• Title/Summary/Keyword: food and nutrition

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Xanthine and Aldehyde Oxidase Inhibitory Activities, and Antihyperuricemic Effects of Fermented Smilax china L. Leaf Extracts and Fractions (발효 청미래덩굴잎 용매 추출물 및 분획물의 xanthine 및 aldehyde oxidase 저해활성과 항고요산혈증 효과)

  • Lee, Sang-Il;Lee, Ye-Kyung;Kim, Soon-Dong;Cheng, Jinhua;Yang, Seung Hwan;Suh, Joo-Won
    • Journal of Applied Biological Chemistry
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    • v.57 no.1
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    • pp.53-59
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    • 2014
  • To evaluate the inhibitory effect of xanthine oxidase (XO) and aldehyde oxidase (AO), and antihyperuricemic effect by Aspergillus oryzae fermented Smilax china L. leaf extracts and fractions, we observed extracted yield by each solvent, the content of total polyphenol and total flavonoid (TF), the activities of XO and AO, and serum uric acid level. Extracted yield (g/kg) by 80% ethanol (EtOH) was 13.56, those of n-hexane, dichloromethane (DICM), ethylacetate (EtOAc) and n-butanol fraction (BuOH) were 1.35-3.33. Furthermore, total polyphenol content (mg/g-extract) of EtOAc fraction, BuOH fraction, DICM fraction and EtOH fraction is 478.07-501.26, 259.49-289.02, 165.03-232.27, 134.02-196.54, respectively. Those of fermented EtOAc and DICM fraction was 4.85 and 40.74% higher than that of non-fermented fraction, respectively, while the other fermented fractions were lower than those of non-fermented fractions. And total flavonoid content (mg/g-extract) of EtOAc fraction was higher than those of other fractions. Additionally, TF of fermented EtOAc and BuOH fraction is 10.56 and 60.17% higher, than that of fermented fraction, respectively, although those of the other fermented fractions was lower than that of non-fermented fractions. On the other hand, XO inhibitory activities of all fermented fractions was significantly higher than that of all non-fermented fraction, while those of fermented EtOAc (75.02%) and BuOH fraction (65.59%) was markedly higher than that of non-fermented fraction (39.42 and 5.34%), respectively. In addition, AO inhibitory activities of DICM and EtOAc fraction was 81.82 and 77.93% higher, respectively, than those of the other fractions, and those of fermented fractions as with XO were significantly higher than that of non-fermented fractions. Meanwhile, serum uric acid level (SU) of hyperuricemic control mice (HC, 6.98 mg/dL) was 1.83 folds higher than that of normal control (NC, 3.82 mg/dL). Furthermore, SU in the group treated with EtOAc fraction decreased in a dose dependent manner compared with the allopurinol control group, although those of fermented fractions were significantly lower than those of non-fermented fractions. This study suggests that fermented Smilax china L. leaf extracts may regulate the XO and AO inhibitory activities and antihyperuricemic effect due to aglycone components from glycoside form flavonoids by fermentation of A. oryzae.

Socheongja and Socheong 2 Extracts Suppress Lipopolysaccharide-induced Inflammation and Oxidative Stress in RAW 264.7 Macrophages through Activating Nrf2/HO-1 Signaling and Suppressing MAPKs Pathway (RAW 264.7 대식세포에서 Nrf2/HO-1 신호 전달계 활성화와 MAPKs 경로 억제를 통한 소청자와 소청2호의 LPS 매개 염증성 및 산화적 스트레스 반응의 억제)

