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Pollen micro-morphological features have proven to be helpful for the plant taxonomists in the identification and classification of plants. The aim of this study was to evaluate the palynological features of family Asteraceae and Lamiaceae from flora of District Bannu, Khyber Pakhtunkhwa, Pakistan using both scanning electron microscopy (SEM) and light microscope (LM) for their taxonomic importance. Pollen of seven Asteraceous species belonging to four genera and four Lamiaceae species categorized into four genera were collected from different localities of research area. The present research work provides detailed information of diverse morpho-palynological characters both qualitatively and quantitatively including pollen shape, type, diameter, P/E ratio, exine sculpturing and thickness. Type of pollen in Asteraceae and Lamiaceae was ranged from tricolporate, tricolpate, trizonocolpate and hexazonocolpate. The maximum polar diameter (40.05 μm) and equatorial diameter (37.66 μm) was observed in the Ajuga bracteoosa while minimum polar and equatorial diameter was noted in Isodon rugosus (11.10 μm) and Erigeron canadensis (13.20 μm) respectively. Sculpturing of exine include; echinate, reticulate scabrate, aerolate, reticulate-verrucate, reticulate-scabrate, perforate and reticulate to perforate. Exine thickness was examined maximum 1.50 μm in Helianthus tuberosus, whereas minimum in Conyza Canadensis (0.16 μm). The pollen fertility was found highest in C Canadensis (83.33%) and lowest in Ajuga bracteosa (58.06%). The observed pollen morphology has many valuable qualitative and quantitative attributes for the better understanding of their taxonomy and play significant role in correct identification.

Recent field exploration and collections has led to the findings of several new species in Pakistan. Here we reported two new species Ajuga reptance L and Sphagneticola trilobata (L.) Prusk for the first time in Pakistan flora as these species were neither listed in any other literature nor identified before in Pakistan. These species were found as a result of taxonomic studies performed in the year 2019 in District Rawalpindi and Islamabad, Pakistan. Microscopic techniques were used for the confirmation of foliar epidermal and pollen micromorphological features. Detailed study (morphological, palynological, and foliar epidermal) was provided for the correct identification and delimitation of the species using both light and scanning electron microscopy. Morphological results were compared with the flora of Taiwan and China.

Two new diterpenoids, ajudecunoid A (1) and ajudecunoid B (14), along with thirteen known diterpenoids, were isolated from the whole plants of Ajuga nipponensis Makino. Their structures were elucidated by the extensive spectroscopic analysis (UV, IR, MS, and NMR). The absolute configurations of ajudecunoid A (1) and ajudecunoid B (14) were defined through analysis of X-ray crystallography. Fifteen compounds were evaluated for inhibition of the formation of osteoclasts in bone marrow-derived macrophages (BMM) cells. Two neo-clerodane diterpenoids ajuganipponin B (5) and (12S)-6α,19-diacetoxy-18-chloro-4α-hydroxy-12-tigloyloxy-neo-clerod-13-en-15,16-olide (12) showed significant inhibition of osteoclastogenesis with IC50 values of 0.88 and 0.79 μM, respectively. Here we firstly reported diterpenoids with anti-osteoclastogenesis activity from A. nipponensis.

Chromosome number changes and karyotype evolution play an important role in plant genome diversification and eventually in speciation. The genus Ajuga L. (Lamiaceae) has approximately 50 species distributed in temperate to subtropical regions. Four of these species are currently recognized in Korea (A. decumbens Thunb., A. multiflora Bunge, A. nipponensis Makino and A. spectabilis Nakai). Understanding the karyotype evolution in Ajuga has been hampered by the small size of their chromosomes and symmetrical karyotypes. Here we used classic Feulgen staining to establish chromosome numbers and construct karyotypes of the four species of Ajuga recognized in Korea and flow cytometry was used to study their variation in genome. The chromosome number of all investigated plants was 2n = 32. Still, the 2C DNA content ranged from 2.18 pg (A. decumbens) to 4.53 pg (A. multiflora). While the chromosome numbers were the same for all investigated species, the genome size variation could potentially be used as a taxonomic marker.

Introduction : Ajuga turkestanica is a plant used in traditional medicine for its high ecdysteroid content, including the presence of the particularly active turkesterone, which possess efficient anabolic activity. Objectives : To isolate and identify minor ecdysteroids present in a semi-purified plant fraction containing ca. 70% turkesterone. Material and Methods : Multi-step preparative HPLC (combining RP- and NP-HPLC systems) was used to purify the different components present in the turkesterone fraction. Isolated compounds were identified by high-resolution mass spectrometry and 2D-NMR. Results : Fourteen ecdysteroids (including turkesterone and 20-hydroxyecdysone) were isolated. Seven of these, all bearing an 11α-hydroxy group, were previously unreported. Conclusion : Ajuga turkestanica ecdysteroids are characterised by the abundance of 11α-hydroxylated compounds and by the simultaneous presence of 24C, 27C, 28C and 29C ecdysteroids. It is expected that even more ecdysteroids are to be found in this plant since the starting material for this study lacked the less polar ecdysteroids. The simultaneous presence of 20-hydroxyecdysone and turkesterone (its 11α-hydroxy analogue) as the two major ecdysteroids suggests that every ecdysteroid is probably present in both 11α-hydroxy and 11-deoxy forms.

