• Title/Summary/Keyword: Liquid Fraction

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Phytochemical Analysis and Anti-cancer Investigation of Boswellia Serrata Bioactive Constituents In Vitro

  • Ahmed, Hanaa H;Abd-Rabou, Ahmed A;Hassan, Amal Z;Kotob, Soheir E
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.16
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    • pp.7179-7188
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    • 2015
  • Cancer is a major health obstacle around the world, with hepatocellular carcinoma (HCC) and colorectal cancer (CRC) as major causes of morbidity and mortality. Nowadays, there isgrowing interest in the therapeutic use of natural products for HCC and CRC, owing to the anticancer activity of their bioactive constituents. Boswellia serrata oleo gum resin has long been used in Ayurvedic and traditional Chinese medicine to alleviate a variety of health problems such as inflammatory and arthritic diseases. The current study aimed to identify and explore the in vitro anticancer effect of B. Serrata bioactive constituents on HepG2 and HCT 116 cell lines. Phytochemical analysis of volatile oils of B. Serrata oleo gum resin was carried out using gas chromatography-mass spectrometry (GC/MS). Oleo-gum-resin of B. Serrata was then successively extracted with petroleum ether (extract 1) and methanol (extract 2). Gas-liquid chromatography (GLC) analysis of the lipoidal matter was also performed. In addition, a methanol extract of B. Serrata oleo gum resin was phytochemically studied using column chromatography (CC) and thin layer chromatography (TLC) to obtain four fractions (I, II, III and IV). Sephadex columns were used to isolate ${\beta}$-boswellic acid and identification of the pure compound was done using UV, mass spectra, $^1H$ NMR and $^{13}C$ NMR analysis. Total extracts, fractions and volatile oils of B. Serrata oleo-gum resin were subsequently applied to HCC cells (HepG2 cell line) and CRC cells (HCT 116 cell line) to assess their cytotoxic effects. GLC analysis of the lipoidal matter resulted in identification of tricosane (75.32%) as a major compound with the presence of cholesterol, stigmasterol and ${\beta}$-sitosterol. Twenty two fatty acids were identified of which saturated fatty acids represented 25.6% and unsaturated fatty acids 74.4% of the total saponifiable fraction. GC/MS analysis of three chromatographic fractions (I,II and III) of B. Serrata oleo gum resin revealed the presence of pent-2-ene-1,4-dione, 2-methyl- levulinic acid methyl ester, 3,5- dimethyl- 1-hexane, methyl-1-methylpentadecanoate, 1,1- dimethoxy cyclohexane, 1-methoxy-4-(1-propenyl)benzene and 17a-hydroxy-17a-cyano, preg-4-en-3-one. GC/MS analysis of volatile oils of B. Serrata oleo gum resin revealed the presence of sabinene (19.11%), terpinen-4-ol (14.64%) and terpinyl acetate (13.01%) as major constituents. The anti-cancer effect of two extracts (1 and 2) and four fractions (I, II, III and IV) as well as volatile oils of B. Serrata oleo gum resin on HepG2 and HCT 116 cell lines was investigated using SRB assay. Regarding HepG2 cell line, extracts 1 and 2 elicited the most pronounced cytotoxic activity with $IC_{50}$ values equal 1.58 and $5.82{\mu}g/mL$ at 48 h, respectively which were comparable to doxorubicin with an $IC_{50}$ equal $4.68{\mu}g/mL$ at 48 h. With respect to HCT 116 cells, extracts 1 and 2 exhibited the most obvious cytotoxic effect; with $IC_{50}$ values equal 0.12 and $6.59{\mu}g/mL$ at 48 h, respectively which were comparable to 5-fluorouracil with an $IC_{50}$ equal $3.43{\mu}g/mL$ at 48 h. In conclusion, total extracts, fractions and volatile oils of B. Serrata oleo gum resin proved their usefulness as cytotoxic mediators against HepG2 and HCT 116 cell lines with different potentiality (extracts > fractions > volatile oil). In the two studied cell lines the cytotoxic acivity of each of extract 1 and 2 was comparable to doxorubicin and 5-fluorouracil, respectively. Extensive in vivo research is warranted to explore the precise molecular mechanisms of these bioactive natural products in cytotoxicity against HCC and CRC cells.

