CONTEXT:Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage. OBJECTIVE:This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV). MATERIALS AND METHODS:Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1 WT. RESULTS:KPB-100 and KPB-200 inhibited HHV-2 at IC50 values of 2.5 and 2.9 μg/mL, respectively, and VACV at IC50 values of 3.1 and 7.4 μg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC50 values of 1.63 and 13.2 μg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20 μg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC50 value of 4.5 μg/mL in CPE inhibition assay and HHV-1 at an IC90 of 3.0 μg/mL in VYR assay. DISCUSSION AND CONCLUSION:Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.

Medicinal plants are important components in the primary health care of villagers in Guatemala. However, an area often overlooked is the effect of medicinal plants on oral hygiene. Acetone and methanol extracts from 63 medicinal plant species from 31 families were bioassayed against breast, cervical, skin and tongue cancers, and the following microorganisms: Staphylococcus aureus, Escherichia coli, Streptococcus mutans, Lactobacillus acidophilus and Candida albicans. Half-maximum inhibitory concentrations (IC50) and half-maximum cytotoxicity concentrations (CC50) were determined against cancerous and non-cancerous cell lines, respectively. Minimum inhibitory concentrations (MIC) were determined against microbes. Based on levels of inhibition by extracts, IC50 values, CC50 values, and MIC values, seven species (Bursera simaruba Sarg., Burseraceae; Eriobotrya japonica (Thumb.) Lindl., Rosaceae; Litsea guatemalensis Mez, Lauraceae; Mirabilis jalapa L., Nyctaginaceae; Pithecellobium dulce (Roxb.) Benth., Fabaceae); Rubus villosus Thunb., Rosaceae; Thevetia peruviana K. Schum., Apocynaceae) were recommended for additional investigation. With regard to oral hygiene four species (Achillea millefolium L., Asteraceae; Crotalaria longirostrata Hook. and Arn., Fabaceae; P. dulce; Spondias purpurea L., Anacardiaceae) may merit further fractionation and testing against oral diseases. Key words: Anticancer, antimicrobial, IC50, CC50, MIC, oral hygiene, Guatemala.

Context: Essential oils are prevalent in many medicinal plants used for oral hygiene and treatment of diseases.Objective: Medicinal plant species were extracted to determine the essential oil content. Those producing sufficient oil were screened for activity against Staphylococcus aureus, Escherichia coli, Streptococcus mutans, Lactobacillus acidophilus, and Candida albicans.Materials and methods: Plant samples were collected, frozen, and essential oils were extracted by steam distillation. Minimum inhibitory concentrations (MIC) were determined using a tube dilution assay for those species yielding sufficient oil.Results: Fifty-nine of the 141 plant species produced sufficient oil for collection and 12 species not previously reported to produce essential oils were identified. Essential oil extracts from 32 species exhibited activity against one or more microbes. Oils from eight species were highly inhibitory to S. mutans, four species were highly inhibitory to C. albicans, and 19 species yielded MIC values less than the reference drugs.Discussion: Results suggest that 11 species were highly inhibitory to the microbes tested and merit further investigation. Oils from Cinnamomum zeylanicum Blume (Lauraceae), Citrus aurantiifolia (Christm.) Swingle (Rutaceae), Lippia graveolens Kunth (Verbenaceae), and Origanum vulgare L. (Lamiaceae) yielded highly significant or moderate activity against all microbes and have potential as antimicrobial agents.Conclusion: Teas prepared by decoction or infusion are known methods for extracting essential oils. Oils from 11 species were highly active against the microbes tested and merit investigation as to their potential for addressing health-related issues and in oral hygiene.

Medicinal plants are used by rural Guatemalan villagers to treat a variety of ailments, and a better understanding of their effectiveness against common diseases is warranted. Acetone and methanol extracts of 73 medicinal plant species from 44 families were bio-assayed against breast, cervical, skin, and tongue cancers, and Staphylococcus aureus, Escherichia coli, Streptococcus mutans, Lactobacillus acidophilus, and Candida albicans. Half-maximum inhibitory concentrations (IC50) and half-maximum cytotoxicity concentrations (CC50) were determined against cancerous and non-cancerous cell lines, respectively. Minimum inhibitory concentrations (MIC) were determined for active extracts. Bursera simaruba (L.) Sarg. (Burseraceae), Byrsonima crassifolia (L.) Kunth (Malpighiaceae), Guazuma ulmifolia Lam. (Malvaceae), and Quercus acatenangensis Trel. (Fagaceae) were inhibitory to one or more cancer cell lines and yielded promising IC50 and CC50 values. Eucalyptus globulus Labill. (Myrtaceae), Liquidambar styraciflua L. (Altingiaceae), Pelargonium hortorum L.H. Bailey (Geraniaceae), and Psidium guajava L. (Myrtaceae) were inhibitory to one or more microbes and had MIC’s of 250 µg/ml or less against one or more microbes. The activity of these species against cancer and pathogenic microbes indicates that they are valuable resources that should be conserved and considered for future research. Key words: Anticancer, antimicrobial, IC50, CC50, minimum inhibitory concentrations (MIC), Guatemala.

