• Title/Summary/Keyword: $Red{\beta}$

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Whitening and inhibiting NF-κB-mediated inflammation properties of the biotransformed green ginseng berry of new cultivar K1, ginsenoside Rg2 enriched, on B16 and LPS-stimulated RAW 264.7 cells

  • Xu, Xing Yue;Yi, Eun Seob;Kang, Chang Ho;Liu, Ying;Lee, Yeong-Geun;Choi, Han Sol;Jang, Hyun Bin;Huo, Yue;Baek, Nam-In;Yang, Deok Chun;Kim, Yeon-Ju
    • Journal of Ginseng Research
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    • v.45 no.6
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    • pp.631-641
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    • 2021
  • Background: Main bioactive constituents and pharmacological functions of ripened red ginseng berry (Panax ginseng Meyer) have been frequently reported. Yet, the research gap targeting the beneficial activities of transformed green ginseng berries has not reported elsewhere. Methods: Ginsenosides of new green berry cultivar K-1 (GK-1) were identified by HPLC-QTOF/MS. Ginsenosides bioconversion in GK-1 by bgp1 enzyme was confirmed with HPLC and TLC. Then, mechanisms of GK-1 and β-glucosidase (bgp1) biotransformed GK-1 (BGK-1) were determined by Quantitative Reverse Transcription-Polymerase Chain Reaction and Western blot. Results: GK-1 possesses highest ginsenosides especially ginsenoside-Re amongst seven ginseng cultivars including (Chunpoong, Huangsuk, Kumpoong, K-1, Honkaejong, Gopoong, and Yunpoong). Ginseng root's biomass is not affected with the harvest of GK-1 at 3 weeks after flowering period. Then, Re is bioconverted into a promising pharmaceutical effect of Rg2 via bgp1. According to the results of cell assays, BGK-1 shows decrease of tyrosinase and melanin content in α-melanocyte-stimulating hormone challenged-murine melanoma B16 cells. BGK-1 which is comparatively more effective than GK-1 extract shows significant suppression of the nuclear factor (NF)-κB activation and inflammatory target genes, in LPS-stimulated RAW 264.7 cells. Conclusion: These results reported effective whitening and anti-inflammatory of BGK-1 as compared to GK-1.

Effect of SAL5 on chronic ethanol-induced fatty liver model (흰쥐에서 SAL5의 알코올성 지방간 형성에 미치는 영향)

  • Kim, Bok-Kyu;Yang, Won-Kyung;Park, Yang-Chun;Jung, Ga-Young;Shin, Eun-Ju;Do, Seon-Gil;Kim, Seung-Hyung
    • The Korea Journal of Herbology
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    • v.33 no.1
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    • pp.17-26
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    • 2018
  • Objective : In this study, we investigated the effect of SAL5(mixing extracts of Schisandra chinensis Baillon, Artemisia capillaris Thunb., and Aloe vera Linne) on chronic ethanol-induced fatty liver model. Methods : Sprague-Dawley male rats were fed Liber-DeCarli (normal), ethanol liquid diet (control), SAL5 (200 mg/kg). We administrated the SAL5 on chronic ethanol-induced fatty liver model for 5 weeks. We measured alkaline phosphtase (ALP), alanine transminase (ALT), aspartate transminase (AST) and ${\gamma}-glutamyl$ transpeptase (${\gamma}-GTP$) in serum and triglyceride (TG), superoxide dismutase (SOD), catalase, glutathione (GSH) and malondialdehyde (MDA) level in liver. Liver histopathology was examined by Hematoxylin-eosin and Oil red O staining of the fixed liver tissues. Real-time PCR was performed to measure the mRNA expression of inflammatory cytokines and MMP-2, MMP-9. Results : SAL5 administration resulted in significantly decreased liver marker enzymes activities of alanine transminase (ALT), ${\gamma}-glutamyl$ transpeptase (${\gamma}-GTP$) in serum and triglyceride (TG) activities in liver. The control group decreased the activities of superoxide dismutase (SOD), catalase (CAT) with the reduced level of glutathione (GSH) in liver. On the other hand, SAL5 group increased the activities of SOD, CAT and the level of GSH. SAL5 delayed the development of an alcoholic fatty liver by reversing fat accumulation in the liver, as evidenced in histological observations. The gene expression of mRNA were significantly decreased at the $IL-1{\beta}$, $TNF-{\alpha}$, NOS-II and MMP-2 by SAL5. Conclusions : These results indicate that SAL5 might have protective effect chronic ethanol-induced fatty liver models.

Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity

  • Chih-Hsin Lin;Yu-Shao Hsieh;Ying-Chieh Sun;Wun-Han Huang;Shu-Ling Chen;Zheng-Kui Weng;Te-Hsien Lin;Yih-Ru Wu;Kuo-Hsuan Chang;Hei-Jen Huang;Guan-Chiun Lee;Hsiu Mei Hsieh-Li;Guey-Jen Lee-Chen
    • Biomolecules & Therapeutics
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    • v.31 no.1
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    • pp.127-138
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    • 2023
  • Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

Anti-Melanogenic, Anti-Wrinkle, Anti-Inflammatory and Anti-Oxidant Effects of Xylosma congesta leaf Ethanol Extract (산유자 잎 에탄올 추출물의 미백, 주름억제, 항염증 및 항산화 효능)

  • Lee, Jae Yeon;Ahn, Eun-Kyung;Ko, Hye-Jin;Cho, Young-Rak;Ko, Woon Chul;Jung, Yong-Hwan;Choi, Kyung-Min;Choi, Mi-Rae;Oh, Joa Sub
    • Journal of Applied Biological Chemistry
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    • v.57 no.4
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    • pp.365-371
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    • 2014
  • In the present study, we investigated the biological activities of Xylosma congesta leaf ethanol extract (XCO) using a variety of in vitro and cell culture model systems for anti-melanogenic, anti-wrinkle, anti-inflammatory and anti-oxidant activities. First, XCO markedly inhibited ${\alpha}$-melanocyte stimulating hormone-stimulated melanin synthesis in B16F10 cells. Secondly, XCO marginally induced procollagen synthesis in CCD-986SK cells. Thirdly, XCO dose-dependently suppressed lipopolysaccharide-induced nitric oxide (NO) production in RAW 264.7 cells. XCO did not affect cell viability at different concentrations used in this study, indicating that XCO-mediated inhibition of melanin, procollagen and NO synthesis is not mediated by cytotoxicity. Finally, XCO was found to exert anti-oxidant effect. Taken together, these findings demonstrate for the first time that XCO possesses anti-melanogenic, anti-wrinkle, anti-inflammatory and anti-oxidant activities, and suggest further evaluation and development of XCO as a functional supplement or cosmetic that may be useful for whitening skin, reducing wrinkles and treating inflammatory responses.

CORRELATION BETWEEN VASCULAR ENDOTHELIAL GRWOTH FACTOR SIGNALING AND MINERALIZATION DURING OSTEOBLASTIC DIFFERENTIATION OF CULTURED HUMAN PERIOSTEAL-DERIVED CELLS (배양된 인간 골막기원세포의 조골세포 분화과정에서 골기질 형성정도와 혈관내피세포성장인자 신호와의 상관관계)