  • Kwon, Da Hye;Choi, Eun Ok;Hwang, Hye-Jin;Kim, Kook Jin;Hong, Su Hyun;Lee, Dong Hee;Choi, Yung Hyun
    • Journal of Life Science
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    • v.28 no.2
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    • pp.207-215
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    • 2018
  • Inflammatory response and oxidative stress play critical roles in the development and progression of many human diseases. Therefore, a great deal of attention has been focused on finding functional materials that can control inflammation and oxidative stress simultaneously. The purpose of this study was to investigate the effects of Socheongja and Socheong 2, Korean black seed coat soybean varieties, on the inflammatory and oxidative stress induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages. Our data indicated that the extracts of Socheongja (SCJ) and Socheong 2 (SC2) significantly suppressed LPS-induced production of nitrite oxide (NO) and prostaglandin $E_2$, key pro-inflammatory mediators, by suppressing the expression of inducible NO synthase and cyclooxygenase-2. It was also found that SCJ and SC2 reduced the LPS-induced secretion of pro-inflammatory cytokines, such as tumor necrosis $factor-{\alpha}$ and $interleukin-1{\beta}$, which was concomitant with a decrease in the protein levels. In addition, SCJ and SC2 markedly diminished LPS-stimulated intracellular reactive oxygen species accumulation, and effectively enhanced nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase (HO)-1 expression. Furthermore, LPS-induced activation of mitogen-activated protein kinases (MAPKs) was abrogated by SCJ and SC2. Taken together, these data suggest that SCJ and SC2 may offer protective roles against LPS-induced inflammatory and oxidative responses in RAW 264.7 macrophages through attenuating MAPKs pathway, and these effects are mediated, at least in part, through activating Nrf2/HO-1 pathway. Given these results, we propose that SCJ and SC2 have therapeutic potential in the treatment of inflammatory and oxidative disorders caused by over-activation of macrophages.

Effects of Different Exercise Intensity on Excess Post Exercise Oxygen Consumption (EPOC), Resting Metabolic Rate (RMR), and Biochemical Variables in Obese and NIDDM Patients (운동 강도의 차이가 제2형 당뇨병 환자와 비만인의 초과산소 섭취량, 안정 시 대사량 및 생화학적 변인에 미치는 영향)

  • Kwak, Yi-Sub;Ku, Woo-Young;Yoo, Byung-In;Jin, Young-Wan;Choi, Kyung-Suk;Cho, Joon-Yong;Woo, Jin-Hee;Hwang, Hye-Jin
    • Journal of Life Science
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    • v.18 no.10
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    • pp.1455-1463
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    • 2008
  • The purpose of this study was to evaluate the effect of various exercise intensity on Excess post exercise energy expenditure (EPEE), Resting Metabolic Rate (RMR),thyroid hormonal changes and biochemical variables in obese and NIDDM patients. The subject of the present study were divided into four groups and four periods: trained (T; n=10), control (C; n=10), obese (O; n=10) and NIDDM (N; n=10) group. And the periods were divided as follows; Resting (RE), Maximal (MA), High intensity (HI), and Low intensity (LI). There was significant difference in RMR among different intensity of exercise. in the T (p<0.05) not in the C, O, and N groups. however, there was no significant different percent body fat among all groups. In the energy expenditure, there was significant different among C, O, N groups compare to T in HIEE (high intensity exercise energy expenditure), LIEE (low intensity exercise energy expenditure), HIEEPE (high intensity exercise energy expenditure post exercise) and LIEEPE (low intensity exercise expenditure post exercise). In the hormonal level, there was significant different in T4 level in the T group at LI period and there was also significant difference in T4, Free T3, & Free T4 levels in T group at LI period, however there was no significant different in the O and N groups except LI period. In the fatigue variables, there was significant different in lactate and ammonia levels in the N group in the period of HI compare to C. The present cross-sectional study was design to investigate the relationship between exercise intensity and RMR in four groups. The focus of this investigation was to compare RMR in aerobically trained (T), control (C), obese (O) and NIDDM (N) group. The relationship among RMR, exercise intensity and percent body fat would best be investigated using Meta Lyzer 3B, MMX3B and body composition analyzer. Each subject completed measurement of percent body fat, RMR, hormone in the period of maximal oxygen uptake exercise (MA), high intensity exercise (HI), and low intensity exercise (LI). From the results, High and Low intensity of exercise, there was a trend for an increased RMR (kcal/day) in the trained groups and control group (in case of LI) not for the obese and N groups. This is best explained not by the reduced percent body fat but by the highly induced energy expenditure (during exercise and post exercise energy expenditure) and increased T4, Free T3, and Free T4 hormonal levels in the low intensity exercise for the T group and sometimes C group.