In the early 1960s recognition of the adverse effects of environmental contamination due to industrial, pesticide, and agricultural pollution led to the emergence of the field of ecotoxicology (Ramade, 1992). Today, marine estuary and inshore ecosystems continue to be negatively impacted by environmental contamination (Short & Wyllie-Echeverria 1996; Orth et al., 2006; Osborn & Datta, 2006). In order to reduce these negative impacts, biosurveillance programs are needed to monitor environmental conditions so that changes in ecosystem processes, structure, and the physiological condition of species can be assessed (Blandin, 1986; Tett et al., 2007). An important characteristic of these programs is that indicator species must be capable of rapidly detecting significant changes in the ecosystem so that the cause of deterioration can be addressed early (e.g. Hemminga & Duarte, 2000).

CONTEXT:Thirty-one medicinal plant species from Hawaii, Morocco, and the Sonoran Desert, USA have been shown in past studies to be highly inhibitory to pathogenic bacteria, fungi, and certain cancer cell lines. However, none were tested for antiviral activity.OBJECTIVE:Acetone and methanol extracts from these species were bio-assayed for antiviral activity against herpes simplex virus types 1 and 2, and for cytotoxicity to the Vero C1008 cell line.MATERIALS AND METHODS:Extracts from these species were tested in vitro for antiviral activity using an immunoperoxidase mini-plaque reduction assay to detect viral structural protein synthesis. A 50% inhibitory concentration (IC(50)) was computed. Sulforhodamine B and neutral red assays were used to qualitatively and quantitatively assess the cytotoxicity of extracts to C1008 cells, and to compute a 50% cytotoxic concentration (CC(50)) using a dose response curve.RESULTS:Eight of the 31 plant species assayed showed significant antiviral activity against HSV 1 and HSV 2 viruses. The acetone extract of Kalanchoe pinnata Pers. (Crassulaceae) produced an IC(50) of 0.025 mg/mL and a CC(50) of 1.25 mg/mL yielding a therapeutic index of 50. Additionally, this extract reduced plaque numbers to zero or near zero at a concentration of 0.1 mg/mL when added 30 min before or 30 min after virus infection.DISCUSSION AND CONCLUSION:The mechanism of inhibition against HSV 1 and HSV 2 viruses is now being investigated, along with fractionation of the acetone extract in search of the active compound or compounds.

The lack of solubility and the high degree of volatility of essential oils present significant problems to determining the biological effects of these oils. The activity of 10 essential oils and 4 essential oil blends against a Gram-positive bacterium, Staphylococcus aureus , and a yeast, Candida albicans , was compared using a dilution assay and two diffusion methods. The tube dilution assay, using a 0.2% agar solution to provide a stable homogeneous dispersion of oils, was used to measure minimum inhibitory concentrations (MICs). The relative merits of using p-iodonitro tetrazolium dye (INT) or optical density (OD) in measuring MIC of essential oils also were evaluated. All 14 oils were active against both microorganisms. Thyme, mountain savory, and Turkish oregano were the most active oils against S. aureus with MICs of 0.31-0.42 mu l/ml. Thyme, Turkish oregano, and mountain savory also were the most active against C. albicans with MICs of 0.31-0.42 mu l/ml. The MIC values as determined by INT or OD methods are strongly correlated for both microorganisms, and both give an accurate estimation of MIC. In the disk diffusion assay, thyme, Turkish oregano, Melissa, mountain savory, and the Exodus II (E2) blend were most active against S. aureus . In the hole-plate assay, the same oils were active against S. aureus except for Melissa. For C. albicans , thyme, Melissa, mountain savory, Turkish oregano, rosewood, E2 blend, and the T1 blend were most active in the disk diffusion assay. The same oils were active against C. albicans in the hole-plate diffusion assay except for the T1 blend. Of the methods tested, the tube dilution assay best addresses the solubility and volatility concerns of essential oils. We favor the determination of MIC using INT because of several eliminated steps. Although there were differences in the way in which some essential oils responded in the two diffusion methods, the correlations between the disk and hole-plate diffusion methods were high and yielded comparable results. However, results from the dilution assay were weakly correlated with agar diffusion results. Diffusion assays are useful as a qualitative assessment of biological activity of essential oils but are not appropriate in assessing quantitative effects.

