Phytochemistry (v.62, #1)

Adverts (XI-XIV).

Saponins in cereals by Anne E Osbourn (1-4).
Saponins are a diverse family of secondary metabolites that are produced by many plant species, particularly dicots. These molecules commonly have potent antifungal activity and their natural role in plants is likely to be in protection against attack by pathogenic microbes. They also have a variety of commercial applications including use as drugs and medicines. The enzymes, genes and biochemical pathways involved in the synthesis of these complex molecules are largely uncharacterized for any plant species. Cereals and grasses appear to be generally deficient in saponins with the exception of oats, which produce both steroidal and triterpenoid saponins. The isolation of genes for saponin biosynthesis from oats is now providing tools for the analysis of the evolution and regulation of saponin biosynthesis in monocots. These genes may also have potential for the development of improved disease resistance in cultivated cereals.Oats (Avena spp.) are unusual amongst the monocots in that they produce antimicrobial triterpenoid saponins (avenacins). Genes for avenacin biosynthesis may have potential for the development of disease resistance in cultivated cereals. Progress in this area is considered here.
Keywords: Saponins, Triterpenoids; Cereals; Avena; Oats; Disease resistance; β-Amyrin; Avenacins;

Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme by Miguel A Duarte-Vázquez; Blanca E Garcı́a-Almendárez; Arturo Rojo-Domı́nguez; John R Whitaker; C Arroyave-Hernández; Carlos Regalado (5-11).
A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50–85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K m and V max for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability.A neutral peroxidase isozyme purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated. The glycoprotein fraction contained (w/w basis): 37% mannose, 31% fucose, 16% galactose, and 15% GlcNAc. The carbohydrate moiety of TP is important for peroxidase activity and stability.
Keywords: Brassica napus L. var. purple top white globe; Cruciferae; Turnip peroxidase; Enzymatic deglycosylation; Chemical deglycosylation;

Biochemical characterization of sap (latex) of a few Indian mango varieties by K. Saby John; S.G. Bhat; U.J.S. Prasada Rao (13-19).
Mango sap (latex) from four Indian varieties was studied for its composition. Sap was separated into non-aqueous and aqueous phases. Earlier, we reported that the non-aqueous phase contained mainly mono-terpenes having raw mango aroma (Phytochemistry 52 (1999) 891). In the present study biochemical composition of the aqueous phase was studied. Aqueous phase contained little amount of protein (2.0–3.5 mg/ml) but showed high polyphenol oxidase (147–214 U/mg protein) and peroxidase (401–561 U/mg protein) activities. It contained low amounts of polyphenols and protease activities. On native PAGE, all the major protein bands exhibited both polyphenol oxidase and peroxidase activities. Both polyphenol oxidase and peroxidase activities were found to be stable in the aqueous phase of sap at 4 °C. Sap contained large amount of non-dialyzable and non-starchy carbohydrate (260–343 mg/ml sap) which may be responsible for maintaining a considerable pressure of fluid in the ducts. Thus, the mango sap could be a valuable by-product in the mango industry as it contains some of the valuable enzymes and aroma components.Mango sap contained very little protein but most of the protein was associated with polyphenol oxidase and peroxidase activities. Sap also contained a large amount of non-dialysable, non-starchy carbohydrate which may be responsible for maintaining the sap at considerable pressure in the duct system.
Keywords: Mango; Sap; Polyphenol oxidase; Peroxidase; Carbohydrate; Polyphenols; Protein;

Three α-amylases (E.C. 3.2.1.1) were purified to apparent homogeneity from 72 h finger millet malt by three step purification via fractional acetone precipitation, DEAE-Sephacel ion exchange and Sephacryl S-200 gel permeation chromatographies with a recovery of 6.5, 2.9, 9.6% and fold purification of 26, 17 and 31, respectively. α-Nature of these amylases was identified by their ability to rapidly reduce the viscosity of starch solution and also in liberating oligosaccharides of higher D.P. and were accordingly designated as amylases α-1(b), α-2 and α-3, respectively. These amylases, having a molecular weight of 45±2 kDa were found to be monomeric. The pH and temperature optima of these α-amylases were found to be in the range of 5.0–5.5 and 45–50 °C, respectively. K m values of these amylases for various cereal starches varied between 0.59 and 1.43%. Carbodiimide (50 mM) and metal ions such as Al3+, Fe2+, and Hg2+ (5 mM) have completely inhibited these enzymes at 45° C. Amino acid analysis of these enzymes indicated high amounts of glycine which is an unusual feature of these enzymes.
Keywords: α-Amylases; Finger millet; Ragi; Eleusine coracana; Purification; Partial characterization;

