호효과를 증가시킨 결과라고 풀이 된다.흰쥐에서 간조직의 지질농도를 분석한 결과, betaine 또는 더덕열수 추출물이 간조직내 알코올에 의한 지질 축적을 효과적으로 감소시킴을 관찰할 수 있었다. 만성 알코올섭취에 따른 간기능 지표인 혈장 GOT 및 GPT 활성을 분석한 결과, EC군은 ND군에 비해 각각 33%와 40% 유의하게 증가하였다 (p<0.05). BE군의 혈장 GOT 활성은 EC군에 비해 유의적으로 감소하여 간기능 개선 효과를 보였다 (p<0.05). 혈장 및 간조직의 지질과산화물 농도를 분석한 결과, EC군이 ND군에 비해 유의적인 수치는 아니였지만 증가한 경향을 보였으며, AE군과 CE군은 EC군에 비해 각각 혈장 및 간조직에서 지질과산화물 농도 (TBARS)가 유의하게 감소하여 지질과산화물 생성을 낮추는 효과를 나타내고 있다 (p<0.05). 간 조직을 Hematoxylin & Eosin법으로 염색하여 얻은 histological image를 분석한 결과, 정상군에 비하여 에탄올대조군에서 지방간병변이 확인되었다.인진쑥열수추출물 섭취는 간조직에서 cholesterol metabolism(LDLR) 및 absorption(ABCA1, ABCG5), fatty acid synthesis(PRKAA2), fatty acid oxidation (MLYCD), 그리고 transcription factor(PPARα, RXRα, 그리고 LXRα)관련 유전자를 조절하여 알코올성 고지혈증 및 지방간을 개선시키는 것으로 나타났다.더덕열수추출물 섭취는 간조직에서 cholesterol metabolism(LDLR) 및 absorption(ABCA1, ABCG5), fatty acid synthesis(SCD1, PRKAA2), fatty acid oxidation(MLYCD, CPT1a), SREBP-1 target lipogenic genes(FASN), 그리고 transcription factor(PPARα, LXRα)관련 유전자를 조절하여 알코올성 고지혈증 및 지방간을 개선시키는 것으로 나타났다.이상의 결과를 종합해 보면, 만성 알코올 섭취시, 인진쑥열수추출물과 더덕열수추출물의 첨가는 tannic acid또는 betaine보다 체내 지질대사와 간기능 개선을 유도하였으며, 간조직의 다양한 유전자의 발현 변화를 초래하였다. 따라서 두 가지 물질은 알코올성 고지혈증 및 알코올성 지방간 개선에 효과가 있음을 짐작할 수 있다. The causal association between alcohol intake and the development of alcoholic liver disease has been well demonstrated. Recently, molecular biological investigations have become a predominant methodology applied to the study of alcohol-induced liver disease. Nevertheless, the mechanism and nutrient balance of functional food components underlying alcohol-induced liver injury remain poorly understood. The functional food components of this study have been mainly investigated in human and animal models induced by high fat diet or carbon tetrachloride with ethanol extract, single compound or powder. The aim of this study was to examine the effects on the improvement of the alcoholic lipid metabolism, hepatic function and gene expression patterns in the livers of rats chronically treated with functional food supplemented ethanol diet.treated with functional food supplemented ethanol diet.Male Sprague-Dawley rats were randomly divided into six groups, and fed one of the following diets for 8 weeks; normal diet (ND), ethanol control diet (ED), 0.5% tannic acid-supplemented ethanol diet (TC), 0.5% betaine-supplemented ethanol diet (BE), 0.2% Artemisia iwayomogi-supplemented ethanol diet (AE), 0.5% Codonopsis lanceolata-supplemented ethanol diet (CE).The ethanol diet group exhibited a significantly lower cumulative body weight gain than in the normal diet group rats (P<0.05). The final body weight of the ethanol diet group rats was lower 23%, 26%, 43% versus BE, AE, and CE rats (P<0.05). AE and CE groups was significantly higher food efficiency ratio than that of rats fed EC (P<0.05). The relative liver weights, relative kidney weights, and relative spleen weights were significantly raised by ethanol diet group (p<0.05). The relative liver and kidney weights of 0.5% Codonopsis lanceolata-supplemented ethanol diet groups (CE) were significantly reduced by 12%, 9% versus ethanol diet group (EC) (p<0.05).The triglyceride and total cholesterol levels was lower 33%, 