BACKGROUND: Ecdysteroids are steroid hormones that control moulting and govern several changes during metamorphoses in arthropods. The discovery of the same molecules (phytoecdysteroids) in several plant species displayed a wide array of rather beneficial agricultural impact. Many representatives of the genus Ajuga plants contain phytoecdysteroids with a 5β-7-ene-6-one system exhibiting physiological activities in insects. RESULTS: By means of chromatographic (silica gel column, TLC) and LC-MS, two major ecdysteroids (20-hydroxyecdysone and cyasterone) have been isolated and identified from Israeli carpet bugle Ajuga iva (L.) Schreber (Lamiales: Lamiaceae) plants. Ajuga iva extract fractionated on the silica gel column yielded two fractions that showed high activity against the sweetpotato whitefly Bemisis tabaci and the persea mite Oligonychus perseae. A dose of 5 mg AI L−1 of the purely identified A. iva ecdysterone significantly reduced fecundity, fertility and survival of these pests, while commercial 20-hydroxyecdysone at the same dose had lesser effects. CONCLUSION: The results demonstrate considerable efficacy of natural phytoecdysteroids against major agricultural pests, and suggests that these materials should be considered for potential development of friendly control agents. Copyright © 2011 Society of Chemical Industry

The effect of squeezed juices and crude extracts of Ajuga species on feeding activity and metamorphosis of Epilachna varivestis (Col., Coccinellidae) The effect of fresh, squeezed plant juices and that of crude extracts from dried parts of Ajuga reptans and A. remota on the feeding activity and metamorphosis of Epilachna varivestis was investigated by means of various testing methods. Squeezed plant juices and crude plant extracts cause a strong antifeedant effect. The causal agents are most probably ajugarins, at least in case of A. remota. Furthermore, the metamorphosis is strongly influenced by the extracts, leading to high larval and pupal mortalities, the appearance of pupal-adult specimens and of beetles showing more or less deformed wings. The growth-regulating effect is presumedly caused by ecdysoids, primarily ecdysterone. Zusammenfassung Die Wirkung von Pressaften aus frischen und von Rohextrakten aus getrockneten Teilen von Ajuga reptans und A. remota auf die Frasaktivitat und Metamorphose von Epilachna varivestis wurde mit Hilfe verschiedener Testmethoden untersucht. Pressafte und Rohextrakte bewirken eine deutliche Frashemmung, die zumindest bei A. remota wahrscheinlich auf Ajugarine zuruckgefuhrt werden konnen. Auserdem wird die Metamorphose stark beeinflust, was zu hoher Larven- und Puppenmortalitat sowie zur Entstehung sog. pupal-adulter Tiere und zu Kafern mit mehr oder weniger stark deformierten Flugeln fuhrt. Die metamorphosestorende Wirkung ist mit groser Wahrscheinlichkeit auf die in den Ajuga-Arten enthaltenen Ecdysoide, insbesondere das Ecdysteron zuruckzufuhren.

The effect of squeezed fresh juices and crude extracts of Ajuga remota on the two-spotted spider mite Tetranychus urticae Koch The effect of freshly squeezed plant juices and that of methanolic extracts of Ajuga remota on Tetranychus urticae was investigated in the laboratory by means of various methods. Squeezed plant juices and crude methanolic extracts provoked a strong repellent effect in females of T. urticae. They also caused a decrease of fecundity. The postembryonic development was negatively influenced after the treatment of fresh eggs. High mortality rates and morphological abnormalities were observed in first instar larvae, especially after spraying of a methanolic root-extract on eggs. The possible causes of repellent and growth-regulating effects are discussed. Zusammenfassung In Laborversuchen wurde die Wirkung von Frischpressaften und Methanol-Rohextrakten aus A. remota auf T. urticae mit verschiedenen Testmethoden untersucht. Pressafte und Methanolextrakte erwiesen sich als stark abschreckend auf Weibchen von T. urticae und bewirkten eine Reduktion der Eiablage. Auserdem wurde die postembryonale Entwicklung nach Eibehandlung beeintrachtigt, so das es zu erhohten Mortalitatsraten und auch zu morphologischen Abnormitaten durch den Methanolextrakt kam. Diese Effekte lassen sich wahrscheinlich zumindest teilweise auf die Einwirkung hormonell aktiver Substanzen zuruckzufuhren. Die perkutane Wirkung der Extrakte war nur sehr schwach ausgepragt.