  • Park, Bong-Wook;Byun, June-Ho;Ryu, Young-Mo;Hah, Young-Sool;Kim, Deok-Ryong;Cho, Yeong-Cheol;Sung, Iel-Yong;Kim, Jong-Ryoul
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.29 no.3
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    • pp.197-205
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    • 2007
  • Angiogenesis is a essential part for bone formation and bone fracture healing. Vascular endothelial growth factor (VEGF), one of the most important molecules among many angiogenic factors, is a specific mitogen for vascular endothelial cells. VEGF-mediated angiogenesis is required for bone formation and repair. However, the effect of VEGF on osteoblastic cells during osteogenesis is still controversial. In recent days, substantial progress have been made toward developing tissue-engineered alternatives to autologous bone grafting for maxillofacial bony defects. Periosteum has received considerable interest as a better source of adult stem cells. Periosteum has the advantage of easy harvest and contains various cell types and progenitor cells that are able to differentiate into a several mesenchymal lineages, including bone. Several studies have reported the bone formation potential of periosteal cells, however, the correlation between VEGF signaling and cultured human periosteal cell-derived osteogenesis has not been fully investigated yet. The purpose of this study was to examine the correlation between VEGF signaling and cultured human periosteal-derived cells osteogenesis. Periosteal tissues of $5\;{\times}\;20\;mm$ were obtained from mandible during surgical extraction of lower impacted third molar from 3 patients. Periosteal-derived cells were introduced into the cell culture and were subcultured once they reached confluence. After passage 3, the periosteal-derived cells were further cultured for 42 days in an osteogenic inductive culture medium containing dexamethasone, ascorbic acid, and ${\beta}-glycerophosphate$. We evaluated the alkaline phosphatase (ALP) activity, the expression of Runx2 and VEGF, alizarin red S staining, and the quantification of osteocalcin and VEGF secretion in the periosteal-derived cells. The ALP activity increased rapidly up to day 14, followed by decrease in activity to day 35. Runx2 was expressed strongly at day 7, followed by decreased expression at day 14, and its expression was not observed thereafter. Both VEGF 165 and VEGF 121 were expressed strongly at day 35 and 42 of culture, particularly during the later stages of differentiation. Alizarin red S-positive nodules were first observed on day 14 and then increased in number during the entire culture period. Osteocalcin and VEGF were first detected in the culture medium on day 14, and their levels increased thereafter in a time-dependent manner. These results suggest that VEGF secretion from cultured human periosteal-derived cells increases along with mineralization process of the extracellular matrix. The level of VEGF secretion from periosteal-derived cells might depend on the extent of osteoblastic differentiation.

Studies on the Mechanism of the Ameliorative Activities on Hyperglycemia and Dyslipidemia of Red Ginseng Herbal Acupuncture in C57BL/KsJ db/db Mice (홍삼약침(藥鍼)이 제2형 당뇨병 동물모델의 항고혈당(抗高血糖)및 항고지질(抗高脂質) 기전(機轉)에 미치는 영향(影響))