Skin Protection Effect of Grape Pruning Stem Extract on UVB-induced Connective Tissue Injury (포도전정가지 추출물이 UVB로 유도된 결합 조직 손상에 미치는 피부 보호 효과)

  • Kim, Joung-Hee;Kim, Keuk-Jun
    • Journal of Life Science
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    • v.28 no.2
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    • pp.141-147
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    • 2018
  • This study aimed to analyze the contents of rutin, procyanidin B3, quercetin, and kaempferol, known to have antioxidant, anti-inflammatory, and anti-carcinogenic effects, among the polyphenol types contained in grape pruning stem extracts (GPSE). It utilized grape stems discarded after harvest to measure the effects of GPSE on skin moisture, inhibition of skin cell proliferation, and anti-inflammatory activity on the damaged skin of HR-1 mice induced with ultraviolet B (UVB), and to verify the applicability of GPSE as a material for functional food and functional cosmetics. The polyphenol was extracted from grape pruning stems with 80% EtOH, and then the extract was used while storing at $-20^{\circ}C$, after filtering, concentrating, and freeze-drying it. The content of an active ingredient of GPSE was analyzed using high performance liquid chromatography (HPLC). From 53 kg of the grape pruning stem specimen, 2.34 kg of the EtOH fraction extracts were extracted to achieve a 4.42% yield ratio. Analysis of the active ingredients showed 0.28 mg/g of procyanidin B3, 12.81 mg/g of rutin, 0.51 mg/g of quercetin, and 8.24 mg/g of kaempferol. After UVB irradiation on the dermis, to confirm the degree of inhibition of collagen synthesis, we examined the protein expression of MMP-9 using immunohistochemical staining. The results of this study confirm the existence of active polyphenol types, such as rutin, kaempferol, quercetin, and procyanidin B3, in GPSE. Moreover, the study found that GPSE has anti-collagenase effects and it decreases the effects of UV damage on skin barrier function. GPSE is a functional ingredient with a potential for skin protection effects, and it has high utilization potential as an ingredient for functional cosmetics.

Optimization of fractionation efficiency (FE) and throughput (TP) in a large scale splitter less full-feed depletion SPLITT fractionation (Large scale FFD-SF) (대용량 splitter less full-feed depletion SPLITT 분획법 (Large scale FFD-SF)에서의 분획효율(FE)및 시료처리량(TP)의 최적화)

  • Eum, Chul Hun;Noh, Ahrahm;Choi, Jaeyeong;Yoo, Yeongsuk;Kim, Woon Jung;Lee, Seungho
    • Analytical Science and Technology
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    • v.28 no.6
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    • pp.453-459
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    • 2015
  • Split-flow thin cell fractionation (SPLITT fractionation, SF) is a particle separation technique that allows continuous (and thus a preparative scale) separation into two subpopulations based on the particle size or the density. In SF, there are two basic performance parameters. One is the throughput (TP), which was defined as the amount of sample that can be processed in a unit time period. Another is the fractionation efficiency (FE), which was defined as the number % of particles that have the size predicted by theory. Full-feed depletion mode (FFD-SF) have only one inlet for the sample feed, and the channel is equipped with a flow stream splitter only at the outlet in SF mode. In conventional FFD-mode, it was difficult to extend channel due to splitter in channel. So, we use large scale splitter-less FFD-SF to increase TP from increase channel scale. In this study, a FFD-SF channel was developed for a large-scale fractionation, which has no flow stream splitters (‘splitter less’), and then was tested for optimum TP and FE by varying the sample concentration and the flow rates at the inlet and outlet of the channel. Polyurethane (PU) latex beads having two different size distribution (about 3~7 µm, and about 2~30 µm) were used for the test. The sample concentration was varied from 0.2 to 0.8% (wt/vol). The channel flow rate was varied from 70, 100, 120 and 160 mL/min. The fractionated particles were monitored by optical microscopy (OM). The sample recovery was determined by collecting the particles on a 0.1 µm membrane filter. Accumulation of relatively large micron sized particles in channel could be prevented by feeding carrier liquid. It was found that, in order to achieve effective TP, the concentration of sample should be at higher than 0.4%.

