Phytochemistry (v.63, #5)

Vanillin by Nicholas J. Walton; Melinda J. Mayer; Arjan Narbad (505-515).
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an important flavour and aroma molecule, but is also of interest because of its biogenetic relationship to the phenylpropanoid pathway and to other molecules of physiological significance, notably salicylate. Recent progress towards characterisation of the biosynthesis of vanillin is reviewed. In Vanilla, there is some evidence that the route to vanillin-β-d-glucoside may proceed from 4-coumaric acid via 4-hydroxybenzaldehyde, with glucoside formation occurring not necessarily as the final step, and possibly with the involvement of 4-hydroxybenzyl alcohol β-d-glucoside tartrate bis-esters as “shunt” metabolites. This appears to be given tentative support by the recent partial characterisation of a 4-hydroxybenzaldehyde synthase from Vanilla. On the other hand, a well-characterised, CoA-dependent, non-oxidative chain-shortening mechanism to produce vanillin from ferulic acid, occurring as part of a pathway of hydroxycinnamate degradation in Pseudomonas, may not be representative of hydroxycinnamate chain-shortening mechanism(s) occurring in Vanilla and other plants. Nevertheless, by expression of the Pseudomonas enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), attempts have been made to introduce a direct capacity for vanillin formation into model plants by diversion of the phenylpropanoid pathway. The results obtained have emphasised the obstacles to achieving the desired oxidation level (aldehyde) and ring substitution (4-hydroxy-3-methoxyphenyl), even when a substantial metabolic diversion is successfully achieved. Finally, the significance of the latest biosynthetic and biotechnological developments is reviewed briefly in relation to authentication of vanillin.Graphic
Keywords: Vanillin; Vanilla; Benzaldehydes; Ferulic acid; Phenylpropanoid; Metabolic engineering;

Upon centrifugation, rubber latex is divided into a layer of rubber particles, the cytosol, and the lutoid-body fraction, which is of vacuolar origin. One of the proteins isolated from the lutoid-body fraction is a protein with a molecular mass of 43 kDa, which has esterase activity on p-nitrophenylpalmitate and which shows significant sequence similarity with patatin, a vacuolar protein with esterase activity from potato (Solanum tuberosum). This protein is a major allergen in rubber latex products (Hev b 7) and can also be isolated from the cytosol fraction of rubber latex. The mature protein isolated from lutoid-bodies has no structural features expected for a vacuolar protein: the N-terminal methionine in the cDNA-derived sequence is cleaved off, the second residue is N-acetylated, and the C-terminal sequence is identical to that in the cDNA-derived sequence. Thus the patatin-like protein in Hevea brasiliensis is not a vacuolar protein, but may be associated with not yet characterized particles in the cytoplasm, which either sediment with lutoid-bodies or remain in the cytosol fraction, depending on the centrifigation conditions.Patatin is a vacuolar protein isolated from potatoes. The homologous protein isolated from the lutoid-body fraction of latex of Hevea brasiliensis has not the structural features expected for a vacuolar protein.
Keywords: Hevea brasiliensis; Euphorbiaceae; Rubber latex; Lutoid-bodies; Vacuoles; Patatin;

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds by Ashok P. Giri; Abhay M. Harsulkar; Maurice S.B. Ku; Vidya S. Gupta; Vasanti V. Deshpande; Prabhakar K. Ranjekar; Vincent R. Franceschi (523-532).
Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28–6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman–Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman–Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.Seven major trypsin inhibitors (TIs) isolated from winged bean seed revealed differential inhibition when individually assessed for their potential to inhibit the Helicoverpa armigera gut proteinases, which is a mixture of six major and several minor trypsin/chymotrypsin-like serine proteinases. We suggest that such inhibitors are a good target for future work aimed at enhancing resistance of crop plants to H. armigera, a major devastating field pest.
Keywords: Lepidoptera; Noctuidae; Leguminosae; Helicoverpa armigera; Psophocarpus tetragonolobus; Winged bean; Insect gut proteinases; Proteinase inhibitors; Two-dimensional gel electrophoresis; Activity visualization;