This study evaluates the potential benefits of using ecology-based theories of plant chemical defense when screening plants for pharmaceutical activity. Extracts from 63 Sonoran Desert plant species representing 26 families were screened in microtiter/optical density–based assays against a cervix epithelial carcinoma cell line (HeLa) and mouse fibroblast cells (3T3). In HeLa cytotoxicity assays, 34 of 63 species (54%) showed significant inhibitory effects. Nine of these active extracts were significantly less toxic to a noncancerous mouse fibroblast cell line (3T3) and, thus, showed potentially cancer specific activity. Using the plant apparency/predictability hypothesis as a theoretical basis, this study used plant growth habit (e.g., annual, herbaceous perennial, woody perennial, evergreen) as an indicator of plant predictability, and hence likelihood of having well-established chemical defenses. This approach led to predictions that may be useful in locating plant species that produce biologically active secondary metabolites. As predicted, extracts from evergreen species exhibited the highest activity with 41.7% of extracts showing inhibition, followed by woody perennials (37.3%), herbaceous perennials (35.7%), and annuals (23.3%). Average percent inhibition was significantly higher in the evergreen and woody perennial groups as compared to other growth forms. These results support predictions made by the plant predictability hypothesis and illustrate the potential value of incorporating ecological theories of plant chemical defenses in plant screening efforts.

The feedbacks between plant and soil processes play an important role in driving forest succession. One poorly understood feedback mechanism is the interaction between plant secondary chemicals and soil microbes. In the Alaskan taiga, changes in nutrient cycling caused by balsam poplar (Populus balsamifera) secondary chemicals may affect the transition from alder (Alnus tenuifolia) to balsam poplar on river floodplains. We examined the effects of four poplar condensed tannin fractions on N cycling in alder and poplar soils. Tannins were added to forest floor samples from both poplar and alder sites. Samples were incubated for 1 month in the laboratory with soil respiration rates measured over the course of the incubation. At the end of the incubation we measured both net and gross nitrogen mineralization and nitrification, microbial biomass C and N, and the activity of various exoenzymes. In all soils, tannin additions reduced N availability, however, the mechanisms differed depending on the molecular weight of the tannin and the native soil microbial community. Low molecular weight tannin fractions served as a labile C source in poplar Oi, poplar Oe, and alder Oe horizons but were toxic to microbes in alder Oi. High molecular weight tannin fractions appeared to act primarily by binding extracellular substrates and thus limiting C and N mineralization, with the strongest effects observed in the alder soils.

The vegetation mosaic of the Alaskan taiga is produced by patterns of disturbance coupled to well-defined successional patterns. In primary succession on river floodplains, one of the critical transitions in succession is that from thinleaf alder (Alnus tenuifolia) to balsam poplar (Populus balsamifera). This is the shift from a N2-fixing shrub to a deciduous tree. Through this transition there are major changes in N cycling including a decrease in N2-fixation, mineralization, and nitrification. Most models of plant effects on soil processes assume that these changes are caused by shifts in litter quality and C/N ratio. This paper reviews several studies examining the effects of balsam poplar secondary chemicals on soil nutrient cycling. Balsam poplar tannins inhibited both N2-fixation in alder, and decomposition and N-mineralization in alder soils. Other poplar compounds, including low-molecular-weight phenolics, were microbial substrates and increased microbial growth and immobilization, thereby reducing net soil N availability. Thus, substantial changes in soil N cycling through succession appear to have been mediated by balsam poplar secondary chemicals.

Current literature indicates that the decrease in willow stature and productivity on the winter range in Yellowstone National Park is due to a complex interaction among abiotic and biotic factors. Lack of change in soluble carbohydrates, total phenolics and tannins when willows were mechanically clipped in exclosures in Yellowstone National Park show that willows were not able to respond physiologically to these treatments. These data are supportive of the premise that the willow decline is due to unfavorable growing conditions on the northern winter range. Willow physiology, growth, and recruitment are impacted adversely by ungulate browsing, but experimental manipulations within exclosures indicate that browsing may not be the only important factor. The trend toward aridity, lowered water tables, reduced stream flow, lack of flooding, and absence of beaver are suggested to be important factors in reduced willow production, reproduction, and lower levels of primary and defensive chemicals.