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-δ-cadinene synthase by Gail S. Martin; Jinggao Liu; Chauncey R. Benedict; Robert D. Stipanovic; Clint W. Magill (31-38).
Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of δ-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from Ro, To, and T1 plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T2 cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T1 leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.The integration of antisense δ-cadinene synthase into cotton genome has a negative impact on the biosynthesis of cadinane sesquiterpenoids.
Keywords: Gossypium arboreum; Cotton; Malvaceae; Antisense cDNA; δ-Cadinene synthase activity; Gossypol;

Expression of a Streptomyces 3-hydroxysteroid oxidase gene in oilseeds for converting phytosterols to phytostanols by Mylavarapu Venkatramesh; Balasulojini Karunanandaa; Bin Sun; Catharine A Gunter; Sekhar Boddupalli; Ganesh M Kishore (39-46).
Plant sterols and their hydrogenated forms, stanols, have attracted much attention because of their benefits to human health in reducing serum and LDL cholesterol levels, with vegetable oil processing being their major source in several food products currently sold. The predominant forms of plant sterol end products are sitosterol, stigmasterol, campesterol and brassicasterol (in brassica). In this study, 3-hydroxysteroid oxidase from Streptomyces hygroscopicus was utilized to engineer oilseeds from rapeseed (Brassica napus) and soybean (Glycine max), respectively, to modify the relative amounts of specific sterols to stanols. Each of the major phytosterols had its C-5 double bond selectively reduced to the corresponding phytostanol without affecting other functionalities, such as the C-22 double bond of stigmasterol in soybean seed and of brassicasterol in rapeseed. Additionaly, several novel phytostanols were obtained that are not produced by chemical hydrogenation of phytosterols normally present in plants.The C-5 specific hydrogenation of phytosterols into phytostanols by 3-hydroxysteroid oxidase from Streptomyces sp. strain A19249 is reported.
Keywords: Glycine max; Leguminosae; Brassica napus; Cruciferae; Transgenic; Sitosterol; Campesterol; Stigmasterol; Sitostanol; Campestanol; Stigmastanol; 3-Hydroxysteroid oxidase;

Isolation and characterization of two fructokinase cDNA clones from rice by Huawu Jiang; Weimin Dian; Feiyan Liu; Ping Wu (47-52).
Two cDNA clones, OsFKI and OsFKII, encoding fructokinase (EC 2.7.1.4) were isolated from immature seeds of rice (Oryza sativa L.) by PCR. OsFKI cDNA encoded a deduced protein of 323 amino acids that was 59–71% identical to previously characterized plant fructokinases. In contrast, OsFKII cDNA encoded a deduced protein of 336 amino acids that shared only 64% amino acid identity with OsFKI. The deduced proteins both possessed an ATP-binding motif and putative substrate recognition site sequences that were previously identified in bacterial fructokinases. Genomic DNA blot analysis also revealed that each fructokinase gene exists as a single copy in the rice genome. The identity of OsFKI and OsFKII as fructokinases was confirmed by the expression of enzyme activity in E. coli. Although both OsFKI and OsFKII utilized fructose as substrate, only OsFKII activity was strongly inhibited at a high fructose concentration. The mRNA corresponding to OsFKII accumulated at high levels in developing rice grains, whereas there were only low levels of OsFKI transcripts in immature seeds. These results indicate that fructokinase in rice endosperm is encoded by two divergent genes, which play different roles in rice grains for starch storage based on their sensitivity to substrate inhibition and level of transcripts in endosperm.Fructokinase in rice endosperm is encoded by two divergent genes, which possess different sensitivity to substrate inhibition and different level of transcripts in rice endosperm.
Keywords: Fructokinase; Rice (Oryza sativa L.); Gramineae;

