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Total methanolic extracts of Saponaria vaccaria seed derived from several varieties, as well as various purified components obtained through successive chromatographic separations of total extracts were evaluated for their growth inhibitory activity in WiDr (colon), MDA-MB-231 (breast), NCI-417 (lung) and PC-3 (prostate) human cancer cells as well as the non-tumorigenic fibroblast BJ (CRL-2522) cell line using MTT colorimetric assay. Purified bisdesmosidic saponins segetoside H and I were further examined using microscopy and apoptosis assays. Bisdesmosidic saponins exhibited dose-dependent growth inhibitory and selective apoptosis-inducing activity. Growth inhibitory effects were particularly strong in a breast (MDA-MB-231) and a prostate (PC-3) cancer cell line. Total extracts exhibited a different preference being most active against a colon cancer cell line (WiDr). In a comparison of varieties, all of the total seed extracts exhibited similar dose-dependent activities, but with some variation in potency. Monodesmosidic saponins vaccarosides A and B, phenolic vaccarin, and cyclopeptide segetalin A, co-occurring seed substituents, did not exhibit activity. The non-tumorigenic fibroblast cell line BJ (CRL 2522) was growth inhibited but did not undergo apoptosis when treated with bisdesmosidic saponins at low micromolar concentrations. Saponin-rich extracts from Kochia scoparia seed and Chenopodium quinoa were also evaluated alongside Saponaria saponins but did not exhibit activity. Closely related Quillaja saponins exhibited activity but were less potent.

*SUMMARY Cyclic peptides (CPs) are produced in a very wide range of taxa. Their biosynthesis generally involves either non-ribosomal peptide synthases or ribosome-dependent production of precursor peptides. Plants within the Caryophyllaceae and certain other families produce CPs which generally consist of 5–9 proteinogenic amino acids. The biological roles for these CPs in the plant are not very clear, but many of them have activity in mammalian systems. There is currently very little known about the biosynthesis of CPs in the Caryophyllaceae. A collection of expressed sequence tags from developing seeds of Saponaria vaccaria was investigated for information about CP biosynthesis. This revealed genes that appeared to encode CP precursors which are subsequently cyclized to mature CPs. This was tested and confirmed by the expression of a cDNA encoding a putative precursor of the CP segetalin A in transformed S. vaccaria roots. Similarly, extracts of developing S. vaccaria seeds were shown to catalyze the production of segetalin A from the same putative (synthetic) precursor. Moreover, the presence in S. vaccaria seeds of two segetalins, J [cyclo(FGTHGLPAP)] and K [cyclo(GRVKA)], which was predicted by sequence analysis, was confirmed by liquid chromatography/mass spectrometry. Sequence analysis also predicts the presence of similar CP precursor genes in Dianthus caryophyllus and Citrus spp. The data support the ribosome-dependent biosynthesis of Caryophyllaceae-like CPs in the Caryophyllaceae and Rutaceae.

Pressurized low polarity water (PLPW) extraction of cow cockle seed was carried out to determine the effect of extraction conditions (temperature (125–175 °C), time (15–180 min)), and sample pre-treatment on saponin yield and composition. Accelerated solvent extractions (ASE) and ultrasonic extractions (USE) using water, methanol and ethanol (50%, 80%, 100%) were also carried out to determine the effect of extraction solvent and method on saponin recovery. A higher saponin yield was obtained by ASE compared to USE using pure and aqueous ethanol and methanol. The highest saponin yield, which was obtained by ASE using 80% ethanol, was used to calculate saponin recoveries. The saponin yield of PLPW extracts increased with extraction temperature and time. While only 33.2 wt% of total saponins was extracted from ground seeds at 125 °C in 3 h, 60.2 wt% was recovered in the first 15 min at 175 °C. Total extraction (1 h) of whole seeds yielded more saponins than ground seeds at 125–160 °C. Saponin concentration of the extracts was affected by the extraction solvent and method, sample pre-treatment and to a lesser extent by the time and temperature of PLPW extraction. The highest saponin concentration of PLPW extract was obtained at 125 °C using whole seeds (12%). The saponin composition of water extracts differed from that of aqueous ethanol and methanol extracts.

Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a β-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria.