Monoterpenes, sesquiterpenes, and phenolic and flavonoid glycosides typical of the current year's foliage of Douglas fir (Pseudotsuga menziesii) were bioassayed using agar diets to determine the effect of these compounds on natural and colony populations of western spruce budworm (Choristoneura occidentalis). Several terpenes adversely affected budworm larval growth, supporting previous field and laboratory studies. However, larvae collected from a population in southeastern Idaho were significantly more tolerant of the monoterpenes than those from a Montana population. For the Montana population, agar diet studies showed that camphene, myrcene, terpinolene, bornyl acetate, and tricyclene adversely affected larval growth rate and pupal weight. For the Idaho population, agar diet studies showed that only terpinolene and bornyl acetate adversely affected larval growth and pupal weights. For both populations bornyl acetate was the most toxic, and this compound in addition to other monoterpenes represent defensive mechanisms in the current year's growth of Douglas fir. Sesquiterpene and phenolic-flavonoid glycoside fractions extracted from Douglas fir current year's growth also were bioassayed using agar diets. The sesquiterpene fraction showed a significant negative effect on budworm larval growth, but phenolics and flavonoid glycosides had no effect. Sesquiterpenes, in combination with tricyclene, camphene, myrcene, limonene, terpinolene, and the acetate fraction appear to represent an effective mixture of defensive compounds against the budworm.

The C-13 and H-1 resonances of the alkaloids, obscurinervine (1) and obscurinervidine (2), are assigned using high-field NMR experiments and computerized data analysis procedures. A 2D INADEQUATE analysis of 26 mg of 2 was performed with a high-sensitivity carbon probe and the data interpreted using the spectral analysis program, CCBOND, to provide unambiguous C-13 assignments. Although all signals are visually undetectable, CCBOND determined 20 of the 22 carbon-carbon bonds present. Corresponding H-1 chemical shift assignments are made from HETCOR data. Proton-proton couplings are determined from DQF-COSY data using the new analysis program, HHCORR. Since HHCORR models signals as AB spin systems, the determined coupling constants are fairly independent of higher order effects, linewidths and digital resolution. Also a significant sensitivity improvement over visual interpretation of DQF-COSY data is observed. The obtained coupling constants are interpreted through the Karplus relationship to provide conformational details. These novel software analysis techniques allow accurate and more routine analysis of INADEQUATE and DQF-COSY data providing nonspecialists access to these powerful experiments. Absolute stereochemistry of 2 is determined by a comparison with the ORD curve of (-)-O-methylaspidolimine. Stereospecific H-1 assignments are obtained from proton-proton couplings and molecular mechanics simulations. The C-13 and H-1 chemical shift assignments for the related alkaloid, obscurinervine 1, are determined from CCBOND processed 2D INADEQUATE, HHCORR processed DQF-COSY, and HETCOR data. Differences in the rigidity of 1 and 2 in dimethyl sulfoxide (DMSO) are quantified by variable-temperature H-1 NMR spectroscopy. Complete conformations of all ring systems are obtained from molecular mechanics using dihedral angles derived from proton-proton couplings as a check on the quality of the model. All conformational conclusions are independently supported by the X-ray structure of 1.

The transition from alder (Alnus tenuifolia) to balsam poplar (Populus balsamifera) is a critical turning point in primary succession on river floodplains in interior Alaska. Associated with the change in plant species are large changes in N cycling. N-fixation and nitrification decrease and the system becomes N-limited, with NH4+ dominating the inorganic N pool. Balsam poplar leaves contain large quantities of tannins and low molecular weight phenolic compounds. We evaluated the effect of these compounds on microbial respiration and N cycling in laboratory assays on soils from an alder-dominated site. Plant compounds were purified and applied to silica gel as an inert carrier. Both tannins and phenolics caused net N-immobilization over a 30-day assay. However, tannins inhibited respiration while phenolics stimulated it. There were no specific effects on nitrification. Thus, tannins acted as a general microbial inhibitor, while phenolics acted as a growth substrate. By inhibiting mineralization while stimulating immobilization, poplar secondary compounds may reduce soil N-availability during the transition betwen alder and poplar stages in succession. Keywords: respiration, mineralization, tannins, secondary chemicals, succession, plant–microbe interactions.