19% versus EC rats by Artemisia iwayomogi supplement (P<0.05). The plasma total cholesterol concentration of rats fed ethanol diet was highly increased by 29% in comparison with that of ND rats (p<0.05). In addition, the plasma HDL cholesterol concentration of rats fed ethanol diet was highly increased by 37% compared with that of ND rats (p<0.05). Especially, feeding 0.5% betaine-supplemented ethanol diet resulted in the increase of HDL cholesterol concentration by 17% or 61% in comparison with that of EC rats or ND rats (p<0.05). These results have been suggested as a cardioprotective behavior in animals chronically loaded with ethanol.There were no significant differences of both hepatic triglyceride and cholesterol concentration in the liver between normal diet and ethanol diet group. We believe was probably related to the daily low fluid intake and ethanol concentration level. But the greatly decrease of hepatic triglyceride level by 38.3% or by 35.5% in BE group or CE group was observed, compared with those levels in EC group. The betaine supplements significantly lowered the values of the hepatic cholesterol levels within ethanol treated groups by 58.7% (p<0.05).On the other hand, the preventive effects of Artemisia iwayomogi include an attenuation of the elevations of plasma GOT and GPT produced by ethanol feeding. Additionally, our results suggested that intake of betaine take effects to significantly decrease plasma GOT level (p<0.05).The intake of a single supplementation with tannic acid or betaine even induced the plasma and hepatic lipid peroxidation (TBARS) in rat fed ethanol diet, while an addition of Artemisia iwayomogi or Codonopsis lanceolata resulted in the reduction of lipid peroxidation increased by ethanol diet, compared with that for EC group. Histological examination of liver tissue showed lipid accumulation greater in rats, which fed the ethanol containing diets for 8 weeks, compared to ND rats.The protective effect of Artemisia iwayomogi on fatty liver development is accompanied by changes in hepatic gene expression involved in cholesterol metabolism (LDLR), absorption (ABCA1 and ABCG5), fatty acid synthesis ((PRKAA2), fatty acid oxidation (MLYCD) and transcription receptors (PPAR?, RXR?, and LXR?\).The protective effect of Codonopsis lanceolata on fatty liver development is accompanied by changes in hepatic gene expression involved in cholesterol metabolism (LDLR), absorption (ABCA1, ABCG5), fatty acid synthesis (SCD1, PRKAA2), fatty acid oxidation (MLYCD, CPT1a), SREBP-1 target lipogenic genes(FASN), and transcription receptors (PPAR?, LXR?\).Overall, in this study, Artemisia iwayomogi or Codonopsis lanceolata water extract consumption rather than tannic acid or betaine supplementation has additive effects on the regulation of lipid metabolism, hepatic function. The present study also demonstrated that the prolonged ingestion of ethanol is associated with changes in the expression of a large number of genes. These results provide new insight into possible mechanisms for the potential efficiency of Artemisia iwayomogi or Codonopsis lanceolata in reducing risk of lipidemia and alcohol-induced fatty liver.