Nine quillaic acid and five gypsogenin bisdesmosides were isolated from roots of Saponaria officinalis L. (Caryophyllaceae). Seven of the quillaic acid saponins possessed a 3-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl unit, but differed from each other in oligosaccharide units linked to the C-28 ester. The five gypsogenin saponins isolated from the roots all possessed the 3-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl unit, with their oligosaccharide units linked to the C-28 ester differing. Structures were elucidated by extensive 1D and 2D NMR spectroscopy and mass spectrometry. The saponins were evaluated for growth inhibitory activity in two human cancer cell lines and hemolytic activity in sheep red blood cells.

The structural and conformational requirements of a carrier responsible for the saturable component of the uptake of the plant hormone (+)-abscisic acid in barley suspension culture cells have been probed through the use of a defined series of optically pure ABA analogues. Two analogues showed a tenfold increase over (+)-ABA in inhibiting the uptake of radiolabelled (+)-ABA. Results from different studies (molecular modelling, low temperature NMR spectroscopy and assays with sterically rigid analogues) have shown the likely conformation of ABA in the binding site of the carrier is that with the sidechain in an equatorial-like orientation. Structure/activity studies show that the C-1 carboxylic acid is essential for binding, the C-4 carbonyl is moderately important while the C-1′ hydroxyl group is not important for binding to the carrier.

In vivo measurements of the enzymatic hydroxylation of the phytohormone (+)-abscisic acid to hormonally inactive ( -)-phaseic acid in corn cell cultures revealed that (+)-abscisic acid 8' hydroxylase activity was induced by (+)-abscisic acid treatment. This induction was blocked by the protein synthesis inhibitor cycloheximide and by the transcription inhibitor cordycepin. Following an induction treatment with abscisic acid, the amount of induced enzyme was measured by addition of both cycloheximide, to prevent further induction, and fresh abscisic acid as substrate for the induced enzyme. Phaseic acid production was related to the amount of enzyme induced. The experimental system was optimized for studying enzyme induction and the substrate specificity of the induced enzyme. The induced 8' hydroxylase was specific for (+)-abscisic acid, whereas the low basal activity also hydroxylated (-)-abscisic acid at the 7' position to form 7'-hydroxy abscisic acid. After induction, the 8' hydroxylase was rapidly degraded with a half-life of approximately 2 h. Phaseic and (-)-abscisic acid were weak inducers; however, the unstable oxidation product 8'-hydroxy abscisic acid exhibited 42% of the activity of (+)-abscisic acid. When corn cells were placed under water stress by increasing concentrations of mannitol in the culture medium, 8' hydroxylase induction was suppressed. The experimental system described will be useful for further studies of the physiological and hormonal factors that modulate abscisic acid metabolism and for testing potential enzyme inhibitors and hormone analogues.

Abscisic acid (ABA) was functionalized at the 3′-position by reaction of its α-2′,3′-epoxide with 1,4-butanedithiol to afford the corresponding derivative having a six-atom linker arm containing a terminal aulphhydryl group. This derivative was coupled with epoxy activated Sepharose 6B affording a gel containing ca 1.4 mg of (+)-ABA ml−1. This gel was shown to bind specifically an anti-ABA antibody from hybridoma culture broth, and its binding capacity was determined. The (+)-ABA analogue possessing the thiol linker arm was also derivatized by reaction with a fluorescent derivatizing agent and these compounds were shown to inhibit cress seed germination.

Unnatural phaseic acid (U-PA) was identified by chiral GC as a minor component of the metabolite mixture obtained from the medium of a maize cell suspension culture treated with unnatural (−)( R )-abscisic acid (U-ABA). When U-[3′,5′,5′,7′,7′,7′]-hexadeutero-ABA was fed to the cell suspension, d 6 -PA was obtained, establishing its origin from the exogenously-added U-ABA. The major metabolise produced was identified as U-7′-hydroxyabscisic acid (U-7′-HOABA [ d 5 -7′-HOABA from d 6 -U-ABA]. Confirmation of the assignments was obtained by HPLC/LC/CFSIMS analysis of the metabolise mixtures. This result corroborates a previous report of the formation of U-PA from racemic ABA in avocado mesocarp tissue.

