Isolation of furanoterpene-containing particles from Ceratocystis fimbriata-infected sweet potato root tissue

Furanoterpene-containing particles were isolated by centrifugation on a discontinuous Ficoll density gradient from a homogenate of the non-infected tissue adjacent to the infected region of Ceratocystis fimbriata-infected sweet potato root tissue. The particles were recovered at a relatively high ratio in the 2% Ficoll fraction, in which there was no contamination by mitochondria and only little by endoplasmic reticulum judging from the distribution of the activities of their marker enzymes and electron micrographs. Each particle was enveloped in a single membrane, 7-10 nm thick.     

Isolation of microbodies from, sweet potato root tissue and their development during aging of slices

Microbodies were isolated from, sweet potato root tissue bydifferential and linear sucrose density gradient centrifugation.When the tissue was homogenized in the presence of PolyclarAT, the microbodies sedimented together with the mitochondriathrough the sucrose gradients. The microbodies had a densityof 1.25 g/cm³, and contained catalase and urate oxidase, butnot malate dehydrogenase, isocitrate lyase, glycolate oxidase,hydroxypyruvate reductase and the cyanide-insensitive palmitoylCoA-oxidation system. A small amount of o-diphenol oxidase alsoseemed to be present. Catalase, but not urate oxidase, activity in the crude extractincreased during aging of the sliced tissue. A similar resultwas obtained with the microbody fraction after linear sucrosedensity gradient centrifugation. We propose that microbodiescontaining only catalase develop during aging of sliced sweetpotato root tissue. ¹ This work was supported in part by a Grant-in-Aid (No. 311908)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received June 20, 1979; )     

Isolation and structural elucidation of a dihydroubiquinone-9 from the fungus Aureobasidium pullulans

Abstract A naturally occurring member of ubiquinone (Q) group, a dihydroubiquinone-9 (Q-9 (H₂)), has been isolated as a minor ubiquinone component from the fungus Aureobasidium pullulans . By ultraviolet absorption, mass and nuclear magnetic resonance spectrometric studies, the structure of Q-9 (H₂) was found to be 2,3-dimethoxy-5-methyl-6-IX-dihydromultiprenyl⁹-1,4-benzoquinone (I).     

Biosynthesis of ethylene in sweet potato root tissue

The biosynthetic pathway of ethylene in freshly cut and blackrot-diseased tissues of sweet potato roots was investigated.Glucose-U-¹⁴C administration gave labeled ethylene in both freshand diseased tissues, but at the early stage of infection, therewas ethylene production which was not derived from the fed ¹⁴C-glucose.Acetate-1-¹⁴C and acetate-2-¹⁴C were equally incorporated intoethylene produced from fresh tissue, but acetate-2-¹⁴C was preferentiallyincorporated into ethylene from diseased tissue. Pyruvate-3-¹⁴Cwas more efficient as a precursor than was acetate or glucosein fresh tissue, while its efficiency was the same as that ofacetate in diseased tissue. Monofluoroacetate promoted pyruvate-3-¹⁴Cincorporation in fresh tissue but inhibited incorporation indiseased tissue. We concluded that the TCA cycle is involvedin the case of diseased tissue but not in fresh tissue; thus,showing different pathways for ethylene production in each tissue.In addition, in diseased tissue, ethylene is assumed to be producedfrom some cellular component(s), not easily synthesized fromglucose through fungus infection, but is degraded as soon asinfection commences. ¹This paper constitutes Part 85 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury ²Present address: The Institute for Biochemical Regulation,Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464,Japan (Received April 20, 1970; )     

Biosynthetic correlation of various phytoalexins in sweet potato root tissue infected by Ceratocystis fimbriata1

In sweet potato root tissue infected by Ceratocystis fimbriata,changes in a one hourincorporation of radioactivities from 2-¹⁴C-acetateinto furano-terpene phytoalexins were examined during incubation.The data showed that radioactivities in the CHCl₃ fractions(containing lipid) were increased after 15 hr of incubation,and were found in furano-terpenes as visualized by the TLC radioautograms.Pulse and chase feeding of 2-¹⁴C-acetate at low temperaturewas also performed using tissue discs incubated for 36 hr withthe infected region. Changes in the radioactivities of the individualcomponents were investigated with TLC. Several components suchas OHDHMy were labeled rapidly, then followed a decrease inthe label. These changes were accompanied by increases in theradioactivities of other components such as DHIp, Ip, IpOH andComponent B₁. These results are further evidence for the hypotheticalpathway that has been proposed previously, with some modifications. (Received January 11, 1979; )     