Development of Chloroplast-based DNA Markers to Analyze the Genetic Diversity of Korean Codonopsis lanceolata

이재복 충북대학교 2019 국내석사

더덕(Codonopsis lanceolata)은 한국을 포함하여 동아시아 국가에서 오랫동안 널리 사용되어 온 약용 작물이다. 그러나 더덕은 가치와 중요성에 비해 엘리트 품종의 육성에 대한 근본적인 연구가 부족하다. 선행연구에서 육종을 위하여 개발한 SSR 마커를 이용한 분석에서는 더덕의 유전자형이 너무 다양하여 유전자원을 구분하는데 어려움이 있었다. 엽록체 DNA는 재조합률이 낮고 모계 유전에 의해 후대에 전달되기 때문에 분자 마커의 다형성 값을 감소시킬 수 있다. 게다가 현재 한국의 한약재 시장에서 중국을 원산지로 하는 더덕이 수입되기 시작하면서, 국내산 더덕과 중국산 더덕이 혼용되어 사용될 우려가 제기되고 있다. 본 연구에서는 전국에서 수집한 야생 더덕 유전자원을 기반으로 한 한국 더덕의 유전적 다양성 분석 및 한국 더덕의 유전적 독자성을 확인하기 위해 엽록체 DNA 기반의 분자 마커를 개발하였다. 그에 따라, 총 87개의 야생의 더덕을 수집하였고 26개의 자원에 대하여 차세대 염기서열 분석을 수행하였다. 그 결과, 26개의 야생 더덕 엽록체 서열로부터 29개의 InDel 마커를 개발하였다. 그 다음, 유전적 다양성 분석을 위해 한약재 시장에서 중국을 원산지로 하여 판매되고 있는 20개체의 중국산 더덕을 추가적으로 수집하였다. 그리고 개발된 마커를 이용하여 32개체의 한국 더덕과 20개체의 중국산 더덕의 유전적 다양성을 분석하였으며, 유전적 분화도에 근거한 수집 더덕들의 계통도를 구성하였다. 이 연구에서 개발 된 마커는 향후 미래에 더덕의 분자 육종과 엘리트 품종 개발에 사용될 수 있을 것이다. 또한 본 연구에서 개발된 엽록체 DNA 기반의 분자마커는 국내 더덕의 모계혈통에 기반한 기원추론 및 품종 개발 연구에 사용 될 수 있을 것이라 사료된다. Codonopsis lanceolata has been widely used as a medicinal crop for a long time in East Asian countries, including Korea. However, in spite of its value and importance, C. lanceolata lacks fundamental research for the breeding of elite cultivars. In the analysis using simple sequence repeat (SSR) markers developed for breeding in previous research, C. lanceolata genotypes were very diverse and it was difficult to group genetic resources. Because chloroplast DNA is low in recombination rate and its sequence is preserved by maternal inheritance, it is possible to reduce the polymorphism value of the molecular marker. In addition, C. lanceolata has begun to be imported from foreign countries and distributed to the Korean herb market, raising concerns about the mixing of Korean C. lanceolata and foreign C. lanceolata. In this study, chloroplast-based molecular markers were developed for analysis of the genetic diversity and to establish the genetic identity of Korean C. lanceolata based on the sequences of wild C. lanceolata genetic resources collected from all over the country. A total of 87 wild C. lanceolata genetic resources were collected and next generation sequencing was performed on 26 of these. In addition, for the analysis of C. lanceolata genetic diversity, 20 C. lanceolata samples that originated from China were collected from the Korean herb markets. As a result, 29 InDel markers were developed from the chloroplast sequences from 26 wild C. lanceolata. Next, the genetic diversity of 32 Korean C. lanceolata and 20 Chinese C. lanceolata plants were analyzed using the markers developed in this study. A phylogenetic tree was constructed based on degree of genetic differentiation of C. lanceolata genetic resources. The markers developed in this study can be used for the molecular breeding and breeding of elite cultivars of C. lanceolata in the future. Use of the chloroplast-based molecular markers developed in this study made it possible to infer the geographical origin of Korean C. lanceolata based on maternal inheritance.

더덕(Codonopsis lanceolata) 재배지에서의 당근뿌리혹선충(Meloidogyne hapla) 피해 및 방제에 관한 연구

정도철 安東大學校 大學院 2003 국내박사

This study has been conducted to assess plant-parasitic nematodes infesting Codonopsis lanceolata, to analyze host damage due to the major nematode species, and to develop optimal control tactics using cultural and biological agents. The results are summarized as followings: 1.Ten kinds of plant-parasitic nematode geni including Meloidogyne and Pratylenchus were identified in 34 localities of C. lanceolata fields in three provinces in Korea. Meloidogyne, a dominant genus, was found in 97% localities and showed high average density as 1,700 nematodes per 300㎖ soil. Its average density was 10 times higher in upland field than in paddy field. 2.As the density of the root-knot nematode, M. hapla, increased, early development of C. lanceolata was significantly inhibited in pot assay: 60% decrease in vine growth and more than 54% decrease of rootgrowth. In field assay, such inhibitory effect of M. hapla on C. lanceolata was not significant presumably due to low densities of the nematodes. 3.Germination of C. lanceolata was susceptible to fosthiazate, but not to Bacillus thuringiensis (Bt) or a plant extract. 4.In pot assay, the inhibitory effect of two microbial agents, Bt and Paecilomyces lilacinus, on M. hapla were significant, but less than that of fosthiazate. The plant extract also had significantly inhibitory effect on M. hapla. 5.In field assay, treatments of P. lilacinus and fosthiazate resulted in maximal yields and qualities of C. lanceolata. The effect of the plant extract on the yields of C. lanceolata was also better than no treatment. The nematode-occurring condition of the fields before transplanting had significant effect on development of C. lanceolata; nematode-occurring field type gave less yields than nematode-free field type. These results clearly indicate that Melodogyne is the dominant plant-nematode genus in C. lanceolata fields and suggest that a cultural control technique using paddy field, microbial pesticides using Bt or P. lilacinus, and the plant extract are the promising control tactics against M. hapla in C. lanceolata fields. As a field manual to decrease economical damage of C. lanceolata due to M. hapla, this study suggests that C. lanceolata can be cultured directly in paddy field or in upland field after nematode control using microbial agents or the plant extract.