Benzodioxane chains and the new 5-hydroxyguaiacyl-glycerol unit were identified in isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase. Alfalfa deficient in caffeoyl coenzyme A 3-O-methyltransferase exhibited a 30% increase in cellulose:lignin over wild-type alfalfa.Isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase contained benzodioxanes resulting from the incorporation of the novel monomer, 5-hydroxyconiferyl alcohol. Due to the high level incorporated into the soluble lignin fraction and the use of sensitive NMR instrumentation, unique structural features were revealed. A new type of end-unit, the 5-hydroxyguaiacyl glycerol unit, was identified. It was possible to establish that coniferyl alcohol, sinapyl alcohol, and the novel 5-hydroxyconiferyl alcohol can cross-couple with the 5-hydroxyguaiacyl units that are formed in the lignin, the latter giving rise to extended chains of benzodioxane units. There is also evidence that 5-hydroxyconiferyl alcohol couples with normal (guaiacyl or syringyl) lignin units. Lignin in the alfalfa deficient in caffeoyl CoA 3-O-methyltransferase was structurally similar to the control lignin but the transgenic exhibited a dramatic decrease in lignin content (∼20%) and modest increase in cellulose (∼10%) reflecting a 30% increase in cellulose:lignin ratio. The compositional changes in both transgenics potentially allow enhanced utilization of alfalfa as a major forage crop by increasing the digestibility of its stem fraction.
Keywords: Alfalfa (Medicago sativa L.); Fabaceae; Lignin; O-Methyltransferase; Benzodioxane; 5-Hydroxyguaiacyl glycerol; NMR; Transgenic plants;

The biotransformation of the diterpene 2β-hydroxy-ent-13-epi-manoyl oxide by Gibberella fujikuroi by Braulio M Fraga; Pedro González; Melchor G Hernández; Sergio Suárez (67-70).
Incubation of the diterpene 2β-hydroxy-ent-13-epi-manoyl oxide with Gibberella fujikuroi afforded in good yield 2β,6β-dihydroxy-ent-13-epi-manoyl oxide, 2β,12β-dihydroxy-ent-13-epi-manoyl oxide and 2β,20-dihydroxy-ent-13-epi-manoyl oxide, confirming that although ent-13-epi-manoyl oxide is a final metabolite of a biosynthetic branch in this fungus, more polar derivatives of this compound can be transformed by this micro-organism.Incubation of the diterpene 2β-hydroxy-ent-13-epi-manoyl oxide with Gibberella fujikuroi produces metabolites hydroxylated at the 6β, 11β and 20 positions, confirming that polar derivatives of ent-13-epi-manoyl oxides can be transformed by this fungus.
Keywords: Gibberella fujikuroi; Diterpenes; 2β-Hydroxy-ent-13-epi-manoyl oxide;

The spore-derived mycobionts of the lichen Graphis prunicola, G. cognata and G. scripta were cultivated on a malt–yeast extract medium supplemented with 10% sucrose and their metabolites were investigated. Graphislactones A–D were isolated from the cultures of G. prunicola, while alternariol and graphislactones A and C were isolated from those of G. cognata. From the cultured mycobionts of G. scripta, a new 6H-dibenzo[b,d]pyran-6-one derivative, graphislactone E with graphislactones A and C was obtained. On the other hand, cultivation of the mycobionts of G. prunicola on a malt–yeast extract medium supplemented with 2.5% sucrose and 0.25% sodium acetate produced two new metabolites, graphislactones E and F. Their structures were determined by spectroscopic methods. The biogenetic origin of the carbon skeleton in both compounds was verified by administering sodium [1-13C]-acetate and sodium [1,2-13C2]-acetate.
Keywords: Graphis prunicola; G. cognata; G. scripta; Lichen; Isolated mycobiont; 6H-Dibenzo[b,d]pyran-6-one; Graphislactones; Biogenesis;