Use of plant cell cultures to study graminicide effects on lipid metabolism by Lindsey J Price; Derek Herbert; David J Cole; John L Harwood (533-541).
Tissue cultures from grass species have proven very useful as test systems to study graminicide activity.Graminicides belonging to the cyclohexanedione and aryloxyphenoxypropionate classes are well established to act by disrupting acyl lipid biosynthesis via specific inhibition of acetyl-CoA carboxylase. Species of grass inherently resistant to such herbicides, or biotypes of grassy weed species which display acquired resistance to recommended rates of graminicide application, are known to possess an altered plastidic multifunctional acetyl-CoA carboxylase showing reduced sensitivity to these herbicides in vitro. Studies reported here demonstrate that cell suspension cultures of maize, a graminicide-sensitive species and Poa annua, a graminicide-insensitive species, display a similar differential sensitivity of acyl lipid biosynthesis as tissue from corresponding intact plants. Acyl lipid biosynthesis in P. annua can be inhibited if sufficiently high concentrations of graminicide are used. The major plastidic form and the minor cytosolic forms of acetyl-CoA carboxylase were successfully purified from maize cell suspensions, were compared to those from leaf tissue and were shown to be differentially inhibited by graminicides in a similar manner to their counterparts from leaf tissue. These studies demonstrate that cell suspensions are useful for studying the mode of action of graminicides, especially in view of the limited amount of material obtainable from many grassy species which are very fine-growing.
Keywords: Graminicides; Acetyl-CoA carboxylase; Tissue cultures; Lipid synthesis; Test system for herbicides;

It has been hypothesized that ferulates are only deposited in the primary cell wall of grasses. To test this hypothesis, the fourth elongating, above-ground internode of maize (Zea mays l.) was sampled from three maize hybrids throughout development. Cell wall composition was determined by the Uppsala Dietary Fibre method. Ester- and ether-linked ferulates were determined by HPLC analysis of ferulic acid released from the internodes by low and high temperature alkaline treatments. Internode length increased from 9 to 152 mm over 96 days of growth, with elongation being complete in the first 12 days. More than half of the cell wall material in the maize internodes accumulated after elongation had ended. Deposition of cell wall material appeared to reach its maximum extent 40 days after sampling began, well before physiological maturity of the maize plants. Galactose and arabinose began to accumulate early in cell wall development which was presumed to be associated with primary wall growth during internode elongation. The major secondary wall constituents (analyzed as glucose, xylose, and Klason lignin) did not begin to accumulate rapidly until shortly before internode elongation ended. Ferulate ester deposition began before ferulate ethers were observed in the cell wall, but both forms of ferulate continued to accumulate in secondary cell walls, long after internode elongation had ceased. These data clearly show that contrary to the hypothesis, ferulate deposition was not restricted to the primary wall and that active lignin/polysaccharide cross-linking mediated by ferulates occurs in the secondary wall.The deposition of ester- and ether-linked ferulates in maize cell walls was quantified during internode elongation. Ferulates were incorporated into both primary and secondary walls of maize stem tissues.
Keywords: Zea mays; Poaceae; Maize; Development; Cell wall; Ferulates;

Rice seedlings release momilactone B into the environment by Hisashi Kato-Noguchi; Takeshi Ino (551-554).
Since the growth inhibitor momilactone B was found recently in root exudates of rice (Oryza sativa L.), 3-day-old rice seedlings were transferred to hydroponic culture and the level of momilactone B released into the environment from the seedlings was measured. At day 15 after transfer, the level of momilactone B in the culture solution was 1.8 nmol per seedling compared with endogenous levels of 0.32 and 0.63 nmol per root and shoot, respectively, suggesting that rice seedlings actively releases momilactone B into the culture solution. This release must occur from the roots because only rice roots were immersed in the culture solution. Momilactone B inhibited the growth of ten cress (Lepidium sativum L.) seedlings at concentrations greater than 3 μM. Ten rice seedlings were incubated with ten cress seeds in a Petri dish containing 1 ml of medium, the medium contained 18 nmol of momilactone B, which came to 18 μM. This level of momilactone B was enough to reveal growth inhibition of the cress seedlings. Release level of momilactone B and its effectiveness as a growth inhibitor suggest that it may play an important role in rice allelopathy.Rice seedlings may actively release momilactone B into the neighboring environment and this release level may be enough to cause growth inhibition of neighboring plants.
Keywords: Oryza sativa; Poaceae; Allelopathy; Growth inhibitor; Momilactone B; Rice; Root exudate;