Pericarp and seed structure was investigated in 13 out of the 16 species of the old Caryopteris s.l. and in a few species of the related Rubiteucris, Amethystea, Trichostema and Ajuga. The pericarp structure was found to be very variable, and the variation largely matches the most recent classification where Caryopteris s.l. is divided into six genera: Caryopteris s.s., Discretitheca, Pseudocaryopteris, Rubiteucris, Schnabelia and Tripora. All the six genera but Rubiteucris and Schnabelia can be distinguished from each other by differences in pericarp structure, and Caryopteris s.s. was found to have a particularly divergent structure. Similarities in pericarp structure also suggest that Schnabelia, Rubiteucris and Tripora may be closely related.

Oxidative stress is a common response of epidermal cells to a variety of noxious stimuli such as ultraviolet (UV) radiation from solar light and proinflammatory cytokines from skin-infiltrating leukocytes. Here, we report that two types of plant-derived antioxidants, the phenylpropanoid glycoside verbascoside as well as the flavonoids rutin and quercetin possess protective effects against UVC-induced cell damage and proinflammatory activation. The molecules under investigation were effective against the loss of cell integrity associated with necrosis in doses consistent with their antioxidant activity, whereas they did not significantly oppose UVC-induced proliferation arrest and apoptosis. By contrast, only verbascoside effectively inhibited cytokine-induced release of proinflammatory mediators in a dose-dependent fashion. Verbascoside and its homologue teupolioside dramatically impaired NF-kappaB and AP-1 DNA binding activity. These results suggest that plant polyphenols with antioxidant properties have distinct mechanisms in the suppression of oxidative stress induced in keratinocytes by different stimuli. Verbascoside and teupolioside are hence of potential interest in the protection of the skin from both environmental and inflammatory insults.

Eleven medicinal plants used by traditional healers in Machakos and Kitui District were screened, namely: Ajuga remota Benth, Aloe secundiflora Engl, Amaranthus hybridus L, Cassia didymobotrya Fes, Croton macrostachyus Del, Entada leptostachya Harms, Erythrina abyssinica DC, Harrisonia abyssinica Oliv, Schkuhria pinnata O. Ktze, Terminalia kilimandscharica Engl and Ziziphus abyssinica Hochst for potential antibacterial activity against four medically important bacterial strains, namely: Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Micrococcus lutea ATCC 9341 and Pseudomonas aeruginosa ATCC 27853. The antibacterial activity of methanol extracts was determined as the minimum inhibitory concentration (MIC). The plant extracts were more active against Gram-positive (G+) than Gram-negative (G-) bacteria. The positive controls were streptomycin and benzylpenicillin for G- and G+ bacteria, respectively, both had a significant MIC at

In the current study, the effect of Ajuga iva extract on blood glucose, lipid profile, hepatic and renal toxicity and antioxidant enzyme activities in alloxan-induced diabetic rats was investigated. Diabetes was confirmed by measuring the glucoserua concentration 15 days after alloxan administration. Ajuga iva extract was administrated orally 3 weeks after alloxan injection. Our results investigate that Ajuga iva extract supplementation increased the levels of both enzymatic antioxidant (superoxide dismutase, catalase and glutathione peroxidase) and metals antioxidants (iron, copper, magnesium, calcium) and decreased lipid peroxidation level (TBARs). Besides Ajuga iva ameliorated diabetes provoked hepatic and renal toxicity appeared by a lower level in total and direct bilirubin, urea, creatinine, triglyceride (TG), cholesterol and a higher level in HDL-cholesterol. Besides, the activities of phosphatase alkalines (PAL), aspartate and lactate transaminase (AST & ALT) were decreased. The benefices effects of phytoecdysteroids of Ajuga iva confirmed by histological observation in pancreatic tissues. In conclusion, Ajuga iva phytoecdysteroids supplements seem to be beneficial for correcting the hyperglycemia and preventing diabetic complications in liver, pancreas and kidneys.

The batch culture of a transgenic plant, Ajuga hairy root, in sucrose and MS medium exhibited glucose and fructose accumulation with culture time and depletion of phosphate ion after 16 days. The use of a monosaccharide instead of sucrose in fed-batch culture avoided the difficulty of estimating the concentration of saccharides. Glucose was the most adequate monosaccharide for attaining a high cell mass. In fed-batch culture using a turbine blade fermentor, 27.2 kg-dry cell m−3 was obtained at 39 days in glucose medium, with the final cell mass and production of 20-hydroxyecdysone increasing 2.6-fold and 2.0-fold, respectively, compared with batch culture results with sucrose in a shake flask. The growth of the hairy root was divided into two phases, an exponential growth phase and linear growth phase. The linear growth phase from 5 through 25 kg-dry cell m−3 predominated over the growth curve. On the basis of the data of the fed-batch culture with glucose, the cell yield, 0.77 g g−1, and maintenance coefficient, 0.105 g g−1 day−1, for Ajuga hairy root were comparable to those of various calluses. The cell yield of the plant cell was larger than the yields of microorganisms and the maintenance coefficient was smaller.