The uptake of [ 3 H]-abscisic acid in barley (Hordeum vulgare L. cv. Heartland) cell cultures was found to be mediated through both non-saturable and saturable components. The kinetic parameters of the saturable component, determined at pH 4.5 and 21° C, showed a K m for natural or (+).ABA of 1.3 ± 0.7 μM and an apparent V max of 7.0 ± 2.8 nmol g −1 cells h −1 . The carrier showed a strong preference for the natural enantiomer of ABA as compared to the unnatural one. Other substances tested, e.g. amino acids, organic acids, and other growth regulators, did not appear to interfere with the carrier-mediated uptake of ABA. At low external concentrations of ABA (below 2.0 μM), the saturable component was greater than the diffusion component

Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the C4-hydroxylation of 2,3-dihydro-3-hydroxy-N(1)-methyltabersonine to the 3,4-dihydroxy derivative. The enzyme showed an absolute requirement for 2-oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). The hydroxylase exhibited specificity for position 4 of various alkaloid substrates. The enzyme exhibited a pH optima between 7 and 8 and an apparent molecular weight of 45,000. The appearance of 4-hydroxylase activity was developmentally regulated and was shown to be inducible by light treatment of seedlings. Substrate specificity studies of this enzyme for indole alkaloid substrate suggested that hydroxylation at position 3 and N-methylation occur prior to hydroxylation at position 4. This is in agreement with previous studies which suggest that C4-hydroxylation is the second to last step in vindoline biosynthesis in Catharanthus roseus.

We report here the synthesis and biological activity of a new persistent abscisic acid (ABA) analog, 8[prime]-methylene ABA. This ABA analog has one additional carbon atom attached through a double bond to the 8[prime]-carbon of the ABA molecule. (+)-8[prime]-Methylene ABA is more active than the natural hormone (+)-ABA in inhibiting germination of cress seed and excised wheat embryos, in reducing growth of suspension-cultured corn cells, and in reducing transpiration in wheat seedlings. The (+)-8[prime]-methylene analog is slightly weaker than (+)-ABA in increasing expression of ABA-inducible genes in transgenic tobacco, but is equally active in stimulating a transient elevation of the pH of the medium of corn cell cultures. In corn cells, both (+)-ABA and (+)-8[prime]-methylene ABA are oxidized at the 8[prime] position. ABA is oxidized to phaseic acid and (+)-8[prime]-methylene ABA is converted more slowly to two isomeric epoxides. The alteration in the ABA structure causes the analog to be metabolized more slowly than ABA, resulting in longer-lasting and more effective biological activity relative to ABA.

The metabolism and effects of (+)-S- and (-)-R-abscisic acid (ABA) and some metabolites were studied in maize (Zea mays L. cv Black Mexican Sweet) suspension-cultured cells. Time-course studies of metabolite formation were performed in both cells and medium via analytical high-performance liquid chromatography. Metabolites were isolated and identified using physical and chemical methods. At 10 [mu]M concentration and 28[deg] C, (+)-ABA was metabolized within 24 h, yielding natural (-)-phaseic acid [(-)-PA] as the major product. The unnatural enantiomer (-)-ABA was less than 50% metabolized within 24 h and gave primarily (-)-7[prime]-hydroxyABA [(-)-7[prime]-HOABA], together with (+)-PA and ABA glucose ester. The distribution of metabolites in cells and medium was different, reflecting different sites of metabolism and membrane permeabilities of conjugated and nonconjugated metabolites. The results imply that (+)-ABA was oxidized to (-)-PA inside the cell, whereas (-)-ABA was converted to (-)-7[prime]-HOABA at the cell surface. Growth of maize cells was inhibited by both (+)- and (-)-ABA, with only weak contributions from their metabolites. The concentration of (+)-ABA that caused a 50% inhibition of growth of maize cells was approximately 1 [mu]M, whereas that for its metabolite (-)-PA was approximately 50 [mu]M. (-)-ABA was less active than (+)-ABA, with 50% growth inhibition observed at about 10 [mu]M. (-)-7[prime]-HOABA was only weakly active, with 50% inhibition caused by approximately 500 [mu]M. Time-course studies of medium pH indicated that (+)-ABA caused a transient pH increase (+0.3 units) at 6 h after addition that was not observed in controls or in samples treated with (-)-PA. The effect of (-)-ABA on medium Ph was marginal. No racemization at C-1[prime] of (+)-ABA, (-)-ABA, or metabolites was observed during the studies.

