Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Analyzing the Antioxidant Activity and Fatty Acid Composition of Monofloral Mullein (Verbascum sp.) Pollen Oil obtained via Various Extraction Techniques.

This study focused on characterizing fatty acids and evaluating the antioxidant properties in oils extracted from mullein (Verbascum sp.) bee-collected pollen, utilizing soxhlet and ultrasound-assisted methods with acetone and hexane solvents. Soxhlet extraction demonstrated high efficiency in mullein bee pollen oil extraction. The highest levels of total phenolic content (TPC), total flavonoid content (TFC), DPPH⋅, and ABTS⋅+ activities (41.07±1.43 mg GAE/g extract; 1.86±0.01 mg QE/g extract; 16.23±0.68 mg TE/g extract; 56.88±0.43 mg TE/g extract, respectively) were observed in oil extracted using the soxhlet method with acetone solvent. Conversely, ultrasound-assisted extraction with hexane yielded oils rich in saturated fatty acids, while acetone extraction contained higher monounsaturated fatty acids. Palmitic, linoleic, and oleic acids were predominant in the extracted oils. This study introduces, for the first time, the identification of fatty acids found in mullein bee pollen oil, along with an examination of their antioxidant properties. The choice of solvent was found to significantly influence compound extraction compared to the extraction method.

Antioxidant, AChE inhibitory, and anticancer effects of Verbascum thapsus extract.

Verbascum thapsus (VT) is a medicinal plant that is used in folk medicine to treat a variety of ailments. For this study, the biological functions of VT methanol extract were determined in vitro. The plant's methanol extract was created through the maceration process. The phytochemical composition of plant extracts was investigated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The antioxidant capacity of the extract was determined using the DPPH (2,2-diphenyl-1-picrylhydrazil) and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) tests and its cytotoxicity was assessed using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)) assay on the Caco-2 (human colorectal adenocarcinoma cells), LNCaP (Lymph Node Carcinoma of the Prostate), and HEK293 cell lines (Human embryonic kidney 293 cells) used to model colon, prostate, and non-cancerous cells. VT extract showed low DPPH and ABTS radical scavenging activities compared to standard antioxidants at 30 mg/ml concentration. In addition, it was determined that VT extract inhibited acetylcholinesterase enzyme.

Ferruginoside†D: A Novel Phenylethanoid from Verbascum leiocarpum.

A novel phenylethanoid, ferruginoside D, together with fourteen known compounds were isolated from the Verbascum leiocarpum for the first time. Chemical structures of isolated compounds were determined by spectroscopic analysis including HR-ESI-MS and NMR spectra. The antioxidant, α-glucosidase inhibitory properties, and antiproliferative activities against multiple cell lines (A172, C6, HeLa, A2780, SW620, HT29, Beas2B, RPE, and HSF) of the isolated compounds were evaluated in vitro. According to the results, iridoids, flavonoids (luteolin and apigenin), and phenylethanoids (poliumoside and isoverbascoside) with strong antiproliferative potential were also found to have cytostatic effects. Furthermore, the investigation revealed that compounds luteolin, poliumoside, verbascoside, isoverbascoside, ferruginoside C, ferruginoside D, and ursolic acid show potent alpha-glucosidase inhibitory activity, while compounds luteolin, verbascoside, and isoverbascoside exhibit substantial antioxidant activity. The new compound (ferruginoside D) was found moderately active against cancer cell lines, with strong alpha-glucosidase inhibitory activity, and moderate antioxidant properties.

Taxonomy, comparative genomics of Mullein (Verbascum, Scrophulariaceae), with implications for the evolution of Verbascum and Lamiales.