Spatial clonal structure and patch colonisation in Ajuga reptans L. (Lamiaceae), a common stoloniferous semi-rosette plant, were studied using a combined morpho-ecological and molecular genetic approach. Within a natural patch from a forest near Diedorf (Thuringia, Germany), the spatial clonal structure was analysed, correlating both datasets: Morphologically, characteristics of clonal growth and clonal reproduction were studied, the spatial distribution of modules was mapped and merigenet relationships were reconstructed. Samples from the patch and its surroundings, and an additional sample from Berlin were then analysed by AFLP fingerprinting using four different primer combinations to identify genets. Most divergence in banding patterns was already obtained for samples from the Diedorf forest. Within the patch, however, most samples had very similar fingerprints, indicating their belonging to the same genet and hence a clonal origin, although they are morphologically separated into three “plants”. Based on AFLP data, the relationships of one sample remained ambiguous; but the correlation with morphological data helped to interpret the pattern and indicated that the sample is probably a dividual of the clone, too. The relevance of the observed vegetative multiplication (clonal growth and subsequent clonal reproduction) for patch colonisation and maintenance are discussed. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Generativer (sexueller) oder klonaler Ursprung? Eine morpho-okologische und molekulare Analyse in einem ,patch' von Ajuga reptans L. (Lamiaceae) Die raumliche klonale Struktur und die ‘patch’ Besiedlung von Ajuga reptans L. (Lamiaceae), einer haufigen Halbrosettenpflanze mit Auslaufern, wurden mit einem morpho-okologischen und molekular-genetischen Ansatz kombiniert analysiert. Die Untersuchung wurde in einem ‘patch’ aus einem Wald bei Diedorf (Thuringen) durchgefuhrt: Morphologisch wurden Merkmale des klonalen Wachstums und der klonalen Reproduktion untersucht, die raumliche Verteilung der Module kartiert und Merigenet-Beziehungen rekonstruiert. Fur Proben aus dem ‘patch’, aus dessen naherer Umgebung und einer Pflanze aus Berlin wurden AFLP Analysen mit vier verschiedenen Primer-Kombinationen durchgefuhrt, um genetische Individuen (Genets) zu identifizieren. Die meisten Unterschiede in den Fragmentmustern zeigten sich bereits zwischen Proben aus Diedorf. Die ‘patch’-Proben (morphologisch aufgeteilt in drei “Pflanzen”) hatten jedoch sehr ahnliche fingerprints, was ihre Zugehorigkeit zum selben Genet und eine klonale Abstammung belegt. Fur eine Probe liesen sich die Verwandtschaftsbeziehungen mit den AFLP Daten nicht sicher klaren. Der Abgleich mit den morphologischen Ergebnissen ermoglichte jedoch eine Interpretation, und deutet auf die Zugehorigkeit zum Klon. Die Bedeutung der “vegetativen Multiplikation” (durch klonales Wachstum und klonale Reproduktion) fur die Besiedlung und dauerhafte Besetzung von ‘patches’ wird diskutiert.

Three new withanolides, bracteosin A (= (22R)-5beta,6beta : 22,26-diepoxy-4beta,28-dihydroxy-3beta-methoxyergost-24-ene-1,26-dione; 1), bracteosin B (= (22R)-5beta,6beta : 22,26-diepoxy-4beta,28-dihydroxy-3beta-methoxy-1,26-dioxoergost-24-en-19-oic acid; 2), and bracteosin C (= (22R)-22,26-epoxy-4beta,6beta,27-trihydroxy-3beta-methoxyergost-24-ene-1,26-dione; 3), have been isolated from the whole plants of Ajuga bracteosa. Their structures were deduced by spectral analysis, including 1D- and 2D-NMR techniques. In addition, dihydroclerodin-1, clerodinin A, lupulin A, and dihydroajugapitin have also been isolated for the first time from this species. Compounds 1-3 exhibited evident inhibitory potential against cholinesterase enzymes in a concentration-dependent fashion.

6 pages, 5 figures, 2 tables.-- PMID: 15532684 [PubMed].-- Printed version published Nov, 2004.

Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an alpha-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (alpha-galactosyl-myo-inositol)-independent alpha-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20 degrees C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10 degrees C/3 degrees C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant alpha-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from alpha-galactosidases; it neither hydrolysed nor transferred alpha-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10-50 mM.

The aim of this study was to investigate the gastric ulcer preventive activity of aqueous extract from Ajuga pantantha Hand-Mazz (AHMAE) on reserpine-induced experimental gastric ulcer in Kunming m...