Establishment of Quality Seedling Production System for Codonopsis lanceolata Benth. et Hook.

곽쯔위 경상대학교 대학원 2019 국내석사

Codonopsis lanceolata Benth. et Hook. is a flowering plant native to East Asia. It has small seeds that die easily, and therefore, good germination is hard to achieve. Seed maturation and germination are the two major stages in the alternation of generations in higher plants. Gibberellins participate in numerous developmental processes. Codonopsis lanceolata seeds were pretreated for 7 days with imbibition in one of the following 100 mL solutions and temperatures: in sterile (autoclaved, double distilled) water at 4℃ (TA) or 24℃ (TB), and in a 300 mg·L-1 GA3 solution at 4℃ (TC) or 24℃ (TD). The results of this study showed that three days after cultures were initiated, the seeds in TC and TD showed signs of germination, and the TC had more pronounced effects on the seedlings as compared to the other treatments. The TC increased the percent germination (PG) by 12.8 and 34.2%, respectively, as compared to TA and TB. Furthermore, the maximum germination potential and germination index were also obtained from seedlings pretreated with TC. The contents of soluble sugar significantly increased when seeds were pretreated with cold solutions, whereas the maximum starch content was recorded in seedlings pretreated with TB. The activity of antioxidant enzymes in seedlings varied with the treatment, but only the peroxidase and superoxide dismutase activities were significantly different. Overall, the results suggest that imbibition in a GA3 solution at 4℃ could be the optimal pretreatment for the germination of C. lanceolata seeds. Codonopsis lanceolata is a vine plant with high medicinal values. The cultivation of this plant still needs further research, especially on producing compact seedlings. It has been reported that difference between day and night temperatures (DIF) could highly influence stem extension. The objectives of this experiment were to find an optimum culture condition to produce high quality plug seedlings, and to try to clear the mechanisms of DIF effect on stem elongation. Hence, the effect of DIF on the extension growth of C. lanceolata seedlings was studied in three precisely-controlled units of plant growth chamber. The seedlings were grown under a constant photoperiod of 12 h per day at 250 μmol•m-2•s-1 photosynthetic photon flux density supplied by white light emitting diodes with the same average daily temperature of 22℃, but different DIFs, such as a positive (26/18℃), negative (18/26℃), and zero (22/22℃) DIFs. Growth characteristics were measured after 20 days of treatments. Results showed that length of shoot, average length of node, number of leaves, leaf length, leaf width, and leaf thickness were positively correlated with treatments. As a consequence, the positive DIF regime exhibited superior results for all these traits. However, seedlings grown in the negative DIF had significantly shorter shoot length and average length of node than the positive or zero DIFs. Among all the treatments, the positive DIF portrays a better performance for shoot fresh and dry weights as well as leaf fresh and dry weights. Furthermore, the contents of chlorophyll a, chlorophyll b, and carotenoid in the leaf decreased when DIF was changed from positive to negative. The activities of peroxidase, catalase, ascorbate peroxidase, and superoxide dismutase enzymes were the greatest in the negative DIF treatment. The results revealed that negative DIF did suppress stem elongation and photosynthesis, affecting the synthesis and accumulation of soluble sugar and starch. High night temperatures as a kind of stress can enhance the activities of antioxidant enzymes, and under this stressful environment, seedlings cannot grow and development well. Iron deficiency chlorosis is a plant nutrient disorder symptom that’s widely observed around the world. The aim of this experiment was to identify the causes of leaf chlorosis, and the optimal medium pH and iron concentration to prevent leaf chlorosis and enhance growth in C. lanceolata. For this purpose, three treatment groups were set up, to investigate how the interaction between the iron supply and medium pH affects the plant growth. Seedlings were grown for 60 days in mediums with an iron supply of 0, 15, 30 mg•L-1, and the medium pH was adjusted to 4.7, 5.7, and 6.7. The results revealed that the chlorosis degree increased with decreasing iron concentration or with increasing medium pH. Regardless of the medium pH, at an iron concentration of 0 mg•L-1, seedlings were chlorotic, and at a medium pH of 6.7, seedlings could not survive. At medium pHs of 4.7 and 5.7, and iron concentrations of 15 and 30 mg•L-1, seedlings maintained green leaves, and no visual symptoms of chlorosis were observed. At a medium pH of 5.7, and an iron concentration of 15 mg•L-1, seedlings had the longest shoot length, the highest fresh weight, and high levels of chlorophyll a, chlorophyll b, and carotenoid. Therefore, a medium pH of 5.7 and an iron concentration of 15 mg•L-1 could be the optimal condition for preventing leaf chlorosis and enhancing growth in C. lanceolata.

