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Aromatase inhibitory activities of standishinal and the diterpenoids from the bark of Thuja standishii.

The absolute stereostructure of a novel skeletal diterpene, standishinal (1), from the bark of Thuja standishii was confirmed by X-ray crystallographic analyses of 1 and its p-bromobenzoate derivative. Aromatase inhibitory activities of standishinal, eight known diterpenes, totarol, 12-methoxyabieta-8,11,13-trien-11-ol, 12-hydroxy-6,7-seco-abieta-8,11,13-triene-6,7-dial, trans-communic acid, labda-8(17),13-dien-12 R,15-olid-19-oic acid, 12 S-hydroxylabda-8(17),13(16),14-trien-19-oic acid, 13-oxo-15,16-dinorlabda-8(17),11 E-dien-19-oic acid and 14-oxo-15-norlabda-8 (17),12 E-dien-19-oic acid from the plant, and four synthetic analogs were evaluated using a recombinant human aromatase. Among them, standishinal and its diacetate derivative had significant inhibitory activities.

Abietanes↗

Potential antitumor promoting diterpenoids from the stem bark of Thuja standishii.

Six diterpenes, including one new natural product, were isolated from a CHCl 3 extract of the stem bark of Thuja standishii. The new compound has been characterized as 15-oxolabda-8(17),13 Z-dien-19-oic acid. The known compounds were identified as ferruginol (2), sugiol (3), isocupressic acid (4), sandaracopimaric acid (5) and 15-oxolabda-8(17),13 E-dien-19-oic acid (6). Compounds 2-5 and the derivatives 4a and 4b were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). Compounds 2, 3, 4 and 5 showed strong inhibitory effect on EBV-EA induction (100 % inhibition at 1000 mol ratio/TPA).

Antineoplastic Agents, Phytogenic↗

Response factor considerations for the quantitative analysis of western redcedar (Thuja plicata) foliar monoterpenes.

A method is described for quantitative analysis of monoterpenes in western redcedar (Thuja plicata) foliage by gas chromatography with flame ionization detection. Response factors for monoterpenes identified in redcedar are evaluated to determine similarities among monoterpene responses. Evaluation demonstrates that redcedar monoterpenes yield detector responses that fall into two groups. One monoterpene from each group is used as a standard for quantitative analysis. Redcedar monoterpenes are quantitated by comparing analyte response with the response factor of one of the standards in single-point calibrations. Homogenized foliage samples are extracted with ethyl acetate and the extracts passed through a solid phase extraction column of graphitized carbon to remove plant pigments. Method bias and repeatability are evaluated by fortifying foliage samples with (1S)-(+)-carvone and (1S)-(+)-2-carene and subjecting the samples to the extraction and analysis procedures. Detection limits are also assessed from fortified samples. Excellent recovery (> 95.0%) and precision (< 5%) are obtained from the analysis of 2-carene from fortified samples. Carvone recovery is approximately 80% with excellent precision (< 4%). The method limits of detection obtained from 2-carene and carvone fortified samples are 4.7 and 13.5 microg/g, respectively.

Monoterpenes↗

Linking deer browsing and terpene production among genetic identities in Chamaecyparis nootkatensis and Thuja plicata (Cupressaceae).

To investigate whether differential herbivore browsing reflects genetic variation in plant defense expression, variation in needle terpenes and damage caused by black-tailed deer (Odocoileus hemionus) was analyzed on yellow-cedar (Chamaecyparis nootkatensis) and western redcedar (Thuja plicata). In a 100-genet yellow-cedar population, three genets that were heavily browsed and had extremely low levels of monoterpenes (0-0.36% dry matter), sesquiterpenes, and diterpenes were compared to unbrowsed genets (0.85-3.83% monoterpenes in dry matter). These differences were maintained in individuals protected from browsing, suggesting genetically based variation in constitutive terpene production. In western redcedar, heavily browsed trees had significantly lower total monoterpene concentrations (1.69% dry matter) than lightly browsed trees (3.32% dry matter). One heavily browsed tree expressed no monoterpenes. No differences were found for diterpenes. In both species, the genotypes with extremely low monoterpene concentrations came from the same open-pollinated families.

Animals↗

Somatic mutations at microsatellite loci in western Redcedar (Thuja plicata: Cupressaceae).