Cyasterone is the main phytoecdysteroid component from cultured plants of Ajuga nipponensis, and common in other Ajuga species. Results showed that when treated with 50 mg/L cyasterone, the egg hatching of diamondback moth was retarded. The total percentage of eggs hatched in treatment was 94.7%, significantly less than that in control, which was 100%. Low toxicity and good antifeeding activity to the 3rd instar larvae were exhibited, which are dependent upon concentration. The growth and development of diamondback moth were affected by cyasterone. It was found that, at concentrations higher than 50 mg/L cyasterone, the larval growth and egg production were inhibited; while promoted at lower concentrations. This dual role of cyasterone might be attributed to its hormonal activity and antifeeding activity, which resulted in poor nutrition. The pupation and eclosion were hindered by treatment of larvae. This insect species was more susceptible to cyasterone than 20-hydroxyecdysone.

New neo-clerodane diterpenes have been isolated from Ajuga reptans, and named ajugatansins, along with the previously reported ajugavensin A and ajugareptansone A. The structures of all the compounds were elucidated by spectroscopic means and comparison with closely related compounds reported in the literature. The new 6 alpha,19-diacetoxy-4 alpha,18-epoxy-1 beta-hydroxy-3 beta-(2-methyl-butanoyloxy)-neo-clerod-13-en-15,16-olide (ajugatansin A1) and 6 alpha,19-diacetoxy-4 alpha,18-epoxy-3 beta-hydroxy-12S-(2-methylbutanoyloxy)-neo-clerod-13-en-15,16-olide (ajugatansin B1) are structurally related to ajugarin I and (11S,13R,16S)-6 alpha, 19-diacetoxy-4 alpha,18:11,16:15,16-triepoxy-3 beta-(2-methylbutanoyloxy)-1 beta-[(E)-2-methyl-2-butenoyloxy]-neo-clerodan-2 beta-ol (ajugatansin D1) to dihydroclerodin.

Hairy roots of Ajuga reptans var. atropurpurea produce clerosterol, 22-dehydroclerosterol, and cholesterol as sterol constituents, and 20-hydroxyecdysone, cyasterone, isocyasterone, and 29-norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously 2H- and 13C-labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24-methylenecholesterol, and 13C2-acetate established the mechanism of the biosynthesis of the two C29-sterols and a newly accumulated codisterol, including the metabolic correlation of C-26 and C-27 methyl groups. In Ajuga hairy roots, 3alpha-, 4alpha-, and 4beta-hydrogens of cholesterol were all retained at their original positions after conversion into 20-hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5beta-H of 20-hydroxyecdysone was found to be C-6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7-dehydrocholesterol 5alpha,6alpha-epoxide. Moreover, 7-dehydrocholesterol, 3beta-hydroxy-5beta-cholest-7-en-6-one (5beta-ketol), and 3beta,14alpha-dihydroxy-5beta-cholest-7-en-6-one (5beta-ketodiol) were converted into 20-hydroxyecdysone. Thus, the pathway cholesterol--7-dehydrocholesterol--7-dehydrocholesterol 5alpha,6alpha-epoxide--5beta-ketol--5beta-k etodiol is proposed for the early stages of 20-hydroxyecdysone biosynthesis. 3beta-Hydroxy-5beta-cholestan-6-one was also incorporated into 20-hydroxyecdysone, suggesting that the introduction of a 7-ene function is not necessarily next to cholesterol. C-25 Hydroxylation during 20-hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.

Raffinose family oligosaccharides (RFOs) are important phloem transport and storage carbohydrates for many plants. Ajuga reptans, a frost-hardy evergreen labiate, ideally combines these two physiological roles and served as our model plant to study the regulation and importance of RFO metabolism. Galactinol is the galactosyl donor for the synthesis of raffinose (RFO-trisaccharide) and stachyose (RFO-tetrasaccharide), and its synthesis by galactinol synthase (GolS) is the first committed step of the RFO biosynthetic pathway. Two cDNAs encoding two distinct GolS were isolated from A. reptans source and sink leaves, designated GolS-1 and GolS-2, respectively. Warm- and cold-grown sink and source leaves were compared, revealing both isoforms to be cold-inducible and GolS-1 to be source leaf-specific; GolS-1 expression correlated positively with GolS activity. Conversely, GolS-2 expression was comparatively much lower and its contribution to the total extractable GolS activity is most probably only minor. These observations, together with results from phloem exudation and leaf shading experiments suggest that GolS-1 is mainly involved in the synthesis of storage RFOs and GolS-2 in the synthesis of transport RFOs. Furthermore, in situ hybridization studies showed GolS-1 to be primarily expressed in the mesophyll, the site of RFO storage, and GolS-2 in the phloem-associated intermediary cells known for their role in RFO phloem loading. A model depicting the spatial compartmentation of the two GolS isoforms is proposed.

Four new iridoid glucosides (I)—(IV) are isolated along with several known substances from the whole plant mentioned in the title.

