Renée J. Grayer

Infraspecific taxonomy and essential oil chemotypes in sweet basil, Ocimum basilicum

Essential oil compositions of fresh and freeze-dried leaves were determined for 16 accessions of Ocimum basilicum belonging to different varieties to see whether they could be used as infraspecific taxonomic characters. One accession of O. x citriodorum was also studied. Some 30 monoterpenoids, sesquiterpenoids and phenylpropanoids were identified, the major components (more than 20% of the total essential oil composition in one or more accessions) being geranial and neral in O. x citriodorum, and linalool, methyl chavicol, eugenol, methyl eugenol and geraniol in O. basilicum. Based on a combination of the latter compounds, five major essential oil profiles could be distinguished in the accessions studied for O. basilicum. These profiles were largely the same for fresh and freeze-dried material of the same plant, although in the dried leaves, methyl chavicol and eugenol concentrations had generally declined in comparison to those of linalool. There appeared to be little correlation between essential oil patterns and varietal classification within O. basilicum. In view of the chemical heterogeneity of O. basilicum and its use as an essential oil-producing crop, culinary herb, medicinal plant and insect-controlling agent, in all of which chemicals play an important role, the infraspecific classification of this taxon should take chemical characters into consideration. A system for the classification of essential oil chemotypes in O. basilicum is proposed.

COMPLEXES OF IRON WITH PHENOLIC COMPOUNDS FROM SOYBEAN NODULES AND OTHER LEGUME TISSUES : PROOXIDANT AND ANTIOXIDANT PROPERTIES

The low-molecular-mass fraction of the soybean nodule cytosol contains Fe capable of catalyzing free radical production through Fenton chemistry. A large portion of the pool of catalytic Fe, measured as bleomycin-detectable Fe, was characterized as complexes of Fe with phenolic compounds of three classes: phenolic acids, cinnamic acids, and flavonoids. Many of these compounds, along with other phenolics present in legume tissues, were used for a systematic structure-activity relationship study. All phenolics tested were able to chelate Fe, as judged from their inhibitory effect on site-specific deoxyribose degradation (minus EDTA assay). However, only those having catechol, pyrogallol, or 3-hydroxy-4-carbonyl groupings were potent chelators and reductants of Fe3+ at pH 5.5. The same phenolics promoted oxidative damage to DNA (bleomycin assay) and to deoxyribose (plus EDTA assay), but inhibited linolenic acid peroxidation by chelating and reducing Fe3+ and by neutralizing lipid radicals. Also, phenolics having a pyrogallol nucleus attenuated the free radical-mediated inactivation of glutamine synthetase, which was used as a model system, by chelating Fe2+. It is reasoned that under the microaerobic (10-20 nM O2) and acidic (pH 5.5-6.4) conditions prevailing in nodules, phenolics are likely to act primarily as antioxidants, decreasing oxidative damage to biomolecules.

Plant–fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants

A brief review is given of some biological, chemical and chemotaxonomic aspects of phytoalexin research. Emphasis is placed on the search for antifungal compounds in the plant families Leguminosae and Rosaceae, and in rice, Oryza sativa. The possible role of phytoalexins in the resistance of rice plants against the fungus Pyricularia oryzae (= Magnaporthe grisea) is discussed, and the future prospects of phytoalexin research are outlined.

The application of atmospheric pressure chemical ionisation liquid chromatography–mass spectrometry in the chemotaxonomic study of flavonoids: characterisation of flavonoids from Ocimum gratissimum var. gratissimum

Liquid chromatography coupled to mass spectrometry (LC-MS), using atmospheric pressure chemical ionisation (APCI), was found to complement data obtained from diode array detection enabling common flavonoids in Ocimum gratissimum var. gratissimum to be characterised at the analytical level without complete purification, or even to be characterised in crudely purified ethanolic extracts. Xanthomicrol, cirsimaritin, rutin, kaempferol 3-O-rutinoside and vicenin-2 were identified as the major flavonoids, whereas luteolin 5-O-glucoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside, vitexin, isovitexin, quercetin 3-O-glucoside and isothymusin were detected as minor constituents. First-order positive ion APCI-MS provided not only the pseudomolecular ion of flavonoid O-glycosides, but also an ion corresponding to the aglycone and an intermediate ion if more than one sugar was attached to the flavonoid. Information on the type and sequence of sugars was therefore obtained without the need to undertake MS2 product ion analyses. Product ion analysis was useful for confirming the glycoside fragmentation observed in first-order MS, especially with co-eluting compounds, and for obtaining fragmentation patterns of either free aglycones or the aglycone moiety of flavonoid O-glycosides; the latter could be used to support the aglycone identification achieved from the UV spectra. Negative ion APCI-MS was found useful for flavone C-glycosides and gave very characteristic product ions due to fragmentation of the C-sugars. For flavonoid O-glycosides analysed in the negative ion mode, there was evidence that the position and nature of the attached sugars could influence the aglycone product ion formed by MS2 analysis and hence its product ion spectrum. APCI is considered to be a more useful ion source than electrospray in the chemotaxonomic study of flavonoids. Copyright