  • Kim, Jong-Deok;Kim, Jong-In;Koh, Hyung-Kyun;Lee, Yun-Ho;Kang, Sung-Keel
    • Journal of Acupuncture Research
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    • v.25 no.2
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    • pp.11-26
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    • 2008
  • 목적 : 홍삼약침(藥鍼)이 고혈당 및 지질대사장애에 미치는 개선효과와 그 기전을 조사하고자 한다. 방법 : 홍삼약침(藥鍼)의 anti-diabetic 활성과 그 기전을 C57BL/KsJ db/db mice를 이용하여 관찰하였다. 실험 동물은 대조군(DC), 홍삼약침(藥鍼)군(RGL, RGH) 및 양성대조군(MET, GPZ, PIO)의 6군으로 나누었다. 홍삼약침(藥鍼)군은 $0.2m{\ell}$의 홍삼약침멸(藥鍼滅)을 각각 100mg/kg(RGL) 및 200mg/kg(RGH)씩 인체의 간유(肝兪)($BL_{18}$)에 상응하는 혈위에 1일 1회 10주간 좌우 혈을 번갈아가며 약침 시술하였다. 양성대조군은 metformin 300mg/kg(MET), glipizide 15mg/kg(GPZ) 및 pioglitazone 30mg/kg(PIO)을 각각 1일 1회 10주간 경구투여 하였다. 체중과 혈당은 매주 측정하였다. 실험 10주 후에는 혈액채취로 혈중 glucose, 당화혈색소(HbAlc), insulin, 중성지방(TG), adiponectin, leptin, non-esterified fatty acid(NEFA)를 측정 하였고, 간 조직을 채취하여 조직학적 검사 및 gene expression 분석을 시행하였다. 결과 : 홍삼약침(藥鍼)(RGL, RGH)은 10주 동안 C57BL/KsJ db/db mice의 체중을 증가시키는 부작용은 나타나지 않았다. 홍삼약침(藥鍼)군(RGL, RGH)의 사료섭취량은 대조군과 비슷하였으나 음수량은 증가하였다. 홍삼약침(藥鍼)(RGL, RGH)은 대조군에 비하여 각각 19.8% 및 18.3% 혈당을 낮추었고, 홍삼약침(藥鍼)(RGL)은 insulin resistance를 27.7% 감소시켰으며, 경구내당능 검사의 혈중 glucose에서는 대조군에 비해 홍삼약침(藥鍼)군(RGL, RGH)과 양성대조군(MET, GPZ, PIO)에서 각각 19.8%, 18.3%, 67.7%, 52.3% 및 56.9% 감소시켰다. 당화혈색소(HbAlc)는 홍삼약침(藥鍼)(RGL, RGH), MET, GPZ 및 PIO군에서 대조군에 비하여 각각 11.0%, 6.4%, 18.9%, 16.1% 및 27.9% 감소시켰으며, 혈중 glucose감소와 유사한 경향을 나타내었다. 홍삼약침(藥鍼)(RGL)은 대조군에 비해 TG와 NEFA를 각각 18.8% 및 16.8% 감소시켰고, adiponectin과 leptin을 각각 20.6% 및 12.1% 증가시켰다. 홍삼약침(藥鍼)(RGL, RGH)은 중성지방의 침착으로 인한 간의 질량비 증가를 억제하지 못하였으나, 지방구를 감소시겼음을 관찰할 수 있었다. Microarray 분석에서는 홍삼약침(藥鍼)(RGL, RGH)이 간에서 glycolysis, gluconeogenesis 및 fatty acid beta-oxidation과 관련된 유전자 발현에 영향을 미치는 것으로 나타나 양성대조군 metformin과 유사한 기전을 나타내었다. 요약 : 홍삼약침(藥鍼)은 T2DM동물모델(C57BL/KsJ db/db mice)에서 항당뇨 및 지질대사 개선활성이 있었다. 홍삼약침(藥鍼)은 C57BL/KsJ db/db mice의 간조직에서 lipogenesis억제 및 fatty acid beta-oxidation활성을 통해 혈당 이용을 높이고, insulin sensitivity를 향상시켰다. 또한 유전자 발현분석을 통해 그 기전이 metformin과 유사함을 확인할 수 있었으므로 향후 홍삼약침(藥鍼)의 새로운 약침 기술 개발 근거가 될 수 있을 것으로 사료된다.

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The Extrapolations to Reduce the Need for Pesticide Residues Trials on Continuous Harvesting Leafy Vegetables (농약 잔류 시험을 위한 연속수확 엽채소류의 외삽)