Effects of Level and Degradability of Dietary Protein on Ruminal Fermentation and Concentrations of Soluble Non-ammonia Nitrogen in Ruminal and Omasal Digesta of Hanwoo Steers

  • Oh, Young-Kyoon;Kim, Jeong-Hoon;Kim, Kyoung-Hoon;Choi, Chang-Won;Kang, Su-Won;Nam, In-Sik;Kim, Do-Hyung;Song, Man-Kang;Kim, Chang-Won;Park, Keun-Kyu
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.3
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    • pp.392-403
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    • 2008
  • Four ruminally fistulated Hanwoo steers were used to determine the effects of level and degradability of dietary protein on ruminal fermentation, blood metabolites and concentration of soluble non-ammonia nitrogen (SNAN) in ruminal (RD) and omasal digesta (OD). Experiments were conducted in a $4{\times}4$ Latin square design with a $2{\times}2$ factorial arrangement of treatments. Factors were protein supplements with two ruminal crude protein (CP) degradabilities, corn gluten meal (CGM) that was low in degradability (rumen-degraded protein (RDP), 23.4% CP) or soybean meal (SBM) that was high in degradability (RDP, 62.1% CP), and two feeding levels of CP (12.2 or 15.9% dry matter). Ruminal fermentation rates and plasma metabolite concentrations were determined from the RD collected at 2-h intervals and from the blood taken by jugular puncture, respectively. The SNAN fractions (free amino acid, peptide and soluble protein) in RD and OD collected at 2-h intervals were assessed by ninhydrin assay. Mean ruminal ammonia concentrations were 40.5, 74.8, 103.4 and 127.0 mg/L for low CGM, high CGM, low SBM and high SBM, respectively, with statistically significant differences (p<0.01 for CP level and p<0.001 for CP degradability). Blood urea nitrogen concentrations were increased by high CP level (p<0.001) but unaffected by CP degradability. There was a significant (p<0.05) interaction between level and degradability of CP on blood albumin concentrations. Albumin was decreased to a greater extent by increasing degradability of low CP diets (0.26 g/dl) compared with high CP diets (0.02 g/dl). Concentrations of each SNAN fraction in RD (p<0.01) and OD (p<0.05) for high CP diets were higher than those for low CP diets, except for peptides but concentrations of the sum of peptide and free amino acid in RD and OD were significantly higher (p<0.05) for high CP diets than for low CP diets. Soybean meal diets increased free amino acid and peptide concentrations in both RD (p<0.01) and OD (p<0.05) compared to CGM diets. High level and greater degradability of CP increased (p<0.001) mean concentrations of total SNAN in RD and OD. These results suggest that RDP contents, increased by higher level and degradability of dietary protein, may increase release of free amino acids, peptides and soluble proteins in the rumen and omasum from ruminal degradation and solubilization of dietary proteins. Because SNAN in OD indicates the terminal product of ruminal metabolism, increasing CP level and degradability appears to increase the amount of intestine-available nitrogen in the liquid phase.