Chemical profiling of Ocimum americanum using external flavonoids by Roberto F. Vieira; Renée J. Grayer; Alan J. Paton (555-567).
The external flavonoids of many herbarium specimens of Ocimum americanum were surveyed by HPLC to establish the flavonoid profiles of this species over the full range of its geographic distribution for authentication purposes.A HPLC survey was undertaken of the external flavonoids in 111 herbarium specimens of Ocimum americanum L. (O. canum Sims), which were largely collected from their natural habitats throughout Africa and Asia. The purpose of this study was to establish the flavonoid profiles of this species over the full range of its geographic distribution in order to use these for authentication purposes. Six different external flavonoid chemotypes were found. The major chemotype, present in circa 80% of the specimens of both var. americanum and var. pilosum collected throughout the distribution area of the species, was characterised by very high levels of nevadensin, slightly lower levels of salvigenin and much lower levels of up to 15 other external flavones. Of the remaining five chemotypes, two were found in var. americanum and three in var. pilosum. All specimens belonging to these chemotypes were collected in South or East Africa and represented by only a few specimens. These samples contained much smaller levels of flavones than present in the major chemotype of O. americanum and all lacked nevadensin. Xanthomicrol, a compound absent from the main chemotype, was the dominant flavone in two of the minor chemotypes. The external flavonoid profiles found in the six chemotypes of O. americanum were compared with those of O.×citriodorum (11 herbarium specimens studied) and seven other closely related species of Ocimum. The main nevadensin/salvigenin pattern present in O. americanum was also found in O.×citriodorum, O. basilicum and some specimens of O. minimum, but there were strong quantitative differences in external flavonoids among these taxa. The other chemotypes of O. americanum showed some similarities in their external flavone profiles to those found in the closely related East African species O. fischeri, O. forskolei, O. kenyense and O. kilimandscharicum, which occur in the same geographic areas. This suggests that the uncommon chemotypes of O. americanum may have originated by an exchange of genes with other Ocimum species, e.g. by introgressive hybridisation. Despite some similarities in profiles, chemical differences were also found among the species, so that it should be possible to authenticate a large proportion of leaf samples of O. americanum on the basis of external flavonoid profiles.
Keywords: Ocimum americanum var. americanum; Ocimum americanum var. pilosum; O.×citriodorum; Lamiaceae; Basil; External flavones; Chemotaxonomy; Infraspecific variability; Authentication;

Antimicrobial action of palmarosa oil (Cymbopogon martinii) on Saccharomyces cerevisiae by Anjali Prashar; Pauline Hili; Robert G Veness; Christine S Evans (569-575).
The essential oil extracted from palmarosa (Cymbopogon martinii) has proven anti-microbial properties against cells of Saccharomyces cerevisiae. Low concentrations of the oil (0.1%) inhibited the growth of S. cerevisiae cells completely. The composition of the sample of palmarosa oil was determined as 65% geraniol and 20% geranyl acetate as confirmed by GC–FTIR. The effect of palmarosa oil in causing K+ leakage from yeast cells was attributed mainly to geraniol. Some leakage of magnesium ions was also observed. Blocking potassium membrane channels with caesium ions before addition of palmarosa oil did not change the extent of K+ ion leakage, which was equal to the total sequestered K+ in the cells. Palmarosa oil led to changes in the composition of the yeast cell membrane, with more saturated and less unsaturated fatty acids in the membrane after exposure of S. cerevisiae cells to the oil. Some of the palmarosa oil was lost by volatilization during incubation of the oil with the yeast cells. The actual concentration of the oil components affecting the yeast cells could not therefore be accurately determined.Geraniol in palmarosa oil led to changes in composition of the yeast cell membrane, with leakage of K+ and Mg2+ ions from cells.
Keywords: Cymbopogon martinii; Graminaceae; Palmarosa oil; Geraniol; Geranyl acetate; Saccharomyces cerevisiae;