차세대 염기서열 분석법을 이용한 더덕의 SSR마커 개발

김세림 충북대학교 2017 국내석사

Codonopsis lanceolata is an herbaceous perennial plant. It is predominantly cultivated in East Asia, and is used as a medicinal plant as well as a vegetable. However, there is a lack of research about genetic studies of C. lanceolata. Therefore, we sequenced genomic DNA using next generation sequencing (NGS) and searched the simple sequence repeats (SSRs) for marker development in C. lanceolata. A total of 250,455 SSRs were identified and dinucleotides and trinucleotides accounted for the majority of all the SSRs. A total of 26,334 primer sets were designed from dinucleotide to octanucleotide motifs. We investigated 2,626 SSRs (trinucleotide to pentanucleotide motifs) and found 573 SSRs showed in silico polymorphism. One-hundred seventy-seven SSR primer sets were selected to confirm that the 573 SSRs showed polymorphism in silico. As a result, 160 primer sets amplified expected amplicons and all of them showed polymorphism. From the results of genotyping using 39 SSR markers, the number of alleles ranged from 2 to 13, and the mean value of PIC was 0.54. In addition, 33 SSR markers were applied to 26 C. lanceolata collections (95 individual plants), and genetic diversity and population structure analysis were performed. Genetic relationship analysis showed that C. lanceolata collected from South Korea were not grouped by collection sites and showed genetic diversity. The results of population structure analysis showed that the members of most of the C. lanceolata collections were not grouped into one population and were considered an admixture. Consequently, we developed SSR markers from C. lanceolata that were polymorphic enough to analyze genetic diversity even though the markers did not distinguish the C. lanceolata by collection sites.