A Study on the Relationship between Milk Consumption, Dietary Nutrient Intake and Physical Strength of Adolescents in Middle and Small-Sized Cities in Korea for Dietary Education of Home Economics Subject at Middle and High Schools (중등학교 가정교과 식생활교육을 위한 청소년의 우유 섭취, 영양 섭취 및 체력과의 관계 연구)

  • Kim, Sun-Hyo
    • Journal of Korean Home Economics Education Association
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    • v.28 no.4
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    • pp.159-167
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    • 2016
  • This study was performed to investigate the relationship between milk consumption, dietary nutrient intake, and physical strength in Korean adolescents for dietary education of home economics subject at middle and high schools. Intake of milk and milk products, that of dietary nutrients, and physical strength records were surveyed for 298 high school students resided in small and medium-sized cities in Korea. Intake of milk and milk products was collected from three-day dietary survey, and daily dairy equivalent of calcium was calculated by dividing daily calcium intake(mg) from milk and milk products by calcium content(200 mg) of 200 mL milk. Dietary survey was performed during three days including two-weekdays and one-weekend by food record method. Dietary nutrient intake was analyzed by CAN-Pro 3.0. Physical strength records were offered from subjects' schools. Subjects were divided into low intake group(${\leq}0.29/d$), medium intake group(0.30-0.78/d) and high intake group(0.79-5.66/d) according to daily mean dairy equivalent of calcium taken by them, and most of them consumed milk and milk products less than '2' of the recommended value. Daily mean dairy equivalent of calcium was significantly related with high records of '50 m running' and 'stand long jump'(p<0.05), however it was not related with other records of physical strength. Intakes of energy, carbohydrate, protein, fat, calcium, phosphorus, iron, zinc, thiamin, riboflavin, niacin and vitamin E were significantly correlated with high record of '50 m running'(p<0.01). Intakes of nutrients were related with '50 m running' except fat were significantly correlated with high record of 'stand long jump'(p<0.05). Intakes of protein, phosphorus, iron, zinc, vitamin A, $B_6$ and E were significantly correlated with high record of 'long distance running'(p<0.05). In conclusion intake of milk and milk products was related with physical strength records('50 m running' and 'stand long jump') and intakes of most nutrients including energy nutrient, vitamin and mineral were correlated with physical strength records('50 m running', 'stand long jump' and 'long distance running') in Korean adolescents. Therefore, optimal consumption of milk and various nutrients for balanced nutrition during adolescents should be emphasized in dietary education of home economics subject for their strong physical strength.