Lipid Composition of Oyster, Arkshell and Sea-mussel (굴, 피조개 및 진주담치의 지질조성에 관한 연구)

  • YOON Ho-Dong;BYUN Han-Seok;CHUN Seok-Jo;KIM Seon-Bong;PARK Yeung-Ho
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.19 no.4
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    • pp.321-326
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    • 1986
  • Oyster (Crassostrea gigas), arkshell (Anadare(Scapharce) broughtonii) and sea-mussel (Mytilus edulis) were investigated as to their lipid classes. Lipid extracts from shellfishes were fractionated into neutral lipid (NL), glycolipid (GL) and phospho-lipid (PL) by column chromatography with silicic acid. The fatty acid compositions of their lipid classes and lipid fractions were determined by gas liquid chromatography (GLC). Total lipid contents of shellfishes were $3.5\%$ in the oyster, $1.4\%$ in the arkshell, $1.0\%$ in the sea-mussel. The major fatty acids of total lipids were palmitic acid, eicosapentaenoic acid and docosahexaenoic acid in the oyster and the sea-mussel, palmitic acid, oleic acid and eicosapentaenoic acid in the arkshell. The lipid composition of neutral lipid fractions in shellfishes was separated and identified as free sterol, free fatty acid, triglyceride, hydrocarbon and esterified sterol by TLC. Of these classes, triglyceride fraction was most abundant, amounting to 55.6, 77.7 and $60.4\%$ in the three samples mentioned above, respectively. The main fatty acids of glycolipid were palmitic acid, eicosaenoic acid and docosahexaenoic acid in oyster, myristic acid, palmitic acid and palmitoleic acid in the arkshell, docosahexaenoic acid, linolenic acid and palmitic acid in the sea-mussel. The major fatty acids of phospholipid were palmitic acid, eicosapentaenoic acid and docosahexaenoic acid in the oyster and sea-mussel, palmitic acid, eicosapentaenoic acid and erucic acid in the arkshell.

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Antibacterial and Antimutagenic Effects of Sweetpotato Tips Extract (고구마 끝순 추출물의 항균 및 항돌연변이 효과)

  • Lee, Joon-Seol;Shin, Mee-Jin;Park, Yang-Kyun;Ahn, Young-Sup;Chung, Mi-Nam;Kim, Hag-Sin;Kim, Jeong-Mok
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.52 no.3
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    • pp.303-310
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    • 2007
  • Sweetpotato shoot tops (leaves, tips and petioles) are known to be very useful parts as vegetables because of their high nutritive values and great biomass yield. In this study, the phenolic compound contents, antibacterial activity, mutagenic activity, and antimutagenic activity were investigated in sweetpotato tips that were 10-15cm of shoot top including stems, petioles and tender leaves after sprout of storage roots. The study was done by extracting sweetpotato tips with 80% ethanol and the ethanol fraction was re-extracted with hexane, chloroform, ethyl acetate, butanol and water. In ethyl acetate and butanol fractions, total phenolic compounds contained 95. 6mg/g extract and 69.3 mg/g extract, respectively, The antibacterial activity was measured using the paper disk method with concentrations of 1, 2, 5 and 10 mg/disk of butanol and ethyl acetate fractions against L. monocytogenes and S. Typhimurium strains. Higher doses of solvent extracts showed the higher antibacterial activities. In addition, 5, 10 and 20 mg/mL of the extracts were tested to determine the antibacterial activity in liquid culture. The sweetpotato leaf extract by ethyl acetate showed 1 log reduction compared to control after 24 hrs on Listeria monocytogenes, but 20 mg/ml of butanol extract completely inhibited the growth of the pathogen after 12 hrs. The extracts from ethyl acetate or butanol on Salmonella Typhimurium did less than 1 log reduction during cultivation compared to control. The numbers of S. Typhimirium TA98 and TA100 revertant colonies were 29-33 and 159-188 CFU/plate, respectively, indicating that solvent extracts were no mutagenic activity. The antimutagenic test was performed by adding direct mutagen 2-NF and MMS, and butanol and ethyl acetate showed antimutagenic effect. Thus, this study showed that sweetpotato tips had high phenolic contents and both antimicrobiol and antimutagenic properties. Sweetpotato tips would be good nutritive source because of their high nutrient content without any toxicity in consuming.