Codonopsis lanceolata의 組織培養에 의한 精油生産

최은정 덕성여자대학교 1993 국내석사

향장 제형들로부터의 더덕 추출물의 방출 특성 연구

정소영 중앙대학교 의약식품대학원 2008 국내석사

Codonopsis lanceolata is well known for excellent therapeutic action in Korean traditional medicine. Among the active ingredients saponins have been considered to be the major therapeutic ingredient. It has pharmaceutical action as surfactant such as emulsification, cleansing, foaming and ability to remove the horny layer of the skin. So it would show great cosmetic character for rough surface of the skin. Therefore, Codonopsis lanceolata containing saponins would probably to influence on topical disorders such as allergic dermatitis or atopic dermatitis. To utilize this effect the active ingredients should be released from the topical dosage forms. The objective of this study was to characterize several topical formulation for enhanced release of Codonopsis lanceolata extract. The experiment was evaluated by producing dosage forms of creams and pack which contained three percent of Codonopsis lanceolata water soluble extract by using immersion method to measure the amount of the extract released utilizing UV/vis spectrometer. The measuring results of each type of four dosage form creams, releasing amounts, were 14%, 44%, 24%, and 18% in order of hydrophilic cream, O/W cream, W/O cream and lipophilic cream. Releasing amount of W/O cream was about half compare to O/W cream. The result of hydrophilic cream generally showed fast release as time increases. So it is possibly assumed after four hours the releasing would keep go on continuously. Lipophilic cream well be in need of oil phase pass process in order to release Codonopsis lanceolata extract from based materials and differences in formulations of oil phase operates on diffuse layer so that would have the function of releasing inhibition. Result of pack's releasing amount was 69% and that was slightly less than hydrophilic cream which showed utmost extraction in cream formulations. Among cream formulations with Codonopsis lanceolata extract the cream formulation which has domain that can include water solution of Codonopsis lanceolata extract. As the characteristics of formulation, pack shows fast releasing within time because its formulation is water soluble. Hence, Codonopsis lanceolata extract which separated in water layer is highly hydrophilic substance so it surely shows that it gets great influence of releasing feature in formulation's hydrophile property and solvent availability. According to this experiment great expectations rely on increasing absorption of hydrophilic cream in case of developing cosmetics which including Codonopsis lanceolata extract and there need to be constant research and development. 더덕은 예부터 한방적으로 약리작용이 탁월하다고 알려져 사용해 오고 있다. 주요 구성 성분인 사포닌 성분은 계면활성제로 유화작용, 세정 작용 및 기포 작용 등과 다양한 작용 외에 피부의 각질층을 깨끗하게 하므로 표면조직이 거친 피부에 좋은 향장학적 특성을 보인다. 그러므로 더덕을 이용한 항알러지 항아토피 기능성 향장품에 사용되는 분획은 주로 사포닌을 함유한 층으로 예상되고, 알러지나 아토피 생성에 대한 억제 효과도 매우 클 것으로 기대한다. 또한 화장품 제제로부터 추출물 방출은 효능에 매우 큰 요인이 되고 제형, 용해도, 유제의 입자 크기 물의 함량 등 많은 요인에 의해 영향을 받는다. 본 연구는 더덕추출물을 이용하여 아토피 화장품으로써의 여러 제형을 설계하고 이에 따른 방출 특성을 연구 하였다. 실험은 3%의 더덕추출물을 함유하는 크림제형과 팩제형을 제조하여 Immersion법을 이용하여 자외가시부흡광도계를 이용하여 방출량을 측정하였다. 각 4가지 제형 크림의 방출량을 측정한 결과, 친수성크림, O/W 크림, W/O 크림, 친유성 크림의 순서로 74%, 44%, 24% 18%의 방출량을 나타내었다. W/O 크림의 방출량은 O/W 크림에 비하여 약 절반의 방출량을 나타내었으며 친수성 크림의 방출 양상의 결과, 대체적으로 시간이 지날수록 증가되는 다소 빠른 방출을 보였으며 4시간 이후로 지속적인 방출이 일어날 것으로 추측해본다. 친유성 크림은 더덕추출물이 기제로부터 방출을 하기 위하여 유상을 통과하는 과정이 필요하게 되는데 제형의 차이에 따라 유상이 확산층으로 작용하여 방출을 억제하는 기능을 하였다. 팩제의 방출량 결과, 69%로 크림제형에서 가장 높은 방출량을 보인 친수성 크림보다 조금 낮은 수치를 나타내었다. 더덕추출물이 함유된 크림제제 중에서 더덕 추출물의 수용성을 포함할 수 있는 영역을 가진 크림제제가 방출에 유리하였다. 제형의 특징으로 볼 때 팩제는 기제가 수용성이므로 빠른 시간에 빠른 방출 양상을 보이는 것을 알 수 있었다. 따라서 수층에서 분리한 더덕추출물은 매우 친수성 물질이기 때문에 제형의 친수성 영역 및 용매 이용률에 방출특성이 크게 영향을 받음을 확인하였고 본 실험을 통해 더덕추출물을 함유하는 향장품으로 개발할 경우, 친수성 크림이 흡수가 증대될 것으로 예상하며 꾸준한 연구 개발이 필요하다고 본다.