External flavones in sweet basil, Ocimum basilicum, and related taxa

Abstract Sixteen accessions of Ocimum basilicum L. belonging to different cultivars and varieties, and one accession each of O. x citriodorum Vis. and O. minimum L., were examined for their external leaf flavonoids. The O. basilicum samples showed the presence of one or two major flavone aglycones, which were identified as salvigenin and nevadensin, and up to 10 minor ones: cirsileol, cirsilineol, eupatorin, apigenin, acacetin, genkwanin, apigenin 7,4′-dimethyl ether, cirsimaritin, ladenein and gardenin B. No trace was found of xanthomicrol, the only lipophilic flavone so far reported from O. basilicum . There were appreciable differences amongst the accessions in total flavonoid concentration and in their nevadensin / salvigenin ratios, which, on the whole, were not correlated with morphological characters. The surface flavonoid profile of O. x citriodorum was very similar to those of the O. basilicum plants studied, but that of O. minimum was significantly different. Only 4′-methyl ethers of apigenin derivatives were detected in the latter taxon, whereas apigenin and luteolin derivatives in which the 4′-hydroxyls are free were absent. Furthermore, the ratio of nevadensin / salvigenin was much higher in O. minimum than in O. basilicum or O. x citriodorum . These differences support the treatment of O. minimum as a separate species rather than as a variety of O. basilicum .

A comparison between chemical and molecular characters for the determination of phylogenetic relationships among plant families: An appreciation of Hegnauer’s “Chemotaxonomie der Pflanzen”

Abstract The views on the natural relationships among plant families belonging to the Asteridae s.l. on the basis of chemical profiles expressed in Hegnauer’s series “Chemotaxonomie der Pflanzen” (1962-present) are compared with the results of cladistic analyses of the rbc L gene DNA sequences in those families. The suggestion, based on chemistry, that the Cornaceae are not closely related to the Apiaceae and Araliaceae, is supported by molecular evidence, and so is the view that the Apiaceae and Araliaceae are closer to the Asteraceae instead. Likewise a close affinity between the Scrophulariaceae and Lamiaceae on the one hand, and between the Solanaceae and Boraginaceae on the other is confirmed, instead of close connections between the Scrophulariaceae and Solanaceae, and between the Lamiaceae and Boraginaceae, which has often been the traditional view. Two small families of water plants with reduced flowers, the Callitrichaceae and Hippuridaceae, which used to be placed in widely different orders by different systematists, show chemical profiles which indicate close relationships to the Scrophulariaceae and Lamiaceae. Again molecular data are congruent with the chemical ones. In turn, unexpected molecular results have been supported by chemical evidence, e.g. the placement of the Polemoniaceae close to the Primulaceae and Theaceae, and that of Eucommia next to Aucuba . However, the chemistry of the Menyanthaceae does not unequivocally support the repositioning of this family from the Gentianales to the affinity of the Asteraceae suggested by molecular data. On the whole, molecular trees based on rbc L DNA sequences appear to provide a very useful framework to assess the comparative merits and usefulness of secondary compound classes as chemotaxonomic characters, and molecular information also can be used as a tool to study the evolution of small chemical compounds. On the other hand, chemical characters can be used as an additional support for DNA trees.

Leaf flavonoid glycosides as chemosystematic characters in Ocimum

Abstract Thirty-one accessions of nine species belonging to three subgenera of Ocimum (basil, family Lamiaceae) were surveyed for flavonoid glycosides. Substantial infraspecific differences in flavonoid profiles of the leaves were found only in O. americanum , where var. pilosum accumulated the flavone C -glycoside, vicenin-2, which only occurred in trace amounts in var. americanum and was not detected in cv. Sacred. The major flavonoids in var. americanum and cv. Sacred, and also in all other species investigated for subgenus Ocimum , were flavonol 3- O -glucosides and 3- O -rutinosides. Many species in subgenus Ocimum also produced the more unusual compound, quercetin 3- O -(6″- O -malonyl)glucoside, and small amounts of flavone O -glycosides. The level of flavonol glycosides produced was reduced significantly in glasshouse-grown plants, but levels of flavone glycosides were unaffected. A single species investigated from subgenus Nautochilus , O. lamiifolium , had a different flavonoid glycoside profile, although the major compound was also a flavonol O -glycoside. This was identified as quercetin 3- O -xylosyl(1‴→2″)galactoside, using NMR spectroscopy. The species investigated from subgenus Gymnocimum , O . tenuiflorum (= O. sanctum ), was characterised by the accumulation of flavone O -glycosides. These were isolated, and identified as the 7- O -glucuronides of luteolin and apigenin. Luteolin 5- O -glucoside was found in all nine species of Ocimum studied, and is considered to be a key character for the genus.