Studies on Change of Lipids Improvement-Meju during the Fermentation (개량(改良)메주의 숙성과정(熟成過程) 중 지질(脂質)조성의 변화(變化)에 관(關)한 연구(硏究))

  • Yang, Soo Dong;Bae, Man Jong;Yoon, Sang Hong;Choi, Cheong
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.12 no.3
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    • pp.189-195
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    • 1983
  • Changes of lipid composition in the Improvement-Meju inoculated with Aspergillus oryzae were examined. To investigate those changes systematically, silicic acid column chromatography was used for analysis of glycolipid, neutral lipid, phospholipid, and gas chromatography to examine the change of those fatty acid content. Following results were obtained. The lipid fraction obtained from soaked soybean and cooked soybean were mainly composed of about 93~94% neutral lipid, whereas phospholipid and glycolipid was 4.0~5.0%, 2.0~2.1% level, respectively. During meju incuvation period, neutral lipid decreased gradually, but glycolipid and phospholipid increased. Among the nonpolar lipids prepared from cooked soybean and soaked soybean, triglyceride content was mainly composed of 88~89%, and the content of sterol ester, free fatty acid, diglyceride and sterol was higher in soaked soybean than in cooked soybean. During meju incuvation period, triglyceride content decreased remarkablely, whereas content of sterol ester, free fatty acid and diglyceride increased gradually. From the soaked soybean and the cooked soybean, the fatty acids content of crude lipid, neutral lipid, glycolipid and phospholipid were composed of linoleic acid 54~70%, oleic acid 20.0~22.6%, palmitic acid 11.0~12.4%, linolenic acid 6.0~7.8% and stearic acid 3.4~4.3% in turn and myristic acid showed the trace, palmitic acid was a little higher in glycolipid and phospholipid than in crude lipid and neutral lipid. During meju incuvation period, the change of fatty acid content showed linoleic acid and linolenic acid reduction gradually in the neutral lipid, glycolipid and phospholipid. On the other hand, palmitic acid, stearic acid and oleic acid increased gradually, the maximum value was at the 4-days. The change of glycolipid fatty acid and phospholipid fatty acid was examined. 9-kinds including traced 3-kinds was detected. It was supposed that traced 2-kinks was occurred for incuvation, and those are the matter investigating in the future.

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Geophysical Implications for Configurational Entropy and Cube Counting Fractal Dimension of Porous Networks of Geological Medium: Insights from Random Packing Simulations (지질매체 공극 구조에 대한 구성 엔트로피와 상자집계 프랙탈 차원의 지구물리학적 의미 및 응용: 무작위 패킹 시뮬레이션 연구)

  • Lee, Bum-Han;Lee, Sung-Keun
    • Journal of the Mineralogical Society of Korea
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    • v.23 no.4
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    • pp.367-375
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    • 2010
  • Understanding the interactions between earth materials and fluids is essential for studying the diverse geological processes in the Earth's surface and interior. In order to better understand the interactions between earth materials and fluids, we explore the effect of specific surface area and porosity on structural parameters of pore structures. We obtained 3D pore structures, using random packing simulations of porous media composed of single sized spheres with varying the particle size and porosity, and then we analyzed configurational entropy for 2D cross sections of porous media and cube counting fractal dimension for 3D porous networks. The results of the configurational entropy analysis show that the entropy length decreases from 0.8 to 0.2 with increasing specific surface area from 2.4 to $8.3mm^2/mm^3$, and the maximum configurational entropy increases from 0.94 to 0.99 with increasing porosity from 0.33 to 0.46. On the basis of the strong correlation between the liquid volume fraction (i.e., porosity) and configurational entropy, we suggest that elastic properties and viscosity of mantle melts can be expressed using configurational entropy. The results of the cube counting fractal dimension analysis show that cube counting fractal dimension increases with increasing porosity at constant specific surface area, and increases from 2.65 to 2.98 with increasing specific surface area from 2.4 to $8.3mm^2/mm^3$. On the basis of the strong correlation among cube counting fractal dimension, specific surface area, and porosity, we suggest that seismic wave attenuation and structural disorder in fluid-rock-melt composites can be described using cube counting fractal dimension.