BACKGROUND: The genus Verbascum L. (Scrophulariaceae) is distributed in Africa, Europe, and parts of Asia, with the Mediterranean having the most species variety. Several researchers have already worked on the phylogenetic and taxonomic analysis of Verbascum by using ITS data and chloroplast genome fragments and have produced different conclusions. The taxonomy and phylogenetic relationships of this genus are unclear. RESULTS: The complete plastomes (cp) lengths for V. chaixii, V. songaricum, V. phoeniceum, V. blattaria, V. sinaiticum, V. thapsus, and V. brevipedicellatum ranged from 153,014 to 153,481 bp. The cp coded 114 unique genes comprising of 80 protein-coding genes, four ribosomal RNA (rRNA), and 30 tRNA genes. We detected variations in the repeat structures, gene expansion on the inverted repeat, and single copy (IR/SC) boundary regions. The substitution rate analysis indicated that some genes were under purifying selection pressure. Phylogenetic analysis supported the sister relationship of (Lentibulariaceae + Acanthaceae + Bignoniaceae + Verbenaceae + Pedaliaceae) and (Lamiaceae + Phyrymaceae + Orobanchaceae + Paulowniaceae + Mazaceae) in Lamiales. Within Scrophulariaceae, Verbascum was sister to Scrophularia, while Buddleja formed a monophyletic clade from (Scrophularia + Verbascum) with high bootstrap support values. The relationship of the nine species within Verbascum was highly supported. CONCLUSION: Based on the phylogenetic results, we proposed to reinstate the species status of V. brevipedicellatum (Engl.) Hub.-Mor. Additionally, three genera (Mazus, Lancea, and Dodartia) placed in the Phyrymaceae family formed a separate clade within Lamiaceae. The classification of the three genera was supported by previous studies. Thus, the current study also suggests the circumscription of these genera as documented previously to be reinstated. The divergence time of Lamiales was approximated to be 86.28 million years ago (Ma) (95% highest posterior density (HPD), 85.12-89.91 Ma). The complete plastomes sequence data of the Verbascum species will be important for understanding the Verbascum phylogenetic relationships and evolution in order Lamiales.

Health-promoting and disease-mitigating potential of Verbascum thapsus L. (common mullein): A review.

Medicinal plants represent rich sources of traditional medicines and numerous currently used medicines are either directly or indirectly derived from plants. Verbascum thapsus L. (great mullein or common mullein), a medicinal herb indigenous to northern Africa, western and central Asia, and Europe, has been brought to the Americas and Australia. It has been used as a medicine for lung, skin and throat disorders and has a long history of therapeutic importance, particularly as an astringent and calming agent. Presently, the dried leaves, flowers, various plant extracts and flower oil are used in several formulations within Indian traditional medicine. An extract taken from the roots is useful in minimizing toothache, and it also relieves stiffness and seizures. V. thapsus contains a wide variety of phytoconstituents, such as flavonoids, iridoid, phenylethanoid and phenylpropanoid glycosides, saponins, as well as vitamin C and minerals. The most valuable constituents are coumarin and hesperidin, which possess healing properties. Emerging literature based on experimental studies on V. thapsus demonstrates various biological and pharmacological properties, including antiviral, antioxidant, analgesic, sedative, anti-inflammatory, hypnotic, antibacterial, antifungal, as well as anticancer activities. The present review provides an updated, comprehensive, and critical evaluation of various health-promoting and disease-mitigating properties of V. thapsus.

Phenolic and flavonoid compounds and antioxidant activity in flowers of nine endemic Verbascum species from Iran.

BACKGROUND: In this study, flowers of nine mullein species (Verbascum erianthum, V. songaricum, V. speciosum, V. szovitsianum, V. stachydiforme, V. sinuatum, V. haussknechtianum, V. cheirantifolium, and V. saccatum) were collected from Iran. The total flavonoid content (TFC), antioxidant activity, total carotenoid (TCC), ß-carotene content and the amount of phenolic compounds were determined in all of the collected mullein species. Quantitative analysis of phenolic compounds was performed using high-performance liquid chromatography with diode-array detection (HPLC-DAD). RESULTS: Verbascum saccatum and V. songaricum, in particular, were found to be rich in total flavonoids and exhibited the highest antioxidant capacity. The HPLC-DAD analysis revealed that V. erianthum possessed the highest amount of caffeic acid (0.022 g kg-1 DW), chlorogenic acid (2.649 g kg-1 DW), p-coumaric acid (0.253 g kg-1 DW), and apigenin (0.066 g kg-1 DW). The highest gallic acid (0.134 g kg-1 DW), rutin (5.254 g kg-1 DW), quercetin (1.303 g kg-1 DW), and cinnamic acid (1.031 g kg-1 DW) content were observed in V. saccatum. Our results demonstrate the considerable variations in the TFC, antioxidant activity, TCC, and ß-carotene content among the mullein species. CONCLUSIONS: The findings of this study provide useful information for breeding strategies, and for choosing the best species with high phenolic compound content to produce natural antioxidants for medical and pharmaceutical use. © 2021 Society of Chemical Industry.

Influence of soil pollution on the morphology of roots and leaves of Verbascum thapsus L.