Production and Quality Parameters of Oat Grown in Conventional/Organic Farming

  • Petr Konvalina;Ivana Capouchova
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.19-19
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    • 2022
  • Hulled and naked oat is a perspective crop for the low input production systems due to its low requirements for soil quality and nutrition. Oats have good competitive ability against weeds and can provide appropriate yield in organic farming in comparison with other cereal species such as wheat or barley. It is a perspective crop from the point of view of use in the food industry too. The aim of our study was to compare the production and quality parameters of naked and hulled oat grown in both organic (OF) and conventional fields (CF). Small plot trials were conducted in two locations in the Czech Republic (České Budějovice, Prague) for four years (2018-2021) in two production systems (OF, and CF). We used four varieties of hulled oat (Korok, Kertag, Raven, Seldon) and one variety of naked oat (Patrik). During the vegetation, agronomically important data were recorded. After harvest samples were processed in the laboratory and analyzed selected quality parameters of grain dry matter (the protein content was determined by the Kjeldahl method, starch content in grain according to Ewers, fat content in grain dry matter by the modified method according to Soxhlet, and ash content in grain dry matter). The data were evaluated using the program STATISTICA version 13.2, StatSoft, Inc., California, USA. It is clear from the results that the number of panicles before the harvest was influenced by the location, cultivation system, year, and, to a lesser extent, the influence of the variety. The number of panicles in OF averaged 340 per square meter, which was 90% of the value of CF. For thousand grain weight (TGW), a significantly predominant effect of year was found. The independent effect of location on TGW was statistically not significant. Grain yield was predominantly influenced by cultivation system and location. In OF, it reached an average of 3.97 t.ha-1, which was 75% of the yield of CF. As part of the evaluation of the basic grain quality indicators, the content of protein, starch, fat, and ash in the dry matter of the grain was evaluated. The content of protein in the dry matter of the grain was predominantly influenced by year, followed by the influence of the variety and a fairly comparable influence of the cultivation system and locality. On average, it achieved 16.05% in OF and 17.01% in CF. The starch content was then related to the protein content, where as a result of the lower protein content in the grain of OF oats, the content of starch and fat was on the contrary increased. The year turned out to be the most significant factor, affecting both the starch content in the dry matter of the grain and the fat content. This was followed again by a fairly comparable influence on the cultivation system and locality. The influence of the cultivation system and location was not statistically significantly applied in the case of ash content in dry matter. Based on our results we can propose both types of oat (hulled and naked) as perspective crops for OF. An organic farmer can expect to achieve stable yields which, in less favorable conditions for the production of cereals in the OF, may be close to the level of conventional yields. In the future, it will be important to change agrotechnology in OF and increase oat yield because this crop has a good potential to grow in areas with low nitrogen input or less fertile soil.

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Hypoxia Inducible Factor-1α Directly Regulates Nuclear Clusterin Transcription by Interacting with Hypoxia Response Elements in the Clusterin Promoter

  • Park, Jeongsook;Park, So Yun;Shin, Eunkyung;Lee, Sun Hee;Kim, Yoon Sook;Lee, Dong Hoon;Roh, Gu Seob;Kim, Hyun Joon;Kang, Sang Soo;Cho, Gyeong Jae;Jeong, Bo-Young;Kim, Hwajin;Choi, Wan Sung
    • Molecules and Cells
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    • v.37 no.2
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    • pp.178-186
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    • 2014
  • Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) bound directly to these sites and activated transcription. Exposure to the hypoxia-mimetic compound $CoCl_2$, incubation under 1% $O_2$ conditions, or overexpression of HIF-$1{\alpha}$ enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with $CoCl_2$ and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-$1{\alpha}$ to HRE sites and is epigenetically controlled by methylation of its promoter region.

Predicting the Nutritional Value of Seafood Proteins as Measured by Newer In Vitro Model 2. C-PER and DC-PER of Marine Crustacea (수산식품 단백질 품질평가를 위한 새로운 모델 설정 2. 해산 갑각류의 C-PER 및 DC-PER)

  • RYU Hong-Soo;LEE Keun-Woo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.19 no.3
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    • pp.219-226
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    • 1986
  • To confirm the application of a newer in vitro assays to determining the nutritional value of marine crustaceans (mainly shrimps and crabs), which have been considered to be highly nutritive depending on their levels of the essential amino acids and digestibility, their C-PERs and DC-PERs were determined and studied the factors influencing their in vitro results. Four species of seawater shrimps and 2 species of seawater crabs were used in this experiment. The in vitro digestibilities showed $83{\sim}86\%$ for raw shrimps and the trypsin indigestibile substrate content (TIS) was ranged from 1.32 to 3.33 mg/g solid expressed quantitatively as mg of purified soybean trypsin inhibitor. The smaller size of shrimps revealed a greater in vitro digestibility and a lower contents of TIS. It was noted that the in vitro digestibility of raw blue crab meat was around $85\%$ while boiled tenner crab meat showed $86\%$ or above, and the leg meat had the greatest in vitro digestibility in the various parts of crab meats. The poor in vitro digestibilities for shrimp's and crab's meat, compared with that of the other seafoods as noted in previous reports, suggest that the drop in pH, due to the change in their freshness during harvesting and frozen storage, resulted in underestimating their digestibilities using four-enzyme digestion technique. The lysine contents in all samples were higher than that of ANRC casein but they contained a slightly lower sulfur-containing amino acids than those in ANRC casein. But the other EAA, such as valine, tyrosine and phenylalanine, were found to be a half as little as that in casein and played a key-factor in calculation of C-PER or DC-PER. It was observed that the value of C-PER and DC-PER for all samples ranged from 2.1 to 2.4, and the predicted digestibilities showed $90\%$ or above in all samples. It was a different results from the fact that the animal proteins bear a higher values and predicted digestibilities than those of C-PER values. The lack of correlation between C-PER and DC-PER values is attributable to the fact that the lower content of valine, tyrosine and phenylalanine, and drop in pH owing to the changes of freshness in marine crustacea proteins. Therefore, if a newer in vitro digestion technique-which are taken into account the pH drop before digestion, TIS content and released free amino acids and/or peptides-developed, C-PER assays can provide more advantages in assessing the protein nutritional value of marine crustacea than any other in vitro assays.