A per-generation somatic mutation rate for microsatellites was estimated in western redcedar (Thuja plicata, Donn ex D. Don.: Cupressaceae). A total of 80 trees representative of the average size and age of reproductive trees were sampled in four natural populations in southwestern British Columbia. Samples of bulked haploid megagametophytes were collected from two or three positions on each tree, assuming that the collections were far enough apart that the same mutant sector was not sampled twice. All samples were genotyped at eight microsatellite loci. A single mutation corresponding to a stepwise increase in one dinucleotide repeat was detected. The estimated mutation rate for microsatellites was 6.3 x 10(-4) mutations per locus per generation (or 3.1 x 10(-4) per allele per generation), with a 95% confidence interval of 3.0 x 10(-5) to 4.0 x 10(-3) mutations per locus. Somatic mutations can contribute to a greater mutational load in trees, as compared to shorter lived plants, and genotypic mosaics within an individual have important implications for plant defense strategies and plant evolution.

Alleles↗

Responses of transpiration and photosynthesis to reversible changes in photosynthetic foliage area in western red cedar (Thuja plicata) seedlings.

Experiments were conducted on 1-year-old western red cedar (Thuja plicata Donn.) seedlings to determine the response of illuminated foliage to reversible changes in total photosynthetic foliage area (L(A)). Reductions in L(A) were brought about by either shading the lower foliage or by reducing the ambient CO2 concentration (c(a)) of the air surrounding the lower part of the seedling. In the latter case, the vapor pressure was also changed so that transpiration rates (E) could be manipulated independently of photosynthetic rates (A). We hypothesized that following such treatments, short-term compensatory changes would occur in stomatal conductance (g(s)) and A of the remaining foliage. These changes would occur in response to hydraulic signals generated by changes in the water potential gradient rather than changes in the distribution of sources and sinks of carbon within the seedling. When a portion of the foliage was shaded, there was an immediate reduction in whole-seedling E and a concomitant increase in g(s), A and E in the remaining illuminated foliage. However, the intercellular CO2 concentration did not change. These compensatory effects were fully reversed after the shade was removed. When the lower foliage A was reduced to < 0 micromol m-2 s-1, by shading or lowering c(a), and E was either unchanged or increased (by adjusting the vapor pressure deficit), there was no significant increase in g(s) and A in the remaining foliage. We conclude that compensatory responses in illuminated foliage occur only when reductions in L(A) are accompanied by a reduction in whole-plant E. The relationship between the reduction in whole-seedling E and the increase in A is highly linear (r2 = 0.68) and confirms our hypothesis of the strong regulation of g(s) by hydraulic signals generated within the seedling. We suggest that the mechanism of the compensatory effects is a combination of both increased CO2 supply, resulting from increased g(s), and a response of the rate of carboxylation, possibly related to the activity of Rubisco.

Carbon Dioxide↗

Leaf longevity of western red cedar (Thuja plicata) increases with depth in the canopy.

Leaf longevity determines the annual cost of replacing foliage biomass and influences water and nutrient budgets. Longevity is readily estimated in most evergreen species by annual bud scars along the shoot. However, some species with indeterminate growth do not show these annual markers, making estimation of longevity difficult. One of these species is the widespread and economically valuable western red cedar (Thuja plicata J. Donn ex D. Don), for which no dependable estimates of leaf longevity exist. In this study, we estimated leaf longevity for western red cedar by counting growth rings in shoots at the point of leaf abscission. Estimates were obtained on 26 dominant or codominant trees growing in natural stands in a montane forest in northern Idaho, USA. Leaf longevity averaged 8.9 (SE = 0.2) years, but it strongly increased with depth in the canopy (0.3 year m(-1); mean crown depth was 15 m), increasing from a mean of 6.8 years in the upper third of the canopy to 10.6 years in the lower third. The increase in longevity with depth in the crown is consistent with many reports showing that longevity increases in resource-limiting environments. Longevity did not vary significantly with altitude or solar insolation in these montane forests. Among stand-level variables, longevity was correlated only with leaf area index: it increased slightly in stands with high leaf area indices. This approach to longevity estimation may be useful for any species that produces annual rings but no obvious bud scars, including many Cupressaceae species.

Altitude↗

Volatile constituents of the fruit and leaf oils of Thuja orientalis L. grown in Iran.

The composition of the hydrodistilled essential oils from the fruits and leaves of Thuja orientalis L. grown in Iran was analyzed by GC/MS. Nineteen and twenty-eight compounds have been identified in the volatile oils of the fruit and leaf, respectively. While the fruit oil contained alpha-pinene (52.4%), delta-3-carene (14.2%), alpha-cedrol (6.5%) and beta-phellandrene (5.1%), the leaf oil contained alpha-pinene (21.9%), alpha-cedrol (20.3%), delta-3-carene (10.5%) and limonen (7.2%) as the main components.