Allelopathic substances in Pueraria thunbergiana by Hisashi Kato-Noguchi (577-580).
Leaves of Pueraria thunbergiana possess allelopathic activity and the putative compounds causing this growth inhibitory effect were isolated from their aqueous methanol extract. The chemical structures of these growth inhibitors were determined by high-resolution MS and 1H NMR spectral data as cis,trans-xanthoxin and trans,trans-xanthoxin. cis,trans-Xanthoxin and trans,trans-xanthoxin inhibited the root growth of cress (Lepidium sativum L.) seedlings at concentrations greater than 0.3 and 3 μM, respectively. The doses required for 50% inhibition on the cress roots were 1.1 and 14 μM for cis,trans- and trans,trans-xanthoxin, respectively. The concentrations of cis,trans- and trans,trans-xanthoxin in P. thunbergiana leaves were 51.4 and 72.5 ng g−1 fresh weight, respectively. The effectiveness of cis,trans- and trans,trans-xanthoxin on the growth inhibition and the occurrence of both xanthoxins in P. thunbergiana suggest that xanthoxins may contribute to the growth inhibitory effect of P. thunbergiana, and may play an important role in the allelopathy of P. thunbergiana after being released into the soil.The putative compounds causing the growth inhibitory effect of P. thunbergiana leaves were isolated and the structures were determined as cis,trans-xanthoxin and trans,trans-xanthoxin.
Keywords: Pueraria thunbergiana; Leguminosae; Allelopathy; Cress; Growth inhibitor; Phytotoxicity; Xanthoxin;

Sesquiterpene constituents from the liverwort Bazzania japonica by Runhua Lu; Claudia Paul; Simla Basar; Wilfried A König; Toshihiro Hashimoto; Yoshinori Asakawa (581-587).
The hydrodistillation products of the liverwort Bazzania japonica were separated by preparative gas chromatography (GC) and investigated by spectroscopic methods. Seven unknown compounds were isolated and identified by GC–MS and NMR. Four of them, the norsesquiterpene hydrocarbons 4-epi-11-nor-aristola-1(10),11-diene (1), 4-epi-11-nor-aristola-1,9,11-triene (2), 4-epi-11-nor-aristola-9,11-diene (3), and one oxygenated sesquiterpene, (−)-aristol-1(10)-en-12-ol (5) are new natural compounds, and one, (+)-himachala-2,4-diene (7), has for the first time been isolated from liverworts. The absolute configurations of 5 and 7 were derived by chemical correlation reactions and/or enantioselective GC using cyclodextrin phases. 1, 2 and 3 have identical absolute configuration.Three norsesquiterpenes 4-epi-11-nor-aristola-1(10),11-diene (1), 4-epi-11-nor-aristola-1,9,11-triene (2) and 4-epi-11-nor-aristola-9,11-diene (3) together with (−)-aristol-1(10)en-12-ol were identified as new natural compounds from the liverwort Bazzania japonica using spectroscopic methodes in conjunction with enantioselective gas chromatography. In addition himachala-2,4-diene and aristol-1(10)-en-12-al were for the first time identified as constituents of liverworts.
Keywords: Bazzania japonica; Liverwort; Norsesquiterpene hydrocarbons; Oxygenated sesquiterpenes;