The impact of inorganic pollutants in the zone of industrial wastewater settling tanks (South of Russia) was studied. The levels of Mn, Cr, Ni, Cu, Zn, Pb, Cd were determined for Verbascum thapsus L., which are part of the mesophilic succession of wild plants in the studied technogenically polluted territory. The bioavailability of heavy metals (HM) for plants from transformed soils has been established. Anatomical and morphological features in the tissues of the plants affected by HM were analyzed using light-optical and electron microscopic methods. Contamination of the soil cover with Mn, Cr, Ni, Cu, Zn, Pb and Cd has been established with maximum content of Zn. It was revealed that the HM content in the V. thapsus plants exceeded the maximum permissible levels (Russian state standard): Zn by 23, Pb by 2, Cr by 31 and Cd by 3 times. The lower level of HM content in the inflorescences of mullein plants in comparison with the root system, stems and leaves indicates the resistance of generative organs to technogenic pollution. In the root and leaves of the V. thapsus, the anatomical and ultrastructural observation were carried out using light-optical and transmission electron microscopy. Changes in the ultrastructure of plants under the influence of anthropogenic impact have been revealed. The most significant changes of the ultrastructure of the polluted plants were found in the cell organelles of leaves (mitochondria, plastids, peroxisomes, etc.) including spatial transformation of the thylakoid system of plastids during the metal accumulation by plants, which may determine the mechanism of plant adaptation to technogenic pollution.

Evaluation of antioxidant, cytotoxic, antibacterial effects and mineral levels of Verbascum lasianthum Boiss. ex Bentham.

The present study aimed to determine the antibacterial, antioxidant, cytotoxic activities and element levels of Verbascum lasianthum Boiss. ex Bentham. The free radical scavenging activity, total phenolic content, total antioxidant capacity, total oxidant capacity levels were analyzed as the antioxidant parameters. Seven bacteria and one yeast strains were used to determine the antimicrobial activity. The cytotoxic effects of plant extracts were determined using A549, MCF-7, HepG2 and SH-SY5Y cell lines. The findings demonstrated that the antioxidant activity increased with an increase in the phenolic content of extracts. This species is rich in bio-elements such as Fe, Cu, Mn, Zn, and Mg. Different concentrations of extracts could have antibacterial activity. This plant had an apparent cytotoxic effect only in the A549 cell line and increased the proliferation in other cell lines. The findings demonstrated that plant could be used alone or as a supplement to the current treatment protocols in diseases due to their antioxidant, antibacterial and cytotoxic effects. However, it is recommended that Verbascum L. species intended for use in therapy should be procured from areas where there is no soil pollution or organic farming is preferred.

Mullen Scales of Early Learning Adaptation for Assessment of Indian Children and Application to Tuberculous Meningitis.

INTRODUCTION: Tuberculous meningitis (TBM) results in significant morbidity and mortality among children worldwide. Associated neurocognitive complications are common but not well characterized. The Mullen Scales of Early Learning (MSEL), a well-established measure for assessment of neurodevelopment, has not yet been adapted for use in India. This study's goal was to adapt the MSEL for local language and culture to assess neurocognition among children in India, and apply the adapted measure for assessment of children with TBM. METHODS: Administration of MSEL domains was culturally adapted. Robust translation procedures for instructions took place for three local languages: Marathi, Hindi and Tamil. Multilingual staff compared instructions against the original version for accuracy. The MSEL stimuli and instructions were reviewed by psychologists and pediatricians in India to identify items concerning for cultural bias. RESULTS: MSEL stimuli unfamiliar to children in this setting were identified and modified within Visual Reception, Fine-Motor, Receptive Language and Expressive Language Scales. Item category was maintained for adaptations of items visually or linguistically different from those observed in daily life. Adjusted items were administered to six typically developing children to determine modification utility. Two children diagnosed with confirmed TBM (ages 11 and 29 months) were evaluated with the adapted MSEL before receiving study medications. Skills were below age-expectation across visual reception, fine motor and expressive language domains. CONCLUSIONS: This is the first study to assess children with TBM using the MSEL adapted for use in India. Future studies in larger groups of Indian children are warranted to validate the adapted measure.

6-O-(3″, 4″-di-O-trans-cinnamoyl)-α-l-rhamnopyranosylcatalpol and verbascoside: Cytotoxicity, cell cycle kinetics, apoptosis, and ROS production evaluation in tumor cells.