Cholesterol lowering activities of Codonopsis lanceolata and Platycodon grandiflorum extracts : Soo Hyun Kim

김수현 Kangwon National University 2009 국내박사

더덕 순의 성분분석 및 항염증 기능성분 분리

이채원 전북대학교 일반대학원 2023 국내석사

Codonopsis lanceolata Trautv (CLT) is a wild perennial herb in the mountains of Asia. This plant has been widely used in traditional medicine to treat many diseases such as diabetes, obesity, and colitis. There have been a variety of CLT research, including phytochemical, antioxidants, anti-inflammatory, anti-diabetes, etc. However, studies on the component and efficacy of Codonopsis Lanceolata Trautv. Buds (CLTB) have not yet been revealed. The purpose of this study was to investigate components, efficacy, and also to evaluate the biological active compounds from CLTB by that using activity targeting isolation proceeding. And this study also aimed to investigate the protective effects and molec ular mechanisms of Codonopsis lanceolata buds-derived chrysoeriol on lipopolysaccharide (LPS)-induced inflammation and apoptosis in RAW 264.7 cells. Chrysoeriol dose-dependently inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines and blocked the expression of inducible nitric oxide (iNOS) and cytlooxygenase-2 (COX-2) by inhibiting the nuclear factor-κB (NF-κB) signaling pathway. Chrysoeriol also stimulated activities of antioxidant enzyme and decreased the intracellular reactive oxygen species (ROS) generation. Furthermore, pretreatment of chrysoeriol prevented the increase of Bcl-2 associated x/B-cell lymphoma2 (Bax/Bcl-2) ratio and mitochondrial membrane potential (MMP) loss. Chrysoeriol also inhibited cleave of caspase-9 and caspase-3. Take together, Codonopsis lanceolata buds and its key compound, chrysoeriol play important role in development of anti-inflammatory activity. 더덕은 아시아 산지에 서식하는 야생 다년생 식물이다. 예로부터 그 효능이 천식, 가래, 기침 및 항염증 및 피로회복에 효과가 있고, 당뇨병, 비만, 대장염 등 다양한 방면에서 치료제로 사용된 바 있다. 더덕유래 화합물의 항산화, 항염증, 항당뇨 등의 연구가 보고되고 있으나 더덕의 새순인 더덕순에 관한 연구는 아직 미미하다. 본 연구의 목적은 활성표적분리법을 이용하여 더덕순의 다양한 성분과 효능을 알아보고 생리활성물질을 규명하는데 있다. 더덕순의 에틸 아세테이트 분획물로부터 칼럼 크로마토그래피를 사용하여 lutein (1), apigenin (2), chrysoeriol (3) 의 3종의 플라보노이드를 분리했다. 분리된 화합물의 구조는 분광학적 방법 (1H NMR 및 13C NMR) 문헌고찰과 비교하여 확인하였다. 클로로필, 카로티노이드(루테인, 알파카로틴, 베타카로틴), 비타민 C, 비타민 B1,2,3 그리고 플라보노이드 MS 분석으로 더덕순의 성분분석을 수행했다. 그 다음 DPPH, ABTS 자유 라디칼 소거능 분석, TPC, TFC 항산화 활성, α-glucosidase, tyrosinase 저해 활성, 항염증 활성을 알아보기 위한 NO 저해 활성을 수행하여 염증 억제 매커니즘을 확인하였다. 또한 RAW 264.7 세포에서 지질다당류(lipopolysaccharide, LPS)로 유발된 염증 및 세포사멸에 대한 더덕 새싹 유래 크리소에리올(chrysoeriol)의 보호 효과 및 분자적 기전을 알아보았다. Chrysoeriol은 용량 의존적으로 산화질소(NO), 프로스타글란딘 E2(PGE2) 및 전염증성 사이토카인의 생성을 억제하고 핵 인자를 억제하여 유도성 산화질소(iNOS) 및 사이토옥시게나아제-2(COX-2)의 발현을 차단하였다. 이 화합물은 또한 항산화 효소의 활동을 자극하고 세포 내 반응성 산소종(ROS) 생성을 감소시켰다. Chrysoeriol의 전처리는 Bcl-2 관련 x/B 세포 림프종2 (Bax/Bcl-2) 비율 및 미토콘드리아 막 전위(MMP) 손실의 증가를 방지했으며, caspase-9와 caspase-3의 절단을 억제했다. 종합하면, 이 결과는 더덕순 유래 Chrysoeriol의 잠재적인 분자 메커니즘이 LPS로 유도된 세포 손상에서 NF-κB 및 Bax/Bcl-2/caspase-3 신호 전달 경로와 관련이 있음을 시사한다. 따라서 더덕순은 그 성분과 효능이 항염증 효능을 가지고 있어 추후 더 많은 관련연구가 필요하다.