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Inhibitory Effects of Asparagus cochinchinensis in LPS-Stimulated BV-2 Microglial Cells through Regulation of Neuroinflammatory Mediators, the MAP Kinase Pathway, and the Cell Cycle (Lipopolysaccharide로 자극된 BV-2 미세교세포에서 신경염증 매개체, MAP kinase경로, 세포주기의 조절에 의한 천문동(Asparagus cochinchinensis)의 저해효과)

  • Lee, Hyun Ah;Kim, Ji Eun;Choi, Jun Young;Sung, Ji Eun;Youn, Woo Bin;Son, Hong Joo;Lee, Hee Seob;Kang, Hyun-Gu;Hwang, Dae Youn
    • Journal of Life Science
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    • v.30 no.4
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    • pp.331-342
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    • 2020
  • The suppression of neuroinflammatory responses in microglial cells can be considered a key target for improving the progression of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Asparagus cochinchinensis has traditionally been used as a medicine to treat fever, cough, kidney disease, breast cancer, inflammatory diseases, and brain diseases. In this study, we investigated the neuroprotective mechanism of an aqueous extract from A. cochinchinensis root (AEAC), particularly its anti-inflammatory effects on lipopolysaccharide (LPS)-activated BV-2 microglial cells. BV-2 cells were treated with four different concentrations of AEAC. No significant toxicity was detected in BV-2 cells treated with AEAC. Nitric oxide (NO), cyclooxygenase-2 (COX-2) mRNA, and inducible nitric oxide synthase (iNOS) mRNA levels were 21% lower in the AEAC+LPS group than in the Vehicle+LPS group. Lower proinflammatory (TNF-α and IL-1β) and anti-inflammatory cytokine (IL-6 and IL-10) levels were also detected in the AEAC+LPS group than in the Vehicle+LPS group, albeit at varying rates. Moreover, the phosphorylation of mitogen-activated protein kinase (MAPK) members after LPS treatment was significantly recovered in the AEAC-pretreated group compared to the Vehicle+LPS group, enhancement of the phosphorylation of mitogen-activated protein kinase (MAPK) members after LPS treatment was significantly recovered in the AEAC-pretreated group, while cell cycle arrest at the G2/M phase caused by LPS treatment was less severe in the AEAC+LPS group. The increase in reactive oxygen species (ROS) generation induced by LPS treatment was also lower in the AEAC-pretreated group than in the Vehicle+LPS group. This is the first study to show that AEAC exerts anti-neuroinflammatory activity against LPS stimulation by regulating the MAPK signaling pathway, the cell cycle, and ROS production.