The aim of this study was to evaluate the impact that 6-O-(3″, 4″-di-O-trans-cinnamoyl)-α- l-rhamnopyranosylcatalpol (Dicinn) and verbascoside (Verb), two compounds simultaneously reported in Verbascum ovalifolium, have on tumor cell viability, apoptosis, cell cycle kinetics, and intracellular reactive oxygen species (ROS) level. At 100 µg/mL and 48 hours incubation time, Dicinn and Verb produced good cytotoxic effects in A549, HT-29, and MCF-7 cells. Dicinn induced cell-cycle arrest at the G0 /G1 phase and apoptosis, whereas Verb increased the population of subG1 cells and cell apoptosis rates. Furthermore, the two compounds exhibited time-dependent ROS generating effects in tumor cells (1-24 hours). Importantly, no cytotoxic effects were induced in nontumor MCF-10A cells by the two compounds up to 100 µg/mL. Overall, the effects exhibited by Verb in tumor cells were more potent, which can be correlated with its structural features, such as the presence of phenolic hydroxyl groups.

Verbascum nigrum: Cytotoxicity Evaluation in A431 Epidermoid Carcinoma Cells and Untargeted LC-HR-MS/MS Metabolite Profiling.

The crude methanolic extract obtained from Verbascum nigrum aerial parts (VNE) and its six fractions (VNF1-VNF6) were initially screened regarding their effects on the viability of immortalized HaCaT keratinocytes and A431 epidermoid carcinoma cells (MTT assay, 24 h). None of the tested samples affected the viability of HaCaT cells in a concentration range of 25-150 µg/mL. VNE and VNF4 exhibited significant cytotoxic effects in A431 cells, with IC50 values of 81.92 and 12.27 µg/mL, respectively; the selectivity index was higher than 10 for VNF4. The untargeted LC/HR-MS/MS metabolite profiling led to the tentative annotation of a total number of 23 compounds. Of these, VNE comprised mainly iridoid glycosides (harpagoside, laterioside, acylated aucubin derivatives), whereas VNF4 showed a high abundance of triterpene saponin glycosides (ilwensisaponins A and C, songarosaponins A and B), constituents known for their selective cytotoxic potential.

HPLC-DAD-ESI-Q-TOF-MS/MS profiling of Verbascum ovalifolium Donn ex Sims and evaluation of its antioxidant and cytogenotoxic activities.

INTRODUCTION: Plant species of Verbascum genus have been intensively investigated in the last decades but most studies focused on evaluation of their biological activities; there are only few studies dealing with their chemical characterisation. OBJECTIVE: Detailed investigation of the qualitative and quantitative chemical composition, antioxidant and cytogenotoxic activities of a previously non-studied Verbascum species (V. ovalifolium Donn ex Sims). METHODS: Qualitative analysis of secondary metabolites was performed by HPLC-DAD-ESI-Q-TOF-MS/MS, whereas quantitative data were obtained through HPLC-DAD. Antioxidant activity was evaluated using in vitro assays; cytotoxic and genotoxic effects were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) and comet assays, respectively. RESULTS: More than 50 secondary bioactive metabolites belonging to various classes (iridoids, phenylethanoids, flavonoids, phenolic acids) were detected in the methanolic extract of V. ovalifolium and its fractions. The fragmentation pathways of acylated catalpol-type iridoid diglycosides are thoughtfully described herein. The extracts showed good free radical scavenging and ferric ion reducing properties correlated with phenolic, flavonoid, chlorogenic acid and verbascoside contents. Moreover, 24 h treatment of SK-MEL-2 cells with V. ovalifolium extracts produced significant changes in terms of tumour cell viability. The crude extract and the ethyl acetate fraction showed no important signs of cytogenotoxicity in non-tumour cells. CONCLUSION: The performed phytochemical and biological analyses contribute to the preclinical knowledge about V. ovalifolium and they could help exploiting it in novel herbal medicinal products.

Biological activities, isolated compounds and HPLC profile of Verbascum nubicum.