Ecological and phytochemical factors potentially affecting winter dietary discrimination by porcupines (Erethizon dorsatum) in the mountain brush zone of Utah were studied. Porcupines utilized gambel oak (Quercus gambelii) as their primary winter food and roosting resource. Big-tooth maple (Acer grandidentatum) was the most common tree species in the study area but was rarely utilized by porcupines. Conifer species were used as a food and roosting resource significantly less often than they occurred in the study area, despite thermal advantages provided by their relatively dense canopies. Oak feed trees were successfully separated from conifer feed trees by discriminant analysis 100% of the time. Oak trees were correctly classified as feed and nonfeed trees 71% of the time. Gambel oak contained higher amounts of crude protein, fiber, and tannins, but was lower in ether extract fractions and fatty acid content than conifers. A layer of adipose tissue used as an energy reserve by porcupines may have relaxed energy intake demands sufficiently to permit them to concentrate on a diet of oak tissue, which is high in protein, rather than a high-fat conifer diet. A diet relatively high in protein may have facilitated digestion of food material high in fiber. Temperature did not affect selection of tree species used for roosting. Rock and snow caves were utilized infrequently and the study population ranged widely. Three of 15 study animals were eaten by predators.

Four oleanne triterpene saponins, 3beta,16beta, 23-trihydroxy-keton-13beta,28-epoxyolean-9(11)-en-3-yl -[beta-D-glucopranosyl(1-->2)]-[beta-D-glucopyranosyl(1-->3)]-beta -D-fucopyranoside [1] (clinopodiside D), 16beta-propionyl-3beta,23-dihydroxyoleana-11-21-dien-3 -yl-[beta-D-glucopyranosyl(1-->2)]-[beta-D-glucopranosyl(1-->3)]-b eta-D-fucopyranoside [2] (clinipodiside E), 3beta,16beta,21alpha,23,28-pentahydroxy-11-methoxyolean++ +-12-en-3-yl-[beta-D-glucopyranolyl(1-->2)]-[beta-D-glucopyranosyl (1-->3)]-beta-D-fucopyranoside [3] (clinopodiside F), and 3beta,16beta,21beta,23,28-pentahydroxyoleana-11,13(18) -dien-3-yl-[beta-D-glucopyranosyl(1-->2)]-[beta-D-glucopyranosyl(1 -->3)]-beta-D-fucopyranoside [4] (clinopodiside G), were isolated and purified from the plant Clinopodium chinensis. Their structures were elucidated on the basis of interpretation of nmr and ms data and from chemical evidence. The results are of potential interest for medicinal reasons.

Monoterpene accumulation during maturation of the current year's growth of Douglas-fir, Rocky Mountain variety, is characterized by a two-phase response. Beginning at or near budbreak, α-pinene and a tricyclene-(1)-camphene-bornyl acetate biogenetic group accumulate 3–10-fold. This is followed by another lesser increase later in the season of the minor components (E-) ocimene, citronellal, and borneol. In contrast, levels of β-pinene, myrcene, and terpinolene, and the sesquiterpenes γ-cadinene, α-bisabolene, and an oxygenated sesquiterpene, did not vary significantly during needle maturation. These increases occur first in the lower canopy and proceed upward toward the mid- and upper-canopy levels shortly thereafter. Implications of these observations with respect to metabolism, host-insect interactions, and silviculture management are discussed.

Three new triterpenoid saponins were isolated from the roots of Gypsophila oldhamiana. Their structures were elucidated, using a combination of homonuclear and heteronuclear 2D nmr and fabms, as 3-O-beta-D-galactopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 3)]-beta-D-glucuronopyranosyl quillaic acid methyl ester [1], 3-O-beta-D-galactopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 3)]-beta-D-glucuronopyranosyl gypsogenin methyl ester [2], adn 3-O-beta-D-galactopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 3)]-beta-D-glucuronopyranosyl quillaic acid 28-[O-beta-D-fucopyranosyl(1 --> 4)-beta-D-glucopyranosyl(1 --> 3)]-alpha-L-rhamnopyranosyl) ester [3].

Two new triterpenoid saponins, 3 beta,16 beta,23-trihydroxy-13,28-epoxyolean- 11-en-3-yl-[beta-D-glucopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->3)]- [beta-D-glucopyranosyl(1-->2)]-beta-D-fucopyranoside[1](clinopodiside B) and 28-O-beta-D-glucopyranosyl-3 beta,23-dihydroxyoleana-12,21-dien-3-yl-beta-D-glucopyranosy l-(1-->3)-beta-D- fucopyranoside [3] (clinopodiside C), together with three known saikosaponins, buddlejasaponins IV, IVa, and IVb, have been isolated from the plant, Clinopodium chinensis, and characterized using chemical evidence and spectroscopic methods, in particular 2D nmr spectroscopy.