Antioxidant, Antimutagenicity and Anticancer Activities of Extracts from Cirsium japonicum var. ussuriense KITAMURA (엉겅퀴 추출물의 항산화성, 항돌연변이원성 및 항암활성 효과)

  • Lee, Hui-Kyoung;Kim, Ju-Sung;Kim, Na-Young;Park, Sang-Un;Kim, Myong-Jo;Yu, Chang-Yeon
    • Korean Journal of Medicinal Crop Science
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    • v.11 no.1
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    • pp.53-61
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    • 2003
  • Cirsium japonicum var. ussuriense were extracted with methanol and then fractionated with nhexane, EtOAc and BuOH to get active fractions. And their antioxidant and antimicrobial activities in each fraction were determined. Ethyl acetate and butanol fraction of Cirsium japonicum var. ussuriense showed strong antioxidant activities, but hexane fraction did not show any activities. But in the antimicrobial test, Ethyl acetate fraction showed strong antimicrobial activities except to Aspergillus awamori, Asperigillus niger. Especially, Ethyl acetate fraction showed the strongest activities against Bacillus subtilis. And aqueous fraction showed the strongest activities against Cladosporium herbarum, Hypocrea nigricans. This study was performed to determine the antimutagenic and cytotoxic effect of Cirsium japonicum var. ussuriense methanol extract on Salmonella typhimurium TA98, TA100 and cancer cell lines using Ames test and cytotoxicity assay, respectively. Cancer cell lines include human lung carcinoma(A549), human breast adenocarcinoma(MCF-7) and human hepatocellular carcinoma (Hep3B). Futher fractionations with hexane, ethyl acetate, butanol and water from methanol extract of Cirsium japonicum var. ussuriense were performed to obtain effective fraction, methanol extract showed 60.14% inhibition effect on the mutagenesis induced by MNNG against TA100, while 77% and 72.5% inhibition was observed on the mutagenesis induced by 4NQO against TA98 and TA100, respectively. and methanol extract showed 82.25% and 73.7% inhibitory effect on the mutagenesis induced by Trp-P-1 against TA98 and TA100, respectively. methanol extract showed the strongest effect against A549, MCF-7 and Hep3B at the same concentration compared to those of other fration.

Study on perception and eating attitude towards vegetables among elementary school children and their parents in Gangwon area (강원지역 초등학생과 학부모의 채소류 섭취에 대한 인식 및 섭취태도 연구)

  • Kim, Mi Kyeong;Oh, Hae Sook;Lee, Myung Hee
    • Journal of Korean Home Economics Education Association
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    • v.27 no.3
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    • pp.19-34
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    • 2015
  • Current study aimed to evaluate elementary school children's perception and eating attitude towards vegetables and analyze environmental factors influencing it. Survey was conducted among elementary school students and their parents in Gangwon district, 410 subjects each(Total 742, 391 students, 351 parents). The results are as follows. Both the children(64.3%) and the parents(84.9%) showed high concern over health. 46.6% of children and 52.4% of parents admitted unbalanced eating behavior, and commonly avoided foods were mostly vegetables such as mushroom, carrot, garlic, sweet pepper-green, welsh onion, kimchi, pumpkin, or beans. Unbalanced eating habit was significantly related to health consideration in choosing what to eat(p<0.01) and interest in health(p<0.05). 70.9% of children with unbalanced eating habit showed intention for correction, and those with high interest in health had stronger intention (p<0.01). Regarding children's perception of 20 kinds of vegetables frequently used in school lunch, swiss chard leaf beet, amaranthus magistratus, and curled mallow were rarely heard of or hardly eaten before. Korean chinese cabbage, radish(62.7%), cucumber(62.1%), perilla leaves(60.4%), lettuce(58.1%), and spinach(54.5%) were among the most frequently eaten. Survey result on children's preference for the vegetables shows lettuce, cucumber, Korean chinese cabbage, perilla leaves, spinach, and radish were highly preferred and stem of garlic, crown daisy, sweet pepper-green, pepper, and curled mallow were the least preferred. There was a significant positive correlation(p<0.01, p<0.001) between children's and their parent's preference for each vegetables, implying that children's preference towards vegetables is greatly influenced by their parent's choice. Children showed negative attitude towards vegetables with strong flavor and tended to avoid vegetables that they remember as not tasty. This suggest that strong flavor and negative prior experience is what determines children's attitude in vegetable consumption. Many children said they try to eat vegetables even if they haven't had it before, and this tendency was significantly correlated with the degree of interest in health(p<0.05) and the degree of consideration of health in choosing what to eat(p<0.001).

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