The phylogenetic position of Wenchengia is considered in the light of new data on its pollen morphology and leaf epidermal anatomy. The pollen is tricolpate, tectate-perforate, suprareticulate and has simple columellae. Leaves are hypostomatic, with mainly diacytic and diallelocytic stomata. Wenchengia appears to be most closely related to Ajuga, Cymaria, or Acrymia. A position close to Scutellaria, Tinnea, and Renschia is only slightly less parsimonious. Although Wenchengia was originally segregated as a monotypic subfamily, assignment to Ajugoideae or Scutellarioideae may be more consistent with a phylogenetic classification of the family.

[3α-2H]-, [4α-2H]- and [4β-2H]-Cholesterols and [3α-2H]- and [5-2H]-3β-hydroxy-5β-cholest-7-en-6-ones were converted with a hairy root culture of Ajuga reptans var. atropurpurea into 20-hydroxyecdysone, in which the deuterium atoms retained their original positions, thus strongly suggesting that 3β-hydroxy-5β-cholest-7-en-6-one is an obligatory intermediate in the biosynthesis of ecdysteroids in the plant.

Feeding of [13C2] acetate to the hairy root culture of Ajuga reptans var. atropurpurea and 13C NMR analysis of the biosynthesized cyasterone and 29-norcyasterone reveal that the lactone carbonyl carbon of these phytoecdysteroids is derived from C-2 of mevalonate, whereas the methyl group on C-25 comes from C-6.

Feeding of deuterium labeled cholesterols including [6- 2 H]cholesterol and [3α,6- 2 H 2 ]-cholesterol to hairy roots of Ajuga reptans var. atropurpurea followed by 2 H-NMR analysis of the biosynthesized 20-hydroxyecdysone revealed that most of the deuterium atom located at C-6 of cholesterol migrated to the C-5 position of 20-hydroxyecdysone.

Feeding of [ 13 C 2 ]acetate to hairy roots of Ajuga reptans var. atropurpurea followed by 13 C-NMR analysis of the biosynthesized cholesterol and 20-hydroxyecdysone indicated that C-25 hydroxylation in 20-hydroxyecdysone biosynthesis proceeds both in retention and inversion mechanisms. Feeding studies of [26- 13 C]- and [27- 13 C]cholesterols established that the ratio of retention and inversion mechanisms is ca. 3:1.

Three new neo -clerodane diterpenoids (14,15-dehydroajugareptansin, 3β-hydroxyajugavensin B and 3α-hydroxyajugamarin F4), have been isolated from aerial parts of Ajuga reptans cv Catlins Giant, together with the known compound, ajugareptansin. The structures were determined by 1 H and 13 C NMR spectroscopy and MS. Insect antifeedant testing of all four compounds revealed that 14,15-dehydroajugareptansin had significant activity against sixth stadium larvae of Spodoptera littoralis .

Two new neo -clerodane derivatives, areptins A and B, have been isolated from the acetone extract of the aerial parts of Ajuga reptans , in addition to the previously known diterpenes, ajugareptansin, ajugorientin, ajugachin A and iridoid glucosides, 8-acetylharpagide and harpagide. The structures of the new compounds were established by chemical and spectroscopic means as: (11 S ,13 R ,16 S )-2 α ,6 α ,19-triacetoxy-3 β -(2-methylbutyryloxy)-4 α , 18:11, 16:15, 16-triepoxy- neo -clerodan-1 β -ol (areptin A); (11 S ,13 S ,16 S ), 6 α -19-diacetoxy-1 β [( E )-2-methyl-2-butenoyloxy]-4 α , 18:11, 16:15, 16-triepoxy- neo -cleroda-14-en-3 β -ol) (areptin B).

A rapid and reliable in vitro bioassay method to test the effects of synthetic and natural compounds on the development of greenhouse whitefly Trialeurodes vaporariorum Westwood (Homoptera Aleyrodidae) is described. Leaf fragments of Nicotiana glauca Graham infested with eggs of whitefly were cultured under aseptic conditions on half-strength Murashige-Skoog medium. The development of the insect in vitro and in vivo were comparable except for a higher mortality rate in the first larval instar. Ecdysteroids from Ajuga reptans L. were applied to leaf fragments by immersion in aqueous solution of the compounds or by addition to the culture medium. A high mortality rate of first larval instars was detected with 29-norsengosterone and ajugalactone treatments. This effect was specially pronounced when the products were incorporated in the medium. A significant increase in mortality was also recorded in the pupal state with a 20-hydroxyecdysone treatment. This effect was never observed before, because all whitefly larvae on A. reptans leaves die in the first larval instar.