더덕(Codonopsis lanceolata) 소기관 게놈의 유전적 다양성 분석 및 유전자원 식별을 위한 분자마커의 개발

조남수 충북대학교 2021 국내박사

Codonopsis lanceolata is one of the few valuable plants among the 42 species of genus Codonopsis that is used as a medicinal herb for traditional medicine practice and as an ingredient in traditional foods such as Deodeok gui (grilled Deodeok) and Jangajji (pickled vegetables) in Republic of Korea. The dried root of C. lanceolata is known as Yangyu (羊乳) or Yangyugeun (羊乳根), and its representative ingredient is lancemaside, one of the triterpenoid saponins. In particular, it is called 'deodeok' in Republic of Korea and is widely used in various fields due to its excellent pharmacological effects such as expectorant, antipyretic, and drainage, as well as unique flavor and taste, but research on its development as a genetic resource is very insufficient. In addition, the herbal medicine name of Adenophora triphylla var. japonica Hara, which is known to have the effect of protecting the liver and inhibiting damage, is Sasam (沙蔘), but the dried root of C. lanceolata was incorrectly labeled as Sasam and marketed, causing problems of mixing and misuse in the use of herbal medicines. Therefore, the aim of this study is to identify the origin of wild and distributed C. lanceolata through genetic analysis, to establish a molecular biology database and to promote development and utilization as a useful genetic resource by using molecular markers to verify incorrect product labeling of oriental medicine resources. Genomic DNA information of 43 C. lanceolata collected nationwide was collected using next generation sequencing (NGS), and 43 chloroplast genomes and 37 mitochondrial genomes were completed based on the obtained libraries. We performed a genetic diversity analysis by applying 10 chloroplast-based insertion-deletion (InDel) markers to 93 C. lanceolata wild genetic resources collected nationwide and 18 herbal medicine resources collected from 4 herbal medicine markets (54 individual plants; 16 C. lanceolata [48 individual plants] and 2 outgroups, A. triphylla [6 individual plants]). As a result of generating the unweighted pair group method with arithmetic mean (UPGMA)-based phylogenetic tree, wild collections were not classified by region, nor did they form a specific community, and thus it was judged that the genetic diversity was very rich. In addition, it has been found that some C. lanceolata products are being sold as Sasam, which is an incorrect product label, rather than Yangyu or Yangyugeun in resources collected from the herbal medicine market. Nucleotide polymorphisms were detected in the complete organelle genomes (43 chloroplast and 37 mitochondrial genomes) in this study. In chloroplast genomes, 190 single nucleotide polymorphism (SNP) loci and 43 InDel loci were found, and 140 SNP and 13 InDel loci were found in mitochondria. Based on the double chloroplast genome search results, 37 SNP-based and 7 InDel-based kompetitive allele specific PCR (KASP) markers were developed. Genetic diversity analysis was performed using 44 KASP markers for wild, herbal medicine, distribution, and foreign resources. Analysis was performed on 144 collections including wild, herbal medicine, distribution, and foreign resources, and when only wild genetic resources were analyzed, three clusters were identified as shown in the results; it was shown that Gyeongsangnam-do, Jeollabuk-do, and Jeollanam-do resources were concentrated in a specific cluster with 66.7%, 71.4%, and 75.0%, respectively. In addition, distribution resources were included in only two out of three clusters, and it was shown that foreign resources had a deeper relationship in a specific cluster. Analysis of the genetic diversity confirmed the close relationship, such as the distribution state and the degree of regional collections. Research in a category similar to this study should be continuously expanded because it can prevent misuse of herbal medicines and increase the value and trust of natural medicines through precise classification of herbal medicines using molecular biological approaches. In addition, the construction of chloroplast and mitochondrial genomes of C. lanceolata, which are highly valuable as genetic resources, and the study of phylogenetic relationships through biological approaches using them will further increase the value of these resources in C. lanceolata breeding. In particular, the KASP markers developed in this study will be helpful in molecular breeding studies for the development of new varieties of C. lanceolata such as genome-wide association study (GWAS), marker-assisted selection (MAS), quantitative trait locus (QTL) analysis, DNA barcoding, and genome mapping. In the meantime, it will establish a basis for molecular breeding of C. lanceolata, which has been insufficiently researched for the development of new varieties, and will greatly contribute to molecular breeding and industrial growth of more domestic medicinal crops.