Synthesis of gibberellin GA6 and its role in flowering of Lolium temulentum by Rod W King; Lloyd T Evans; Lewis N Mander; Thomas Moritz; Richard P Pharis; Bruce Twitchin (77-82).
The induction of flowering by one long day (LD) in the grass Lolium temulentum is most closely mimicked by application of the gibberellins (GAs) GA5 or GA6, both of which occur naturally. These gibberellins promote floral development but have little effect on stem elongation. Endogenous GA5 and GA6 contents in the shoot apex double on the day after the LD and, for GA5 (and we presume for GA6 as well) reach a concentration known to be inductive for the excised shoot apex in vitro. They are, therefore, strong candidates as LD floral stimuli in this grass. The synthesis of GA6 and an examination of its florigenic properties in L. temulentum are described.
Keywords: Lolium temulentum; Gramineae; Darnel; Flowering; Stem elongation; Shoot apex; Gibberellin A5; Gibberellin A6;

Bacterial resistance modifying agents from Lycopus europaeus by Simon Gibbons; Moyosoluwa Oluwatuyi; Nigel C. Veitch; Alexander I. Gray (83-87).
As part of an ongoing project to identify plant natural products which modulate bacterial multidrug resistance (MDR), bioassay-guided isolation of an extract of Lycopus europaeus yielded two new isopimarane diterpenes, namely methyl-1α-acetoxy-7α 14α-dihydroxy-8,15-isopimaradien-18-oate (1) and methyl-1α,14α-diacetoxy-7α-hydroxy-8,15-isopimaradien-18-oate (2). The structures were established by spectroscopic methods. These compounds and several known diterpenes were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing the Tet(K), Msr(A), and Nor(A) multidrug resistance efflux mechanisms. At 512 μg/ml none of the compounds displayed any antibacterial activity but individually in combination with tetracycline and erythromycin, a two-fold potentiation of the activities of these antibiotics was observed against two strains of S. aureus that were highly resistant to these agents due to the presence of the multidrug efflux mechanisms Tet(K) (tetracycline resistance) and Msr(A) (macrolide resistance).Methyl-1α-acetoxy-7α,14α-dihydroxy-8,15-isopimaradien-18-oate and methyl-1α,14α-diacetoxy-7α-hydroxy-8,15-isopimaradien-18-oate were isolated from Lycopus europaeus and assessed for bacterial resistance modifying activity against multidrug resistant Staphylococcus aureus.
Keywords: Lamiaceae; Lycopus europaeus; Staphylococcus aureus; Multidrug resistance; MDR; Modulation; Isopimarane diterpenes;

Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy by Jacob Vaya; Saeed Mahmood; Amiram Goldblum; Michael Aviram; Nina Volkova; Amin Shaalan; Ramadan Musa; Snait Tamir (89-99).
Twenty flavonoid compounds of five different subclasses were selected, and the relationship of their structure to the inhibition of low-density lipoprotein (LDL) oxidation in vitro was investigated. The most effective inhibitors, by either copper ion or 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPH) induction, were flavonols and/or flavonoids with two adjacent hydroxyl groups at ring B. In the presence of the later catechol group, the contribution of the double bond and the carbonyl group at ring C was negligible. Isoflavonoids were more effective inhibitors than other flavonoid subclasses with similar structure. Substituting ring B with hydroxyl group(s) at 2′ position resulted in a significantly higher inhibitory effect than by substituting ring A or ring B at other positions. The type of LDL inducer had no effect in flavonoids with catechol structure. Calculated heat of formation data (ΔΔH f) revealed that the donation of a hydrogen atom from position 3 was the most likely result, followed by that of a hydroxyl from ring B. Position 3 was favored only in the presence of conjugated double bonds between ring A to ring B. This study makes it possible to assign the contribution of different functional groups among the flavonoid subclasses to in vitro inhibition of LDL oxidation.The structures of 20 flavonoids of five different subclasses were found to correlate to their ability to retard low density lipoprotein oxidation and also to correlate to their calculated differences in heat of formation between each flavonoid and its corresponding radicals (ΔΔH f).
Keywords: Flavonoids; Oxidation; LDL; Heat of formation; Free radical;