Context: Genus Verbascum (Scrophulariaceae) comprises about 360 species of flowering plants. Verbascum has been used in traditional medicine as an astringent, antitussive, analgesic and anti-inflammatory. Objective: Nothing was found in the available literature concerning Verbascum nubicum Murb; therefore, the study evaluates the biological activities, isolated compounds and HPLC profile. Materials and methods: Methanol extract (VME) and butanol fraction (VBF) of air-dried powdered V. nubicum were obtained. Four compounds were isolated from VBE and identified by 1H- and 13C-NMR. High-performance liquid chromatography (HPLC) profile was determined for (VME). LD50, in vitro antioxidant, in vivo antiulcerogenic and anti-inflammatory activities as well as hepatoprotective activity were assessed. Anti-ulcerogenic and hepatoprotective activities were supported by histopathological examinations. Results: HPLC analysis of VME revealed the presence of luteolin 7-glucoside (2215.43 mg/100 g), hesperidin (954.51 mg/100 g) and apigenin (233.15 mg/100 g) as major compounds. Four compounds were isolated and confirmed by NMR data, were identified as gentiopicroside, luteolin, aucubin and gallic acid. The LD50 of VME and VBF extracts were calculated to be 8200 and 4225 mg/kg b.w., respectively. IC50 values of VBE and VMF as measured by DPPH·method were 43.6 and 50 µg/mL, respectively. Also, anti-inflammatory effect of VME (400 mg/kg b.w.) and VBF (200 mg/kg b.w.) induced edema model after 120 min were 61.93 and 56.13%, respectively. Antiulcerogenic activity of VME (400 mg/kg b.w.) and VBF (200 mg/kg b.w.) in albino rats were 65.14 and 84.57%, respectively. Conclusions: The V. nubicum extracts displayed safe and promising antioxidant, anti-inflammatory and hepatoprotective properties. It can be also applied in the pharmacy industry, food industry and healthcare.

Inhibitor for FcεRI expression on mast cell from Verbasucum thapsus L.

We found out 2',3'-dihydroxypuberulin from South American medicinal plant, V. thapsus L., as a candidate of an anti-allergic lead which inhibits the expression of high-affinity receptor of IgE (FcεRI) on the surface of mast cells. Furthermore, the analysis of structure-activity relationship by using synthesized 2',3'-dihydroxypuberulin analogs revealed that both hydroxy groups in the side chain and both of methyl moieties on phenolic hydroxy groups were crucial for potent activity, but absolute configuration of C-3' position wasn't. The active principle, 2',3'-dihydroxypuberulin, was disclosed to down-regulate the mRNA level of ß-chain of FcεRI, different from previous reported active natural product reducing γ-chain level.

Genetic transformation of rare Verbascum eriophorum Godr. plants and metabolic alterations revealed by NMR-based metabolomics.

OBJECTIVES: To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. RESULTS: Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. CONCLUSION: Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

Antitrypanosomal activity of Verbascum sinaiticum Benth. (Scrophulariaceae) against Trypanosoma congolense isolates.

BACKGROUND: African Trypanosomiasis is a neglected tropical disease with a large impact on the livelihood of the rural poor in Sub-Saharan Africa. The available drugs for managing this disease are old, expensive and are facing the problem of drug resistance. Thus, the aim of this study was to evaluate in vivo antitrypanosomal efficacy of aqueous and absolute methanol leaf extracts of Verbascum sinaiticum Benth. against Trypanosoma congolense field isolate. METHODS: Verbascum sinaiticum (Local name 'qetetina') is a biennial plant, and 60-150 cm tall. It is traditionally used to treat wound, stomachache, viral infection, cancer, sunstroke, fever, abdominal colic, diarrhea, hemorrhage, anthrax, and hepatitis. The efficacy of aqueous and absolute methanol leaf extracts of V. sinaiticum was evaluated in a randomized experiment with Swiss albino mice infected with T. congolense field isolate. The extracts were administered at doses of 100, 200 and 400 mg/kg by intraperitoneal injection for seven days at 12 Days Post-Infection (DPI) when the peak parasitaemia level was approximately 10(8) trypanosomes/ml. Parasitaemia, Packed Cell Volume (PCV), mean survival time and change in body weight were used as indices for monitoring the efficacy of the extracts. Diminazene (28 mg/kg) was used as a positive control while 2 % Tween was used as the negative control. Phytochemicals screening were conducted following standard methods. RESULTS: The extracts showed no toxicity effect in Swiss albino mice and had LD50 above 2000 mg/kg. The phytochemicals screened in V. sinaiticum were alkaloids, flavonoids, glycoside, saponins, steroids, phenolic compounds, and tannins. The mice treated with absolute methanol leaf extract of V. sinaiticum at 400 mg/kg dose had significantly lower mean parasitaemia (7.20 ± 0.16) (p < 0.001) as compared to the negative control group (8.82 ± 0.12) on day 14 of treatment. Animals treated with the same dose had significant (p < 0.001) higher PCV value and body weight and as well as the highest mean survival time of 40.20 ± 0.31 days as compared to the negative control at the end of the observation period. CONCLUSION: This study established that Verbascum sinaiticum had trypanocidal activity. The crude extracts have partially eliminated trypanosomes in a dose-dependent manner. The study can be a basis for future in-depth analysis of the biologically active chemicals.