The incomplete structure proposed in 1972 for a unique “flavone-polysaccharide” compound, MF-1, from the liverwort Monoclea forsteri, has been re-examined. Rather than the proposed 8-methoxyluteolin structure with polysaccharides attached to the 7- and 4′-hydroxyls, MF-1 has been shown to be primarily a mixture of penta- and hexa-O-glycosides of 6-methoxyluteolin, which are accompanied by their luteolin analogues. MS and NMR evidence is marshalled to define the structure of MF-la as 6-methoxyluteolin 7-O-[2-O-α-rhamnosyl-3-O-α-arabinosyl-β-glucuronide]-4′-O-[2-O-α-rhamnosyl-3-O-β-xylosyl-β-glucuronide], and MF-1b as 6-methoxyluteolin 7-O-[2-O-α-rhamnosyl-β-glucuronide]-4′-O-[2-O-α-rhamnosyl-3-O-β-xylosyl-β-glucuronide]. This report is the first to provide substantive evidence for the existence of flavone penta- and hexa-O-glycosides in nature. The newly defined structure(s) for MF-1 more closely align M. forsteri with the only other species in the order Monocleales, M. gottschei.The “flavone-polysaccharide” (MF-1) from the liverwort, Monoclea forsteri, is shown to be a mixture of penta- and hexa-O-glycosides of 6-methoxyluteolin, and luteolin. MF-1a is 6-methoxyluteolin 7-O-[2-O-α-rhamnosyl-3-O-α-arabinosyl-β-glucuronide]-4′-O-[2-O-α-rhamnosyl-3-O-β-xylosyl-β-glucuronide] and MF-1b its de-arabinosyl equivalent. These are the first flavone penta- and hexa-glycosides to be described.
Keywords: Monoclea forsteri; Monocleales; Liverwort; Flavone-polysaccharide; 6-Methoxyluteolin 7, 4′-O-glycosides; Flavone penta- and hexa-O-glycosides;

An arylbenzofuran and four isoflavonoids from the roots of Erythrina poeppigiana by Hitoshi Tanaka; Tomoko Oh-Uchi; Hideo Etoh; Magoichi Sako; Masaru Sato; Toshio Fukai; Yoichi Tateishi (597-602).
An arylbenzofuran, erypoegin F and four isoflavonoids, erypoegins G–J, together with six known compounds were isolated from the roots of Erythrina poeppigiana, and their structures were elucidated on the basis of spectroscopic evidence. Erypoegin F is a rare 2-arylbenzofuran possessing a formyl group from a natural source, and erypoegin I is the first naturally occurring isoflavonoid with a 2-oxo-3-methylbutyl group.An arylbenzofuran, erypoegin F (1) and four isoflavonoids, erypoegins G–I (25) were isolated from the root of Erythrina poeppigiana.
Keywords: Erythrina poeppigiana; Leguminosae; Arylbenzofuran; Isoflavonoids; 6a-Hydroxypterocarpans; Erypoegins F–J;

Chemical and cytotoxic constituents from Peperomia sui by Ming-Jen Cheng; Shoiw-Ju Lee; Ying-Ying Chang; Szu-Huei Wu; Ian-Lih Tsai; Bolleddula Jayaprakasam; Ih-Sheng Chen (603-608).
Three polyketide compounds, surinone A, surinone B, surinone C and one acylresorcinol, suranone, along with thirty known compounds, were isolated from the whole plant of Peperomia sui. Their structures were elucidated from spectral analysis. Several compounds showed cytotoxic activity against HONE-1 and NUGC-3 cell lines in vitro.Three polyketide compounds, including surinone A (1), surinone B (2), surinone C (3) and one acylresorcinol, suranone (4), along with thirty known compounds, were isolated from the whole plant of Peperomia sui. The structures of 14 were elucidated from spectral analysis. Several compounds showed cytotoxic activity against HONE-1 and NUGC-3 cell lines in vitro.
Keywords: Peperomia sui; Piperaceae; Whole plant; Polyketide; Surinone A; Surinone B; Surinone C; Acylresorcinol; Suranone;