Stabilities of a nthocyanins from Concord grapes, red cabbage, tradescantia and ajuga were studied. RP-HPLC analysis was used to characterize individual anthocyanins and spectrophotometric analysis for all pigments. Anthocyanins from tradescantia were much more stable than the other extracts. Such increased stability was attributed to its intrinsic nature

Campesterol (3) and dihydrobrassicasterol (4), typical C28-sterols in higher plants, are biosynthesized from a steroidal 24-ene precursor (desmosterol 1) via 24-methylenecholesterol (2) and 24-methyldesmosterol (5). A typical plant C29-sterol, sitosterol (6), is produced from 24-methylenecholesterol via isofucosterol (7) and 24-ethyldesmosterol (8). The biosynthetic mechanism, focussing stereochemical features, of these side-chain transformations has been studied in detail by feeding regio- and stereoselectively 13C- or 2H-labeled steroidal substrates to cell cultures of higher plants such as Oryza sativa, Catharanthus roseus and Morus alba. These studies allowed to correlate the metabolic origin of C-26 and C-27 of the intermediate sterols. It has been established that the 1st methylation leading to 24-methylenecholesterol from desmosterol involves a Re-face hydrogen migration from C-24 to C-25 based on unambiguous assignment of the isopropyl pro-R-Me and pro-S-Me of 24-methylenecholesterol. The 2nd methylation leading to isofucosterol was revealed to proceed in a trans-mechanism in which addition of the methyl group and elimination of the C-28 hydrogen occur on opposite faces of the original delta 24(28) plane. The double bond isomerization from delta 24(28) to delta 24(25) was found to proceed in a syn-SE2' mechanism with the pro-S-methyl group of isofucosterol becoming the (E)-methyl of 24-ethyldesmosterol. Finally, feeding studies of [E-Me-13C]- and [Z-Me-13C]-24-methyldesmosterols established that an anti-mode of hydrogen addition is operating in the conversion of 24-methyldesmosterol to campesterol and dihydrobrassicasterol. Similar studies established that 24-ethyldesmosterol is converted to sitosterol in an anti-mode of hydrogen addition. In addition, the mechanism of sterol side-chain formation in hairy roots of Ajuga reptans var. atropurpurea is briefly described.

398. 1975), and later Stace (New Fl. Brit. Isles: 673. 1991) considered it the correct name, but these are the only men tions of A. xpseudopyramidalis in any Flora. In attempting to typify this name, we have searched for Schur materai in all possible herbaria (B, BP, BRNU, E, GOET, K, L, LW, MW, NA, P, PC, PH, PL, W, WU), and have not found any original material for lectotypification. We have therefore chosen as neotype a specimen from Le?n, Spain that agrees with the description of A. pseudopyramidalis in being similar to A. pyramidalis, yet has stolons like A. reptans. The specimen is identifiable with A. *hampeana. Thus both A. ^rotundifolia mdA. xpseudopyramidalis apply to the same hybrid formula, i.e., A. pyramidalis x A. reptans, as A. xhampeana, the name that has been consis tently used for this hybrid for over a century, e.g., by Fiori (Nuov. Fl. Ital. 2: 401. 1926), Fournier (Quatre Fl. Fr.: 813. 1961), Ball (I.e.), Gams (in Hegi, 111. Fl. Mitt.-Eur, ed. 2, 5(4): 2548. 1975), Stace (Le, 1975), De Langhe & al. (Nouv. Fl. B lg. Luxemb. N. France, ed. 2: 474. 1978; ed. 4: 560. 1992), Clapham & al. (Fl. Brit. Isl, ed. 3: 419. 1987). Ajuga ^rotundifolia was never treated as a hybrid, only as a species or a subspecies of A. pyramidalis, and A. xpseudopyrami dal s has only once been accepted in recent literature. How ever, according to ICBN Arts. 11.4 & 11.9, since the names A. ^rotundifolia and A. xpseudopyramidalis have priority over A. *hampeana and both apply to the same hybrid for mula (Art. H.4.1) the earliest of these names (A. xpseudopy ramidalis) would have to be adopted for this nothospecies. However, to preserve nomenclatural stability in accordance with Art. 14.2, and avoid the displacement of a well-es tablished and widespread name, the appropriate solution is t propose A. xhampeana for conservation against both A. xpseudopyramidalis and A. ^rotundifolia to avoid a change in nomenclature that will only cause conflict.

6 pages, 2 figures, 4 tables.-- PMID: 15688959 [PubMed].-- Printed version published Feb 2005.