Isolation and structural elucidation of a growth stimulant for arbuscular mycorrhizal fungus from Laminaria japonica Areschoug

Arbuscular Mycorrhizal (AM) fungi enhance terrestrial plant growth by forming a symbiotic relationship with the roots of its host plant. A growth stimulant for AM fungi was isolated from a brown alga Laminaria japonica Areschoug. The active substance for in vitro AM hyphal growth was isolated from 75% methanol extracts of L. japonica using a succession of chromatographic procedures, including flash chromatography equipped with an octa decyl silane (ODS) column, gel filtration chromatography and HPLC using an ODS column. Spores of Gigaspora margarita Becker & Hall, an AM fungus, were exposed to the compound in vitro, and hyphal growth of G. margarita was measured after two weeks of incubation. At 40 mg L⁻¹, the compound significantly stimulated the in vitro hyphal growth of G. margarita, compared to the control. This compound was elucidated as 5′-deoxy-5′-methylamino-adenosine by EIMS and NMR spectrum.     

Production of ethylene by injured sweet potato root tissue

Ethylene production by sweet potato root tissue was examinedwith special emphasis on tissue injury. The root tissue producedethylene in response to cut injury. Increasing the cut surfacearea increased ethylene production, and the amount was proportionalto logarithm of the surface area. Tissue discs washed with waterbefore incubation produced less ethylene than unwashed discs. When the tissue was treated with chemicals that might destroythe cells, ethylene production remarkably increased. Monoiodoacetamide,trichloroacetic acid and sodium ethylmercurithiosalicylate wereparticularly effective in inducing ethylene production. Here,again, ethylene production was related to the degree of injury.Treatment of the tissue with increasing concentrations of thesechemicals resulted in increasing ethylene production, but concentrationsover a certain limit rather decreased the ethylene production.This may be due to the rapid destruction of the whole tissueused before ethylene production commenced. For thylene production,the presence of injured but still living cells was necessary. Relationship of the injury-induced ethylene production to metabolicactivation is discussed. ¹Part 67 of the Phytopathological Chemistry of Sweet Potatowith Black Rot and Injury. ²Fulbright grantee of 1967. Present address: Department of Biochemistry,University of Wisconsin, Madison, Wisconsin, U.S.A.     

Production of ethylene by sweet potato roots infected by black rot fungus

Ethylene production by sweet potato roots infected by the blackrot fungus, Ceratocystis fimbriata, increased strikingly afterinfection. The fungus grown on potato extract containing 1%sucrose or steamed sweet potato produced no ethylene. Thus,ethylene was proven to be produced from the host tissue affectedby fungus invasion. The ethylene production seemed to be stimulatedby carbon dioxide. Oxygen was essential for production, butexcess oxygen, probably over 80%, was found to be inhibitory.Apparent fungus growth on sweet potato was reduced under a hightension of oxygen, but this was not a cause of reduced ethyleneproduction in excess oxygen. When tissue plugs of infected sweet potato which were activelyproducing ethylene were sliced into thin discs, ethylene productionwas abolished with the exception that the first 1 mm discs atthe 1st and 2nd day stages produced a significant amount ofethylene. Similarly, plugs which were removed from fungus-invadedparts did not produce an appreciable amount of ethylene. Theproduction of ethylene was observed only by tissue plugs whichconsisted of both fungus invaded and noninvaded parts. Infected sweet potato tissue produced ethylene at a rate comparableto that in apples and may provide a goodsystem for the studyof ethylene biosynthesis. ¹Part 72 of the Phytopathological Chemistry of Sweet Potatowith Black Rot and Injury.     