Gas Chromatography-Mass Spectrometry↗

Ecological studies on the snow scale insect, Lineaspis striata (Newstead) (Homoptera: Diaspididae) on Thuja orientalis in Egypt.

The snow scale insect, Lineaspis striata (Newstead) was recorded as an important insect pest of Thuja orientalis in Alexandria Governorate, Egypt. The present study revealed that the total individuals were in high numbers, (143.7 +/- 43; 141.8 +/- 20.7 and 149.0 +/- 48.5) during May, June and January, respectively. The fluctuating numbers of the immature stages have two peaks during June and November (6.9 +/- 0.7) and (20.5 +/- 5.0), respectively. The numbers of adult males reached the maximum (73.9 +/- 7.5) in October, while the females reached the maximum (88.7 +/- 2.2) in January. The insect has two annual generations. It is usually parasitized with Encarsia citrina (Aphelinidae), with maximum parasitization rate of 17.8 +/- 2.8 during August.

Animals↗

Mitogenic activity of high molecular polysaccharide fractions isolated from the cuppressaceae Thuja occidentalis L. enhanced cytokine-production by thyapolysaccharide, g-fraction (TPSg).

Thuja polysaccharide g fraction (TPSg) was shown to be an inducer of the CD4+ fraction of the human peripheral blood T-cell subset (1,2). Furthermore, it could be demonstrated that TPSg is a potent inhibitor of the expression of HIV-1-specific antigens and of the HIV-1-specific reverse transcriptase (3). This report deals with the cytokine pattern induced by TPSg in human peripheral blood lymphocyte (PBL) and purified monocyte/macrophage cultures. In addition, a further characterization of the CD4+ T-cell fraction stimulated by TPSg was performed by FACS analysis. TPSg is induces IL-1 beta, IL-2, IL-3, IL-6, gamma-IFN, G-CSF, GM-CSF, and TNF-beta production in PBL cultures; and IL-1 beta and IL-6 in monocyte/macrophage cultures. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that no IL-4 was produced by PBL cultures under TPSg influence.

Cytokines↗

Larvicidal potential of Nerium indicum and Thuja oriertelis extracts against malaria and Japanese encephalitis vector.

Ethanolic and acetone extracts of Nerium indicum and Thuja orientelis have been studied against III instar larvae of Anopheles stephensi and Culex quinquefasciatus. Ethanolic extract of N. indicum is found more effective than its acetone extract against anopheline larvae with LC50 values of 185.99 and 148.05 ppm for former and 229.28 and 149.43 ppm for the later after 24 and 48 hrs of exposure. The acetone extract with LC50 values of 209.00 and 155.97 ppm is more effective in case of culicine larvae than its ethanolic extract with LC50 494.07 and 194.49 ppm after 24 and 48 hours of treatment. Ethanolic extract of T. orientelis is more effective against both the larval species with LC50 values of 13.10 and 9.02 ppm after 24 and 48 hours for anopheline and 22.74 and 16.72 ppm against culicine larvae. The acetone extract showed LC50 values of 200.87 and 127.53 ppm against anopheline and 69.03 and 51.14 ppm against culicine larvae. Thus ethanolic extract of T. orientelis is an ideal potential larvicide for both types of mosquito larvae.

Acetone↗

Activation of complement by plicatic acid, the chemical compound responsible for asthma due to western red cedar (Thuja plicata).

Plicatic acid, a low-molecular-weight compound responsible for western red cedar (Thuja plicata) asthma was tested for its ability to activate complement and to generate chemotactic activity from pooled normal human serum (NHS). Dose-dependent complement consumption was found as determined by hemolytic assay (CH50). Activation of complement by plicatic acid was also confirmed by the demonstration of conversion of C3 to C3b on immunoelectrophoresis. This activation was completely prevented by pretreating the serum with either edetate (EDTA) or ethylene glycol tetraacetic acid (EGTA), suggesting that complement was activated via the classical pathway. No conversion of factor B was seen in any of the samples. Leukocyte chemotactic activity was also generated when serum was incubated with plicatic acid. The consumption of C3 and CH50 was unimpaired in two samples of serum from patients with severe, untreated hypogammaglobulinemia and thus appears to be immunoglobulin independent. These observations suggest that plicatic acid could activate complement in vivo, thereby inducing an inflammatory response in the airways and contributing to the higher prevalence of industrial chronic bronchitis in exposed subjects. The pathogenetic role of complement activation in red cedar asthma has yet to elucidated.