Two novel and three new sterol glycosides were isolated from the MeOH ext. of the aerial parts of Ajuga salicifolia (L.) Schreber. The structures of the compds. were elucidated as (3R,16S,17S,20R,22S,23S,24S,25S)-16,23:16,27:22,25-triepoxy-3-(beta -D-glucopyranosyloxy)coprostigmast-7-en-17-ol (I), (3R,16S,17S,20R,22S,23S,24S,25S)-16,23:16,27:22,25-triepoxy-3-{[beta -D-glucopyranosyl-(1->2)-beta -D-glucopyranosyl]oxy}coprostigmast-7-en-17-ol (II), (3R,16S,17R,20S,22R,24S,25S)-22,25-epoxy-3,27-bis(beta -D-glucopyranosyloxy)coprostigmast-7-en-16-ol (III), (3R,16S,17R,20S,22R,24S,25S)-22,25-epoxy-3-{[beta -D-glucopyranosyl-(1->2)-beta -D- glucopyranosyl]oxy}-27-(beta -D-glucopyranosyloxy)coprostigmast-7-en-16-ol (IV), and (3R,16R,17S,20R,22S,23S,24S,25S)-22,25-epoxy-3-(beta -D-glucopyranosyloxy)coprostigmast-7-ene-16,17,23,27-tetrol 27-acetate (V) by means of 1D and 2D NMR spectroscopy and HR-MALDI mass spectrometry. The novel compds. which consist of three addnl. ring systems at the coprostigmastane skeleton, were named ajugasalicioside A (I) and B (II), and the new compds. C (III), D (IV) and E (V). In cytotoxicity assays (HeLa cells, Jurkat T cells, and peripheral mononuclear blood cells), ajugasaliciosides A-D specifically inhibited the viability and growth of Jurkat T-leukemia cells at concns. below 10 micro M. Ajugasalicioside A (I; IC50 = 6 micro m) and C (III: IC50 = 3 micro M) were the most active compds. I induced cell-cell contact, inhibited Jurkat T cell proliferation. and up-regulated mRNA levels of the cell-cycle regulator cyclin D1, which might be an indication for cell differentiation. Furthermore, I down-regulated the mRNA levels of the NF-kB subunit p65 in a concn.-dependent manner. These effects were not found for ajugasalicioside B (II), which has an addnl. glucose unit, and the onset of cytotoxicity of II (IC50 = 10 micro M) was delayed by 24 h.

Droplet counter-current chromatography (DCCC.) has been used for the preparative scale separation of pure constituents from crude medicinal plant extracts. Xanthones and the bitter principles have been isolated from North-American Gentiana species. From a crude extract of Ajuga pyramidalisL. (Labiatae), several hundred mg of iridoid glycosides were obtained within eight hours. The total volume of mobile phase employed for the elution was less than 150 ml. All the separations were achieved far more readily than by conventional chromatography. In addition, some general rules for solvent system selection are proposed.

The effect of crude methanolic extracts of Ajuga spp. on postembryonic development of different mosquito species The effects of crude methanolic extracts of Ajuga remota and A. reptans (Labiatae) were tested in the laboratory against the developing stages of Aedes aegypti, A. togoi and Culex quinquefasciatus. The extracts caused severe inhibition of development when young fourth instar larvae were exposed until adult emergence. The rate of mortality was correlated with the extract's concentrations and the duration of exposure. The different mosquito species showed almost the same sensitivity to an extract of A. remota. Extracts of A. reptans (LC90-values from 98 to 128 ppm when tested against A. togoi) were more efficient than those of A. remota (LC90-values from 384 to 700 ppm). Among the different plant parts, effectiveness of the derived extracts was roots > leaves > shoots > flowers. Treatment of developing stages caused a reduced quality of the emerged female adults. The results point out that the ecdysteroid regulation of the mosquitoes is influenced by the effective compounds of the extracts. Zusammenfassung Die Wirkung methanolischer Rohextrakte aus den Labiaten Ajuga remota und A. reptans wurde gegenuber Entwicklungsstadien von A. aegypti, A. togoi und C. quinquefasciatus untersucht. Die Extrakte bewirkten eine ausgepragte Entwicklungshemmung bei einer Exposition junger Viertlar-ven bis zur Imaginalhautung. Die resultierende Mortalitat war positiv korreliert mit der Extrakt-konzentration und der Expositionsdauer. Die einzelnen Stechmuckenarten reagierten mit einer nahezu gleichen Empfindlichkeit auf einen A. remota-Extrakt. Extrakte aus A. reptans waren mit LC90-Werten zwischen 98 und 128 ppm gegenuber A. togoi wirksamer als diejenigen aus A. remota mit LC90-Werten zwischen 384 und 700 ppm. Im Vergleich einzelner Pflanzenorgane nahm die Extraktwirksamkeit in folgender Reihenfolge ab: Wurzeln > Blatter > Stengel > Bluten. Eine Extrakt-Behandlung in den Jugendstadien rief bei den weiblichen Imagines eine Qualitatsminde-rung als Folgeschaden hervor. Die beobachteten Effekte lassen eine Beeinflussung des Ecdysteroidhaushaltes durch die Pflanzeninhaltsstoffe vermuten.

Droplet counter-current chromatography (DCCC.) has been used for the preparative scale separation of pure constituents from crude medicinal plant extracts. Xanthones and the bitter principles have been isolated from North-American Gentiana species. From a crude extract of Ajuga pyramidalisL. (Labiatae), several hundred mg of iridoid glycosides were obtained within eight hours. The total volume of mobile phase employed for the elution was less than 150 ml. All the separations were achieved far more readily than by conventional chromatography. In addition, some general rules for solvent system selection are proposed.