  • Son, Kyeong-Ae;Im, Geon-Jae;Hong, Su-Myeong;Kim, Chan Sub;Gil, Geun-Hwan;Jin, Yong-Duk;Kim, Jinba;Ihm, Yang Bin;Ko, Hyeon Seok;Kim, Jang Eok
    • The Korean Journal of Pesticide Science
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    • v.17 no.4
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    • pp.293-301
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    • 2013
  • This study was carried out to investigate the pesticide residue pattern among different leafy vegetables applied with foliar spraying under greenhouse and to check extrapolating from some residue trial data to other minor crops. Leafy vegetables used in this study were: Mustard greens (Brassica juncea L.), Kale (Brassica oleracea L.), Dacheongchae (a kind of pak-choi (Brassica rapa subsp. chinensis L.)), Leaf broccoli (Brassica oleracea var alboglabra), Perilla leaf (Perilla frutescens (L.) Britton var. Frutescens), Leaf lettuce (Lactuca sativa L.), Swiss chard (Beta vulgaris L. subsp. vulgaris) and Red leaf chicory (Cichorium intybus L. var. foliosum Hegi). These are cultivated all year under indoor or outdoor and cut the leaf from plant continuously during harvest time. The amounts of pesticide deposit in/on the continuous harvesting leafy vegetables were affected by the ratios of leaf area to weight. Ratio of perilla leaf was the largest among crops as 58 $cm^2/g$. The residue levels of 7 pesticides in/on perilla leaf were the highest than those of other crops through the statistical analysis from zero day to fifth day after last application. The representative crop in 8 crops was perilla leaf selected based on the amounts of daily consumption and the high residues. This study suggest that the continuous harvesting leafy greens should be separated from the one time harvesting leafy vegetables for the pesticide recommendations because of different harvesting habits and pre-harvest intervals.

Studies on the Taste of Korean for Cheese (한국인(韓國人)의 치이즈 기호성(嗜好性)에 관한 연구)

  • Kim, Jong Woo;Ko, Keun Hag
    • Korean Journal of Agricultural Science
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    • v.18 no.1
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    • pp.21-32
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    • 1991
  • This experiment was carried out to examine sensory testing for Mozzarella cheese, process cheese, Cheddar cheese and Cheddar cheese made with red pepper, garlic, ginger and welsh onion to develop new cheese varieties which can be prefered by Korean. The chemical composition and sensory testing of cheese were measured. The results were summarized as follows ; 1. Total nitrogen percentages in Cheddar cheese and spiced Cheddar cheeses were similar but those in process cheese and Mozzarella cheese were low. 5% NaCl soluble nitrogen percentages were highest in Cheddar cheese. 5% NaCl soluble nitrogen percentages in each cheese were different. Ripening degree, water soluble nitrogen, TCA soluble nitrogen and SSA soluble nitrogen percentages in each cheese were similiar level. 2. Spiced Cheddar cheeses were more breakdown than other cheese and ${\alpha}_s$-casein breakdowns faster than $\beta$-casein. 3. In the result of sensory evaluation, color score was high in Mozzarella cheese and process cheese. The color score of Cheddar cheese was high in 30's-40's and 50's- 50's. The color score of 10's and 20's was high in Cheddar cheese made with garlic. 4. Odor score was high in Mozzarella cheese and process cheese, too. The odor score of Spiced Cheddar cheeses was high in 10's. 5. Texture score was high in Mozzarella cheese, process cheese and Cheddar cheese. 6. Teste score was high in Mozzarella cheese, process cheese and Cheddar cheese. The taste score of spiced Cheddar cheese was higher in 10's and 20's than that in 30's-40's and 50's-50's.

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Characteristics of Fermented Hot Pepper Soybean Paste (Kochujang) Prepared by Liquid Beni-koji (액체홍국코지를 이용한 고추장의 제조)

  • Kang, Seong-Gook;Park, In-Bae;Jung, Soon-Teck
    • Korean Journal of Food Science and Technology
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    • v.29 no.1
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    • pp.82-89
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    • 1997
  • Monascus anka produces biologically active materials, and liquid M. anka koji was employed for preparing fermented hot pepper soybean paste (kochujang). Three kinds of koji (M. anka, Aspergillus oryzae and mixed koji) were used to prepare kochujang, and changes of physicochemical characteristics were examined during fermentation. A. oryzae koji showed the highest ${\alpha}-amylase$ and protease activities; whereas, M. anka koji showed the highest ${\beta}-amylase$ activity in liquid koji. Water content of kochujang continuously decreased, whereas viscosity increased during fermentation. The water content and viscosity was 46% and $1.4{\times}10^5\;cP$, respectively after 40 days of fermentation. The pH of kochujang with M. anka and/or A. oryzae koji was $4.63{\sim}4.65$ in the beginning and was between 4.53 and 4.67 after 40 days of fermentation. L-, a- and b-values decreased rapidly during fermentation until 20 days of fermentation. After 40 days, L- and b-values of kochujang prepared with M. anka koji showed lower values than A. oryzae, and a-value showed higher values as the amount of M. anka koji increased. Ammoniacle nitrogen content continuously increased during fermentation. Amino nitrogen content was the highest, 241.2 ㎎%, and ammoniacle nitrogen was the lowest, 97.2 ㎎%, in kochujang prepared with A. oryzae. However, amino and ammoniacle nitrogen content showed no difference between M. anka and A. oryzae koji kochujang. Kochujang prepared with M. anka koji showed a potential that kochujang can be manufactured lower level of red pepper than previously used.