Facile [11C]PIB Synthesis Using an On-cartridge Methylation and Purification Showed Higher Specific Activity than Conventional Method Using Loop and High Performance Liquid Chromatography Purification (Loop와 HPLC Purification 방법보다 더 높은 비방사능을 보여주는 카트리지 Methylation과 Purification을 이용한 손쉬운 [ 11C]PIB 합성)

  • Lee, Yong-Seok;Cho, Yong-Hyun;Lee, Hong-Jae;Lee, Yun-Sang;Jeong, Jae Min
    • The Korean Journal of Nuclear Medicine Technology
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    • v.22 no.2
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    • pp.67-73
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    • 2018
  • $[^{11}C]PIB$ synthesis has been performed by a loop-methylation and HPLC purification in our lab. However, this method is time-consuming and requires complicated systems. Thus, we developed an on-cartridge method which simplified the synthetic procedure and reduced time greatly by removing HPLC purification step. We compared 6 different cartridges and evaluated the $[^{11}C]PIB$ production yields and specific activities. $[^{11}C]MeOTf$ was synthesized by using TRACERlab FXC Pro and was transferred into the cartridge by blowing with helium gas for 3 min. To remove byproducts and impurities, cartridges were washed out by 20 mL of 30% EtOH in 0.5 M $NaH_2PO_4$ solution (pH 5.1) and 10 mL of distilled water. And then, $[^{11}C]PIB$ was eluted by 5 mL of 30% EtOH in 0.5 M $NaH_2PO_4$ into the collecting vial containing 10 mL saline. Among the 6 cartridges, only tC18 environmental cartridge could remove impurities and byproducts from $[^{11}C]PIB$ completely and showed higher specific activity than traditional HPLC purification method. This method took only 8 ~ 9 min from methylation to formulation. For the tC18 environmental cartridge and conventional HPLC loop methods, the radiochemical yields were $12.3{\pm}2.2%$ and $13.9{\pm}4.4%$, respectively, and the molar activities were $420.6{\pm}20.4GBq/{\mu}mol$ (n=3) and $78.7{\pm}39.7GBq/{\mu}mol$ (n=41), respectively. We successfully developed a facile on-cartridge methylation method for $[^{11}C]PIB$ synthesis which enabled the procedure more simple and rapid, and showed higher molar radio-activity than HPLC purification method.

Studies on Sclerotium rolfsii Sacc. isolated from Magnolia kobus DC. in Korea (목련(Magnolia kobus DC.)에서 분리한 흰비단병균(Sclerotium rolfsii Sacc.)에 관한 연구)