The chemotaxonomic significance of two bioactive caffeic acid esters, nepetoidins A and B, in the Lamiaceae.

A survey of leaf surface constituents in the family Lamiaceae using HPLC with diode array detection revealed the presence of two characteristic phenolic compounds in many species. The distribution of these phenolics in the Lamiaceae was found to be of taxonomic significance, as they were present in the great majority of species investigated for the subfamily Nepetoideae, including representatives of the well-known genera of culinary herbs, mint, rosemary, sage, thyme and basil. In contrast, they were absent from species of the other subfamilies of Lamiaceae studied and from the related families Verbenaceae, Scrophulariaceae, Acanthaceae and Buddlejaceae. The compounds were isolated from Plectranthus crassus and identified by NMR spectroscopy as the known caffeic acid esters (Z,E)-[2-(3,5-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate and (Z,E)-[2-(3,4-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate, for which the trivial names nepetoidins A and B are proposed. The presence of this pair of caffeic acid esters adds another character to the chemical, palynological and embryological features distinguishing the Nepetoideae from the other subfamilies of Lamiaceae and related families, and supports the view that the Nepetoideae are a specialised and monophyletic group within the family. Nepetoidin B was shown to have a greater antioxidant activity than gallic, rosmarinic and caffeic acids, and showed activity as an insect phagostimulant. Both compounds were antifungal.

Leaf surface flavonoids in iranian species of Nepeta (Lamiaceae) and some related genera

Abstract A HPLC survey of the leaf surface flavonoids of 38 species of Nepeta (Lamiaceae) and four species of the related genera Agastache, Dracocephalum and Lallemantia revealed 14 different flavones, one of which is new (8-hydroxycirsiliol or 5,8,3′,4′-tetrahydroxy-6,7-dimethoxyflavone). In addition, two flavonols (methyl ethers of kaempferol) were found in Dracocephalum kotschyii. The most frequently encountered flavones in Nepeta were cirsimaritin (5,4′-dihydroxy-6,7-dimethoxyflavone); 8-hydroxycirsimaritin (5,8,4′-trihydroxy-6,7-dimethoxyflavone, also called isothymusin) and genkwanin (5,4′-dihydroxy-7-methoxyflavone). Apigenin and the 4′-methyl ethers of cirsimaritin and 8-hydroxycirsimaritin (salvigenin and 8-hydroxysalvigenin, respectively) were also relatively common. The distribution of surface flavones in the four genera provided some valuable data for the phylogenetic relationships at generic level. The presence of surface flavones with a 5-hydroxy-6,7-dimethoxy A-ring (as found in cirsimaritin and salvigenin) and the unusual 5,8-dihydroxy-6,7-dimethoxy A-ring substitution pattern (as found in 8-hydroxycirsimaritin, 8-hydroxysalvigenin and 8-hydroxycirsiliol), can be considered as a characteristic chemotaxonomic feature typical of the genus Nepeta. Cirsimaritin and 8-hydroxycirsimaritin were also detected in the one species examined for Agastache, but the absence of genkwanin and the presence of acacetin in A. barberi and in ten Agastache species studied previously, distinguished Agastache from Nepeta. The presence of methoxylated flavonols and absence of 8-hydroxycirsimaritin and related compounds distinguished species of Dracocephalum and Nepeta, whereas lack of flavonoids with 6- and 8-oxygenation of the A-ring characterised species of the genus Lallemantia.

Leaf flavonoids as systematic characters in the genera Lavandula and Sabaudia

A comprehensive survey of the leaf flavonoids of the genus Lavandula and the related Sabaudia group was carried out using two-dimensional paper chromatography and high-performance liquid chromatography. The flavonoid patterns obtained were found to be systematically informative at the infrageneric level. Three main groupings were identified: the first containing sections Lavandula, Dentata and Stoechas characterised by the accumulation of flavone 7-glycosides; the second containing sections Pterostoechas, Subnuda and Chaetostachys characterised by the accumulation of 8-hydroxylated flavone 7-and 8-glycosides; the third encompassing the Sabaudia group and accumulating both flavone and 8-hydroxylated flavone 7- glycosides. Such a grouping of taxa is congruent with data from other disciplines, although it is not recognised in any present classifications. The taxonomic and evolutionary implications of the flavonoid data are discussed.