Enzymatic synthesis of isopentenyl pyrophosphate in sweet potato root tissue in response to infection by black rot fungus

Cell-free systems were prepared from fresh, cut and black rot-diseasedtissues of sweet potato roots. When incubated in the presenceof mevalonate, all these systems were capable of synthesizing5-phosphomevalonate, mevalonate-5-pyrophosphate and isopentenylpyrophosphate. The time courses for the appearance of the ¹⁴C-labeledmetabolites suggested the following order of synthesis: mevalonate 5-phosphomevalonate mevalonate-5-pyrophosphate isopentenylpyrophosphate. It was also shown; on prolonged incubation, thatisopentenyl pyrophosphate was converted slowly to isopentenylmonophosphate. The activity for synthesis of isopentenyl pyrophosphatefrom mevalonate was higher in diseased tissue than in cut andhealthy tissues. ¹This paper constitutes Part 78 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received June 18, 1969; )     

Isolation and structural elucidation of a triterpenoid saponin from guar,Cyamopsis tetragonoloba

A new saponin has been isolated from the methanolic extract of the seed meal of Cyamopsis tetragonoloba and its structure elucidated as 3-O-{[α-L-rhamnopyranosyl(1 → 2)]-[α-L-rhamnopyranosyl(1 → 4)]-β-D-glucopyranosyl(1 → 2)-     

Isolation, structural elucidation, and neuroprotective effects of iridoids from Valeriana jatamansi

Two new iridoids, jatadoids A (1) and B (2), and two known compounds (3 and 4) were isolated from Valeriana jatamansi. Their structures were elucidated on the basis of extensive spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR). Compound 1 possessed an isovaleroxy group at the C-3 position that has previously been unreported in the class of iridoids. Four compounds were evaluated and compounds 1 and 3 showed moderate neuroprotective effects against MPP⁺-induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells.     

Isolation and structural elucidation of cucurbitaxanthin a and b from pumpkin cucurbita maxima

Two new carotenoids, cucurbitaxanthin A [(3S,5R,6,R3′R)-3,6-epoxy-5,6-dihydro-β,β-carotene-5,3′-diol] and cucurbitaxanthin B [(3S,5R,6R,3′S,5′R,6′S)-3,6,5′,6′-diepoxy-5,6,5′,6′-tetrahydro-β-β-carotene-5,3′-diol] have been isolated from the pumpkin Cucurbita maxima.     

Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract

Traditionally, garlic (Allium sativum) is known to be a significant immune booster. Aged garlic extract (AGE) possesses superior immunomodulatory effects than raw garlic; these effects are attributed to the transformed organosulfur compounds. AGE is also known to contain fructans; the amount of fructans in AGE represents a small fraction (0.22%) of the total fructans in raw garlic. In order to evaluate the biological activity of fructans present in AGE, both high molecular weight (>3.5 kDa; HF) and low molecular weight (<3 kDa; LF) fructans were isolated. The structures of purified HF and LF from AGE determined by (1)H NMR and (13)C NMR spectroscopy revealed that both have (2→1) β-D-fructofuranosyl bonds linked to a terminal glucose at the non-reducing end and β-D-fructofuranosyl branching on its backbone. Biological activity of fructans was assessed by immunostimulatory activity using murine lymphocytes and peritoneal exudate cells (source of macrophages). Both HF and LF displayed mitogenic activity and activation of macrophages including phagocytosis. These activities were comparable to that of known polysaccharide immunomodulators such as zymosan and mannan. This study clearly demonstrates that garlic fructans also contribute to the immunomodulatory properties of AGE, and is the first such study on the biological effects of garlic fructans.     

Some investigations on inactivation of phenylalanine ammonia-lyase in cut-injured sweet potato root tissue

In sweet potato roots, activity of the phenylalanine ammonia-lyase(PAL)-inactivating system in crude enzyme solution increasedmarkedly in response to cut injury after a lag period of about10 hr and reached a maximum after 24 hr of incubation. The resultscoincided with previous results from experiments using a proteinsynthetic inhibitor. The inactivating system could be precipitatedby centrifugation and was distributed in a different patternfrom mitochondrial and microsomal marker enzymes, accordingto data from cellular fractionation by differential and sucrosedensity gradient centrifugation. The optimum pH of the inactivationwas 6.0. Previous studies showed that PAL content changed inparallel with PAL activity in vivo. However, immunochemicalstudies indicated that the inactivation was not due to proteolysis.Furthermore, proteinase activity in sweet potato tissue didnot change in response to cut injury. These results suggestedthat PAL was first inactivated by the inactivating system, thenthe inactivated PAL was rapidly decomposed by the proteinase. ¹ This paper constitutes Part 130 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. This work was supportedin part by a grant from the Ministry of Education. ² Present address: Faculty of Agriculture, Yamaguchi University,Yamaguchi 753, Japan. (Received May 14, 1977; )     