Aqueous extracts of the tissue of succulent young shoots of the pear Pyrus spp. exhibited strong antibacterial activity against the bacterium Erwinia amylovora bv. 4. This activity was investigated quantitatively by a newly developed bioassay method. It was found that the activity changed with the age of the tissue. Extracts of the youngest leaves and stems from the shoot tops showed the strongest activity, and the activity decreased with age of the leaves and stems. The activity also changed with increase in time after preparation of the extract, increasing rapidly in the first hour after preparation, reaching a maximum at about 4 h, and then decreasing slowly. The substance essential for the antibacterial activity was isolated from the extract by steam distillation in vacuo and through charcoal powder column chromatography. It was identified as benzoquinone (2,5-cyclohexadiene-1,4-dione) by NMR-spectra, mass spectra and HPLC analysis. The phenolic metabolism from arbutin to hydroquinone and then to benzoquinone in the aqueous extracts was analyzed quantitatively by HPLC. The changes in the contents of benzoquinone in the extracts of leaves and stems with tissue aging and with increase in time after preparation of the extracts paralleled the changes in antibacterial activity as determined by the quantitative bioassay.Benzoquinone was isolated from young shoots of a pear as the essential substance of the antibacterial activity. It was elucidated by a quantitative bioassay and chemical analysis that benzoquinone is a metabolic product from arbutin.
Keywords: Pear; Pyrus ussuriensis Maxim.; Rosaceae (III); Erwinia amylovora; Antibacterial activity; Benzoquinone; Arbutin; Hydroquinone; Bacterial shoot blight disease;

Thelephantins A, B and C: three benzoyl p-terphenyl derivatives from the inedible mushroom Thelephora aurantiotincta by Dang Ngoc Quang; Toshihiro Hashimoto; Makiko Nukada; Isao Yamamoto; Yuki Hitaka; Masami Tanaka; Yoshinori Asakawa (109-113).
Three benzoyl p-terphenyl derivatives named thelephantins A, B and C were isolated from the ethyl acetate extract of fruit bodies of the Thelephoraceous Basidiomycete Thelephora aurantiotincta. Their structures were elucidated by analysis of high-resolution 2D NMR, MS, IR and UV spectra.Three benzoyl p-terphenyl derivatives named thelephantins A, B and C were isolated from the ethyl acetate extract of fruit bodies of Thelephoraceous Basidiomycete Thelephora aurantiotincta.
Keywords: Thelephora aurantiotincta; Thelephoraceae; Fungi; Basidiomycetes; Thelephantin; Benzoyl p-terphenyls;

Glycosides of 2-C-methyl-d-erythritol from the fruits of anise, coriander and cumin by Junichi Kitajima; Toru Ishikawa; Eiko Fujimatu; Kyoko Kondho; Tomomi Takayanagi (115-120).
Eight glycosides of 2-C-methyl-d-erythritol (1) were isolated from the fruit of anise, and their structures were clarified as 1-O-β-d-glucopyranoside, 3-O-β-d-glucopyranoside, 4-O-β-d-glucopyranoside, 1-O-β-d-fructofuranoside, 3-O-β-d-fructofuranoside, 4-O-β-d-fructofuranoside, 1-O-β-d-(6-O-4-hydroxybenzoyl)-glucopyranoside and 1-O-β-d-(6-O-4-methoxybenzoyl)-glucopyranoside of 2-C-methyl-d-erythritol (29), respectively. Furthermore, 2 and 4 were isolated from the fruit of coriander, and 2, 3 and 4 were isolated from the fruit of cumin. Though the phosphate of 1 was known to be one of the first precursors of isoprenoids in the non-mevalonate pathway, and 1 is considered to be a common constituent in Umbelliferous plants, the glycosides of 1 are found for the first time.Eight glycosides of 2-C-methyl-d-erythritol were isolated from the fruit of anise, two of which were also isolated from the fruit of coriander and three from the fruit of cumin.
Keywords: 2-C-Methyl-d-erythritol glycoside; Anise; Primpinella anisum L.; Coriander; Coriandrum sativum L.; Cumin; Cuminum cyminum L; Umbelliferae;