  • Kim Kichung
    • Korean journal of applied entomology
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    • v.13 no.3 s.20
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    • pp.105-133
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    • 1974
  • The present study is an attempt to solve the basic problems involved in the control of the Sclerotium disease. The biologic stranis of Sclerotium rolfsii Sacc., pathogen of Sclerotium disease of Magnolia kobus, were differentiated, and the effects of vitamins, various nitrogen and carbon sources on its mycelial growth and sclerotial production have been investigated. In addition the relationship between the cultural filtrate of Penicillium sp. and the growth of Sclerotium rolfsii, the tolerance of its mycelia or sclerotia to moist heat or drought and to Benlate (methyl-(butylcarbamoy 1)-2-benzimidazole carbamate), Tachigaren (3-hydroxy-5-methylisoxazole) and other chemicals were also clarified. The results are summarizee as follows: 1. There were two biologic strains, Type-l and Type-2 among isolates. They differed from each other in the mode of growth and colonial appearance on the media, aversion phenomenon and in their pathogenicity. These two types had similar pathogenicity to the Magnolia kobus and Robinia pseudoacasia, but behaved somewhat differently to the soybaen and cucumber, the Type-l being more virulent. 2. Except potassium nitrite, sodium nitrite and glycine, all of the 12 nitrogen sources tested were utilized for the mycelial growth and sclerotial production of this fungus when 10r/l of thiamine hydrochloride was added in the culture solution. Considering the forms of nitrogen, ammonium nitrogen was more available than nitrate nitrogen for the growth of mycelia, but nitrate nitrogen was better for sclerotia formation. Organic nitrogen showed different availabilities according to compounds used. While nitrite nitrogen was unavailable for both mycelial growth and sclerotial formation whether thiamine hydrochlioride was added or not. 3. Seven kinds of carbon sources examined were not effective in general, as long as thiamine hydrochloride was not added. When thiamine hydrochloride was added, glucose and saccharose exhibited mycelial growth, while rnaltose and soluble starch gave lesser, and xylose, lactose, and glycine showed no effect at all,. In the sclerotial production, all the tested carbon sources, except lactose, were effective, and glucose, maltose, saccharose, and soluble starch gave better results. 4. At the same level of nitrogen, the amount of mycelial growth increased as more carbon Sources were applied but decreased with the increase of nitrogen above 0.5g/1. The amount of sclerotial production decreased wi th the increase of carbon sources. 5. Sclerotium rolfsii was thiamine-defficient and required thiamine 20r/l for maximun growth of mycelia. At a higher concentration of more than 20r/l, however, mycelial growth decreased as the concentration increased, and was inhibited at l50r/l to such a degree of thiamine-free. 6. The effect of the nitrogen sources on the mycelial growth under the presence of thiamine were recognized in the decreasing order of $NH_4NO_3,\;(NH_4)_2SO_4,\;asparagine,\;KNO_3$, and their effects on the sclerotial production in the order of $KNO_3,\;NH_4NO_3,\;asparagine,\;(NH_4)_2SO_4$. The optimum concentration of thiamine was about 12r/l in $KNO_3$ and about 16r/l in asparagine for the growth of mycelia; about 8r/l in $KNO_3$ and $NH_4NO_3$, and 16r/l in asparagine for the production of sclerotia. 7. After the fungus started to grow, the pH value of cultural filtrate rapidly dropped to about 3.5. Hereafter, its rate slowed down as the growth amount increased and did not depreciated below pH2.2. 8. The role of thiamine in the growth of the organism was vital. If thiamine was not added, the combination of biotin, pyridoxine, and inositol did not show any effects on the growth of the organism at all. Equivalent or better mycelial growth was recognized in the combination of thiamine+pyridoxine, thiamine+inositol, thiamine+biotin+pyridoxine, and thiamine+biotin+pyridoxine+inositol, as compared with thiamine alone. In the combinations of thiamine+biotin and thiamine+biotin+inositol, mycelial growth was inhibited. Sclerotial production in dry weight increased more in these combinations than in the medium of thiamine alone. 9. The stimulating effects of the Penicillium cultural filtrate on the mycelial growth was noticed. It increased linearly with the increase of filtrate concentration up to 6-15 ml/50ml basal medium solution. 10. $NH_4NO_3$. as a nitrogen source for mycelial growth was more effective than asparasine regardless of the concentration of cultural filtrate. 