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Studies on the Physiological Chemistry of Flower Organ and Seed in Ginseng Plant. IV. Variation of Free Amino Acids in the Flower and Seeds of the $F_1$ Plants of the Combinations Panax ginseng ${\times}$ Panax quinquefolium and Panax ginseng ${\times}$ Panax japonicus. (인삼종자형성에 대한 생리화학적 연구 IV. 고려인삼과 미국인삼 및 고려인삼과 죽절인삼 $F_1$의 화기 및 종자 형성과정에 있어서의 유리아미노산의 소장)

  • Jong-Kyu Hwang;Hee-Chun Yang
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.14
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    • pp.165-172
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    • 1973
  • The sterile phenomenon is frequently found in the inter-species hybrids of ginseng as in other plants. It is known that among the hybrids between Panax Ginseng (PG) and Panax Quinquefolium (PQ), and between Panax Ginseng and Paxax Japonicus (PI), PG${\times}$PI is fertile only very rarely, while PG ${\times}$ PQ is always sterile. Therefore, in order to clarify the relationship between this sterility phenomenon and the metabolism of free amino acids, the changes of free amino acids through the formation of the flower organs and seeds of two hybrids, PG ${\times}$ PQ and PG ${\times}$ PI were investigated by thin layer chromatography. The results are summarized as follows: 1. Distinct differences in the quantity and number of free amino acids were recognized between PG ${\times}$ PQ, PG ${\times}$ PI and their parent plants. From the hybrid PG ${\times}$ PQ, 19 kinds of ninhyrin sensitive substances were detected in all. They were (1) 17 amino acids: alanine, valine, leucine, phenylalanine, proline, hydroxy-proline, serine, threonine, tyrosine, aspartic acid, glutamic acid, lysine, arginine, ${\gamma}$-amino butyric acid, ${\beta}$-alanine, cysteic acid and tryptophan, and (2) two amides: asparagine and glutamine. From the hybrid PG ${\times}$ PI, in addition to the above 19 substances, methionine and one unknown substance were detected. 2. Generally, alanine, as partie acid, glutamic acid, cysteic acid and asparagine were detected in large amounts in the two hybrids as in PG, PG and PJ but it was a noticeable fact concerning these two hybrids that the largest quantity of asparagine was found at microspore satge and pollen mature stage. 3. The decrease of cysteic acid in the two hybrids at the red ripened stage was the same as in PQ and PJ but opposite to the change in PG. The detection of methionine in PG ${\times}$ PJ was worthy of notice. 4. The change of proline was conspicuously different from that in their parent plants. It was detected as a trace of color at the micros pore stage while asparagine was detected in the greatest amount at that time. It is well known that the quantity of proline is closely related to the sterility of plant. This fact was also found true in the formation of ginseng seeds. It was reported as well that asparagine accumulated when proline decreased. 5. The deficiency of proline seemed to be closely related with the sterility of hybrids and with the degradation of pollen in anther. 6. The difference in the changes of free amino acids between the selfed lines of PG, PQ and PJ, and their hybrids seemed to be caused by the transformation of gene-action system by hybridization. On these phenomena along with proline metabolim and its physiological role in seed formation further studies are required.

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