海绵共生真菌产黄青霉LS16抗菌活性物质的分离及结构鉴定

海洋真菌能够产生大量活性独特的次级代谢产物。为了探明海绵共生真菌产黄青霉LS16发酵液中抗副溶血弧菌Vibrio parahemolyticus的活性物质,本实验对副溶血弧菌Vibrio parahemolyticus的抑菌活性进行跟踪,采用VLC（vacuum liquid chromatography）、Sephadex LH-20柱层析、薄层层析和高效液相色谱等技术,从海绵共生真菌LS16乙酸乙酯发酵液中分离纯化得到5个化合物。进一步实验证明,化合物2具有抗副溶血弧菌Vibrio parahemolyticus活性。根据该化合物的波谱数据（¹H NMR、¹³C NMR）对其化学结构进行鉴定,确定其分子式为C₁₅H₁₅NO₃,为生物碱类化合物。     

Isolation and structural elucidation of two new labdane diterpenoids from the aerial part of Roylea cinerea

Two new labdane diterpenoids, cinereanoid A (1) and cinereanoid B (2), along with five known compounds, calyone (3), pilloin (4), 1-methylindole-3-carboxaldehyde (5), β-sitosterol (6) and stigmasterol (7) were isolated from the aerial parts of Roylea cinerea (Lamiaceae). The new structures were determined by using IR, MS, 1D, 2D NMR spectroscopy. The structure of both new compounds was further confirmed by single crystal X-ray crystallographic analysis. In this study we have also reported single crystal X-ray structure of compound 3 which unambiguously confirmed the relative stereochemistry of tertiary hydroxyl and methyl groups, as it was not established by earlier report. Compounds 4 and 5 were isolated for the first time from this plant. In view of very few reports about this species, this report has increased the phytochemical knowledge about R. cinerea.     

Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic

This research was initiated to search for novel antimicrobial compounds produced by food or environmental microorganisms. A new bacterial strain, designated OSY-SE, which produces a unique and potent antimicrobial agent was isolated from soil. The isolate was identified as a Paenibacillus sp. through cultural, biochemical, and genetic analyses. An antimicrobial compound was extracted from Paenibacillus OSY-SE with acetonitrile and purified using liquid chromatography. After analyses by mass spectrometry (MS) and nuclear magnetic resonance (NMR), the antimicrobial compound was determined to be a cyclic lipopeptide consisting of a C(15) fatty acyl (FA) chain and 13 amino acids. The deduced sequence is FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-Val-Lys-Ile. The carboxyl-terminal Ile is connected to Thr by ester linkage. The new compound, designated paenibacterin, showed antagonistic activities against most Gram-positive and Gram-negative bacteria tested, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium. Paenibacterin is resistant to trypsin, lipase, α-glucosidase, and lysozyme. Its antimicrobial activity was lost after digestion by pronase and polymyxin acylase. Paenibacterin is readily soluble in water and fairly stable to exposure to heat and a wide range of pH values. The new isolate and its antimicrobial agent are being investigated for usefulness in food and medical applications.     

Isolation, Structure elucidation, and antimycobacterial properties of dimeric naphtho-gamma-pyrones from the marine-derived fungus Aspergillus carbonarius

Two new dimeric naphtho-gamma-pyrones, compounds 1 and 2, were isolated from the AcOEt extract of the fungal strain WZ-4-11 of Aspergillus carbonarius, together with eight known analogues, including 10,10'-bifonsecin B (3), 6'-O-demethylnigerone (4), nigerone (5), isonigerone (6), fonsecin (7), rubrofusarin B (8), TMC 256A1 (9), and flavasperone (10). Their structures were elucidated by means of UV, CD, IR, and 1D- and 2D-NMR spectroscopy, in combination with HR-MS analysis. The fully assigned (1)H- and (13)C-NMR data of 3, and the (13)C-NMR data of 6 are reported for the first time. Compounds 1 and 2 showed weak antimycobacterial activities against Mycobacterium tuberculosis H37Rv, with MIC values of 43.0 and 21.5 microM, resp.