[Pharmacognosy study of Verbascum species]. / A Verbascum sp. farmakognóziai vizsgálata.

The mullein (Verbascum phlomoides L., V thapsus L., V. thapsiforme Schrad., V. speciosum L.) is a medicinal herb known and used for a long time, especially in traditional Turkish medicine. The aims of our study were to identify the species and study the plant's major active substances both qualitatively and quantitatively, comparing it to data found in scientific literature. The plants were identified as probable hybrids of V. phlomoides and V. thapsiforme. Microscopic analysis of the flowers showed no major difference between the specimens. The diameter of both stomata and pollen we observed was around 15-20 µm. Important flavonoids like rutin and quercetin were identified. Dosage resulted in a 0.135% total flavonoid aglycone content. (expressed as hypericin) and a 1.3% total flavonoid glycoside content (expressed as rutoside). Thin layer chromatography from saponines revealed two spots. A hemolytic index of 13095 was also determined. Repeating the dosage experiment a year later resulted in significantly lower flavonoid aglycone and glycoside content (0.006% and 0.95% respectively) as well as a hemolytic index of approximately 4000.

Leaf architectural, vascular and photosynthetic acclimation to temperature in two biennials.

Acclimation of leaf features to growth temperature was investigated in two biennials (whose life cycle spans summer and winter seasons) using different mechanisms of sugar loading into exporting conduits, Verbascum phoeniceum (employs sugar-synthesizing enzymes driving symplastic loading through plasmodesmatal wall pores of phloem cells) and Malva neglecta (likely apoplastic loader transporting sugar via membrane transport proteins of phloem cells). In both species, acclimation to lower temperature involved greater maximal photosynthesis rates and vein density per leaf area in close correlation with modification of minor vein cellular features. While the symplastically loading biennial exhibited adjustments in the size of minor leaf vein cells (consistent with adjustment of the level of sugar-synthesizing enzymes), the putative apoplastic biennial exhibited adjustments in the number of cells (consistent with adjustment of cell membrane area for transporter placement). This upregulation of morphological and anatomical features at lower growth temperature likely contributes to the success of both the species during the winter. Furthermore, while acclimation to low temperature involved greater leaf mass per area in both species, this resulted from greater leaf thickness in V. phoeniceum vs a greater number of mesophyll cells per leaf area in M. neglecta. Both types of adjustments presumably accommodate more chloroplasts per leaf area contributing to photosynthesis. Both biennials exhibited high foliar vein densities (particularly the solar-tracking M. neglecta), which should aid both sugar export from and delivery of water to the leaves.

Behavioral and electroantennogram responses of plum curculio, Conotrachelus nenuphar, to selected noxious plant extracts and insecticides.

Behavioral and electroantennogram responses of plum curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), adults were tested for several methanolic plant extracts and organically approved insecticides. Plant extracts were evaluated for their potential as antifeedants or oviposition deterrents. These extract responses were also compared to those elicited by the non-neurotoxic, organic irritant-insecticide kaolin clay. Both sexes of plum curculio exhibited antennal response as measured by electroantennogram, which ranged from 0.2 to 1.1 mV, to plant extracts and the organic irritant/insecticide, with the greatest response to the extract of rough cocklebur, Xanthium strumarium L. (1.1 mV). No choice tests were conducted to compare feeding and oviposition by plum curculio on untreated apples or on apples treated with one of the extracts or the insecticide. The insecticide pyrethrum and extracts of X. strumarium and greater burdock, Arctium lappa L., significantly reduced feeding. Also, pyrethrum, A. lappa, Humulus lupulus L. (common hop), X. strumarium, and Verbascum songaricum Schrenk extracts completely inhibited egg deposition. In no-choice assays, the effects of kaolin clay with incorporated plant extracts on plum curculio feeding and oviposition were monitored as complementary tests. A. lappa-kaolin, H. lupulus-kaolin, and X. strumarium-kaolin mixtures significantly reduced the feeding of plum curculio compared to the control or kaolin clay alone. Each of the plant extract-kaolin mixtures evaluated, with the exception of Bifora radians Bieberstein (wild bishop), completely inhibited plum curculio oviposition as compared to controls.