11. In the series of fractionations of the cultural filtrate, mycelial growth occured in unvolatile, ether insoluble cation-adsorbed or anion-unadsorbed substance fractions among the fractions of volatile, unvolatile acids, ether soluble organic acids, ether insoluble, cation-adsorbed, cation-unadsorbed, anion-adsorbed and anion-unadsorbed. and anion-un-adsorbed substance tested. Sclerotia were produced only in cation-adsorbed fraction. 12. According to the above results, it was assumed that substances for the mycelial growth and sclerotial formation and inhibitor of sclerotial formation were include::! in cultural filtrate and they were quite different from each other. I was further assumed that the former two substances are un volatile, ether insotuble, and adsorbed to cation-exchange resin, but not adsorbed to anion, whereas the latter is unvolatile, ether insoluble, and not adsorbed to cation or anion-exchange resin. 13. Seven amino acids-aspartic acid, cystine, glysine, histidine, Iycine, tyrosine and dinitroaniline-were detected in the fractions adsorbed to cation-exchange resin by applying the paper chromatography improved with DNP-amino acids. 14. Mycelial growth or sclerotial production was not stimulated significantly by separate or combined application of glutamic acid, aspartic acid, cystine, histidine, and glysine. Tyrosine gave the stimulating effect when applied .alone and when combined with other amino acids in some cases. 15. The tolerance of sclerotia to moist heat varied according to their water content, that was, the dried sclerotia are more tolerant than wet ones. The sclerotia harvested directly from the media, both Type-1 and Type-2, lost viability within 5 minutes at $52^{\circ}C$. Sclerotia dried for 155 days at$26^{\circ}C$ had more tolerance: sclerotia of Type-l were killed in 15 mins. at $52^{\circ}C$ and in 5 mins. at $57^{\circ}C$, and sclerotia of Type-2 were killed in 10 mins. both at $52^{\circ}C$ or $57^{\circ}C$. 16. Cultural sclerotia of both strains maintained good germinability for 132 days at$26^{\circ}C$. Natural sclerotia of them stored for 283 days under air dry condition still had good germinability, even for 443 days: type-l and type-2 maintained $20\%$ and $26.9\%$ germinability, respectively. 17. The tolerance to low temperature increased in the order of mycelia, felts and sclerotia. Mycelia completely lost the ability to grow within 1 week at $7-8^{\circ}C$> below zero, while mycelial felts still maintained the viability after .3 weeks at $7-20^{\circ}C$ below zero, and sclerotia were even more tolerant. 18. Sclerotia of type-l and type-2 were killed when dipped into the $0.05\%$ solution of mercury chloride for 180 mins. and 240 mins. respectively: and in the $0.1\%$ solution, Type-l for 60 mins. and Type-2 for 30 mins. In the $0.125\%$ uspulun solution, Type-l sclerotia were killed in 180 mins., and those of Type-2 were killed for 90 mins. in the$0.125\%$solution. Dipping into the $5\%$ copper sulphate solution or $0.2\%$ solution of Ceresan lime or Mercron for 240 mins. failed to kill sclerotia of either Type-l or Type-2. 19. Inhibitory effect on mycelial growth of Benlate or Tachi-garen in the liquid culture increased as the concentration increased. 6 days after application, obvious inhibitory effects were found in all treatments except Benlate 0.5ppm; but after 12 days, distingushed diflerences were shown among the different concentrations. As compared with the control, mycelial growth was inhibited by $66\%$ at 0.5ppm and by $92\%$ at 2.0ppm of Benlate, and by$54\%$ at 1ppm and about $77\%$ at 1.5ppm or 2.0ppm of Tachigaren. The mycelial growth was inhibited completely at 500ppm of both fungicides, and the formation of sclerotia was checked at 1,000ppm of Benlate ant at 500ppm or 1,000ppm of Tachigaren. 20. Consumptions of glucose or ammonium nitrogen in the culture solution usually increased with the increment of mycelial growth, but when Benlate or Tachigaren were applied, consumptions of glucose or ammonium nitrogen were inhibited with the increment of concentration of the fungicides. At the low concentrations of Benlate (0.5ppm or 1ppm), however, ammonium nitrogen consumption was higher than that of the ontrol. 21. The amount of mycelia produced by consuming 1mg of glucose or ammonium nitrogen in the culture solution was lowered markedly by Benlate or Tachigaren. Such effects were the severest on the third day after their treatment in all concentrations, and then gradually recovered with the progress of time. 22. In the sand culture, mycelial growth was not inhibited. It was indirectly estimated by the amount of $CO_2$ evolved at any concentrations, except in the Tachigaren 100mg/g sand in which mycelial growth was inhibited significantly. Sclerotial production was completely depressed in the 10mg/g sand of Benlate or Tachigaren. 23. There was no visible inhibitory effect on the germination of sclerotia when the sclerotia were dipped in the solution 0.1, 1.0, 100, 1.000ppm of Benlate or Tachigaren for 10 minutes or even 20 minutes.

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