Animals↗

Cancer chemopreventive agents, labdane diterpenoids from the stem bark of Thuja standishii (Gord.) Carr.

Seven labdane-type diterpenoids from the stem bark of Thuja standishii (Gord.) Carr. (Cupressaceae) and their analogues showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among these compounds, 15,16-bisnor-13-oxolabda-8(17), 11E-dien-19-oic acid was revealed to have the strongest inhibitory effect on the EBV-EA activation, being stronger than that of beta-carotene which has been intensively studied in cancer prevention using animal models. 15,16-bisnor-13-Oxolabda-8(17), 11E-dien-19-oic acid was also found to exhibit the excellent anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.

9,10-Dimethyl-1,2-benzanthracene↗

Anti-tumor promoting diterpenes from the stem bark of Thuja standishii (Cupressaceae).

Three new labdane-type diterpenoids, labda-8(17),13-dien-15,12R-olid-19-oic acid (1), 12S-hydroxylabda-8(17),13(16),14-trien-19-oic acid (2) and 13-ethoxylabda-8(17),11,14-trien-19-oic acid (3), along with known diterpenoids, trans-communic acid (4), totarol (5), 12-methoxyabieta-8,11,13-trien-11-ol (6), and 7 alpha,8 alpha-epoxy-6 alpha-hydroxyabieta-9(11),13-dien-12-one (7) were isolated from the stem bark of Thuja standishii. The structures of 1--3 were established by spectroscopic methods and chemical conversion. These compounds together with standishinal (8), 12-hydroxy-6,7-seco-abieta-8,11,13-trien-6,7-dial (9) and 6 alpha-hydroxysugiol (10) were tested for their inhibitory effects on Epstein--Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. Compound 10 showed strong inhibitory effect on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA), and compounds 2 and 6 showed moderate inhibitory effects on EBV-EA induction. In addition, 15-oxolabda-8(17),11Z,13E-trien-19-oic acid (11) was found to exhibit the anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.

9,10-Dimethyl-1,2-benzanthracene↗

Labdane-type diterpenes and a nordrimane-type sesquiterpene from the stem bark of Thuja standishii.

Two new labdane-type diterpene aldehydes, 15-oxolabda-8(17),11(Z), 13(E)-trien-19-oic acid (1) and 15-oxolabda-8(17),11(Z), 13(Z)-trien-19-oic acid (2), and a new nordrimane-type sesquiterpene, 12-oxo-11-nordrim-8-en-14-oic acid (3), along with a known diterpene, 15-nor-14-oxolabda-8(17),12(E)-dien-19-oic acid (4), were isolated from the stem bark of Thuja standishii. The structures of 1-3 were established by spectroscopic methods.

Cycadopsida↗

Bioactive constituents of Thuja occidentalis.

An ethyl acetate-soluble extract of the combined leaves and twigs of Thuja occidentalis was found to inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) in cultured mouse epidermal ME 308 cells. Bioassay-guided fractionation of this extract led to the isolation of six active constituents (1-6), namely, (+)-7-oxo-13-epi-pimara-14,15-dien-18-oic acid (1), (+)-7-oxo-13-epi-pimara-8,15-dien-18-oic acid (2), (+)-isopimaric acid (3), (1S,2S,3R)-(+)-isopicrodeoxypodophyllotoxin (4), (-)-deoxypodophyllotoxin (5), and (-)-deoxypodorhizone (6). Compounds 1 and 4 are new natural products, and their structures and stereochemistry were determined using spectroscopic methods. Compounds 1-6 were evaluated for inhibition of the transformation of murine epidermal JB6 cells, inhibition of ornithine decarboxylase induction with murine epidermal ME 308 cells, and cytotoxic activity against KB cells.

Animals↗

Effects of an orally applied aqueous-ethanolic extract of a mixture of Thujae occidentalis herba, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix on antibody response against sheep red blood cells in mice.

The influence of the oral administration of an aqueous-ethanolic extract of a mixture of Thujae occidentalis herba, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix, on the immune response in mice was investigated. The data show that the extract significantly enhances the antibody response to sheep red blood cells (SRBC), induces an increase in the numbers of splenic plaque forming cells (PFC) and an increase in the titer of specific antibodies in the sera of treated animals. The long-term application of the extract over several months also stimulated the PFC-response without affecting spleen weight, total cell yield per spleen or white blood cell count in mice.

Administration, Oral↗

Recombinant pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) catalyze opposite enantiospecific conversions.

Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with beta-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.

Amino Acid Sequence↗