Aromatic compound glucosides, alkyl glucoside and glucide from the fruit of anise by Eiko Fujimatu; Toru Ishikawa; Junichi Kitajima (609-616).
From the polar portion of the methanolic extract of the fruit of anise (Pimpinella anisum L.), which has been used as a spice and medicine since antiquity, four aromatic compound glucosides, an alkyl glucoside and a glucide were isolated together with 24 known compounds. The structures of the new compounds were clarified as (E)-3-hydroxyanethole β-d-glucopyranoside, (E)-1′-(2-hydroxy-5-methoxyphenyl)propane β-d-glucopyranoside, 3-hydroxyestragole β-d-glucopyranoside, methyl syringate 4-O-β-d-glucopyranoside, hexane-1,5-diol 1-O-β-d-glucopyranoside and 1-deoxy-l-erythritol 3-O-β-d-glucopyranoside by spectral investigation.(E)-3-hydroxyanethole β-d-glucopyranoside, (E)-1′-(2-hydroxy-5-methoxyphenyl)propane β-d-gluco-pyranoside, 3-hydroxyestragole β-d-glucopyranoside, methyl syringate 4-O-β-d-glucopyranoside, hexane-1,5-diol 1-O-β-d-glucopyranoside and 1-deoxy-l-erythritol 3-O-β-d-glucopyranoside were isolated from the fruit of anise.
Keywords: Anise; Pimpinella anisum fruit; Umbelliferae; Aromatic compound glucoside; Alkyl glucoside; Glucide; Hexane-1,5-diol 1-O-β-d-glucopyranoside; 1-Deoxy-l-erythritol 3-O-β-d-glucopyranoside;

The unmasking of lignin structures in wheat straw by alkali by Nathalie Durot; François Gaudard; Bernard Kurek (617-623).
This study reports on the structural modifications of wheat straw cell wall promoted by potassium carbonate and sodium hydroxide that lead to the unmasking of some lignin structures. The first impact of the treatments was the extraction of a particular fraction of lignin enriched in C–C linked structures compared to the mean composition in reference wheat straw. Concomitantly, an apparent increase in the amount of lignin monomers released by the cleavage of alkyl–aryl ether bonds was observed in alkali-extracted samples. By summing the amount of ether linked monomers analyzed by thioacidolysis in the solubilized lignin to that found in the extracted wheat straw, an excess of up to 37% is apparent, relative to the corresponding amount in the reference wheat straw. Other modifications of the cell wall were also found. Indeed, a fraction of uronic acids was lost during the treatments and a new fractionation pattern of the lignin-carbohydrate complexes was evidenced. It can thus be concluded that a significant proportion of lignin within the cell wall was unmasked after (i) the selective removal of a particular lignin fraction, (ii) a partial saponification of the esterified fraction of lignin with uronic acids and (iii) a modification of the interactions between the cell wall constituents.The unmasking of particular lignin structures after a mild alkali treatment of wheat straw, that are otherwise not available for thioacidolysis analysis, is reported.
Keywords: Wheat straw; Lignin; Lignin carbohydrate complexes; Uronic acids; Alkaline extraction; Fractionation; Abiotic reaction;

Four bisindole alkaloids, viz., 19′(S)-hydroxyconodurine, conodurinine, 19′(S)-hydroxyconoduramine, and 19′(S)-hydroxyervahanine A, in addition to conodurine and ervahanine A, were obtained from the leaf and stem-bark extracts of Tabernaemontana corymbosa. The structures of the new alkaloids were determined using NMR and MS analysis.Four bisindole alkaloids (e.g., conodurinine) were obtained from the leaf and stem-bark extracts of Tabernaemontana corymbosa.
Keywords: Tabernaemontana species; Apocynaceae; Bisindole alkaloids;

The Simple Plant Isoquinolines by Satyajit D Sarker (631-632).

Major Herbs of Ayurveda by Geoffrey A. Cordell (632-633).