Two cell lines of Vinca major L. cv. variegata produced strictosidine lactam as the main alkaloid. Quantities varied from 500–1000 μg/g DW in the high-yielding line and 1–100 μg/g DW in the low-yielding line. Transfer of cells to alkaloid production medium resulted in a 6–8 fold increase in alkaloid production with the high-yielding line, some increase in the low-yielding line and in both lines induced intense greening (up to 200 μg chlorophyll/g DW) indicating chloroplast differentiation. Though in the source plant leaves are the main storage site, no correlation between alkaloid accumulation and chloroplast differentiation could be found.

Robinson annelation of thujone (1) with methyl and ethyl vinyl ketones proceeds stereospecifically to afford the tricyclic enones 6 and 4 respectively. Enone 4 was converted to the glycol 13 which on treatment with 48% hydrobromic acid gave (+)-9-bromo-β-cyperone (14). Reduction with triphenyltin hydride yielded (+)-β-cyperone (3). Enone 6 was converted via standard means to the cyclopropyl alcohol 9, a potential precursor of (+)-junenol (10).

Oxidation of 16,18-dicarbomethoxycleavamine gave the epoxide (6) and the enamide (9). Similar oxidation of 3′,4′-dehydrovinblastine (8) or leurosine (7) gave the natural product catharine (3). Potassium permanganate oxidation of 7 or 8 gave 3R-hydroxyvinamidine (19) whereas similar oxidation of 4′-deoxyleurosidine (29) gave the alkaloid vinamidine (4).

Summary Seeds of Catharanthus roseus (L.) G. Don cv. Little Delicata were germinated in the presence of different dark-light regimes. Alkaloid profiles as well as tryptophan decarboxylase and acetyl-CoA: deacetylvindole 0-acetyltransferase were monitored. Growth of seedlings in the dark resulted in an early ubiquitous accumulation of tabersonine as a major alkaloid and in the subsequent day 5–10 cotyledon restricted accumulation of vindoline and its immediate precursors. Transfer of 5-day old seedlings to the light resulted in rapid loss of vindoline precursors followed by a more gradual disappearance of tabersonine and the subsequent enhanced accumulation of vindoline. Although light enhanced vindoline biosynthesis, it was not essential. This sequence of biogenetic events suggested the following biosynthetic pathway: tabersonine → 16-hydroxytabersonine → 16-methoxytabersonine → 16-methoxy-2,3-dihydro-3-hydroxytabersonine → N(1)-methyl-l6-methoxy-2,3-dihydro-3-hydroxytabersonine (i.e. desacetoxyvindoline) → deacetylvindoline → vindoline. The time course of induction indicated that increase of tryptophan decarboxylase activity coincided with tabersonine accumulation, whereas increase of acetyl-CoA: deacetylvindoline 0-acetyltransferase activity coincided with vindoline accumulation. Results suggested that tabersonine biosynthetic pathway enzymes occur in all plant parts whereas the last 5 steps in vindoline biosynthesis are restricted to aerial parts of the plant and that the whole pathway to vindoline biosynthesis is developmentally regulated.

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.

Syntheses of the C 8 and C 10 olefinic units cis - and trans -5-ethyl-1-iodo-hex-4-enes and cis - and trans -7-ethyl-3-iodo-oct-6-enes are described. The Wittig coupling of such units with derivatives of α- and β-thujaketonic acids to give analogs of insect juvenile hormones is discussed.

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615–767 and 916 could not be induced to produce DAT or NMT enzyme activities.

A large body of research has concerned itself with the production of commercially important phytochemicals by using plant cells in culture. These studies have identified several key areas where basic knowledge is generally limiting exploitation [1,2]. One fundamental problem occurs when non-differentiated tissue cultures are unable to produce and to accumulate complex chemicals originally found in specialized tissues or organs in the intact plant [3,4]. It is this need for specialized organs or tissues for metabolite production that has led to the development of rapidly growing and phytochemical producing organized hairy root cultures following genetic transformation of plants with Agrobacterium rhizogenes [5, and references cited therein]. Such cultures offer the possibility of producing root-derived phytochemicals but not those occurring in other plant tissues [5]. These technical innovations, however, fail to address the fundamental regulatory controls that maintain the specific developmental states which induce the production and accumulation of a given phytochemical.

Robinson annelation of thujone (1) with methyl and ethyl vinyl ketones proceeds stereospecifically to afford the tricyclic enones 6 and 4 respectively. Enone 4 was converted to the glycol 13 which ...