Volume 64, Issue 7-8

The cornerstones of the first fundamental concept of photosynthetic machinery, i.e. the concept of photosynthetic units, are reconsidered and a new logically and experimentally well sustained interpretation of the crucial observations of this hypothesis is presented. The results obtained lead to the conclusion that under low irradiances all chlorophyll molecules are in a state to perform charge separation (photochemical) reaction, while under increased irradiances the essential part of the reaction centres are blocked and only one reaction centre from 500 - 600 remains in functionally active state, i.e. in the range of the value obtained by the founders of the widely accepted concept of photosynthetic units.

Within the framework of our continuous efforts to explore Hypericum species from Jordan, we report the analysis of the major active metabolites, naphthodianthrones and phloroglucinols, in the methanolic extracts of two under-explored Hypericum species; H. empetrifolium Willd. and H. sinaicum Hochst. & Steud. ex Boiss., using LC-(+,-)-ESI-MS (TIC and SIM) and LC-UV/Vis spectroscopy. Based on their LC-UV/Vis profiles, retention times and (+,-)-ESI-MS (TIC and SIM) spectral data, hypericin, protohypericin and pseudohypericin were identified in both of the investigated species. In addition adhyperfirin was only detected in H. empetrifolium, while hyperforin and protopseudohypericin were only detected in H. sinaicum. This is the first report documenting the presence of hypericin, protohypericin, pseudohypericin, protopseudohypericin, and hyperforin in H. sinaicum, and adhyperfirin in H. empetrifolium.

The pyrimidine thione derivatives 2a - d were prepared by the reaction of thiourea, ethyl cyanoacetate and several aromatic aldehydes. The acyclic thioglycosides 4a - 7d were prepared by the reaction of the synthesized pyrimidine thiones 2a - d with different alkyl halides, whereas the reaction of 2a - d with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide afforded the cyclic thioglycosides 8a - d whose deprotection afforded 9a - d. The obtained compounds were tested for their antischistosomal and antiviral activity against hepatitis B virus (HBV). Compounds 5a, 5d, 7a showed high activity against HBV using the MTT assay; moreover compounds 5c, 6d, 7a, 9a, 9c exhibited high activity as antischistosomal agents.

The present study describes the antiproliferative properties of Iris pseudopumila flowers and rhizomes extracts and fourteen constituents isolated from them. The in vitro cytotoxic activity assay against two human cancer cell lines, large lung carcinoma (CORL-23) and amelanotic melanoma (C32), showed that the most antiproliferative extract was the MeOH extract from flowers with a percentage of inhibition of 50.9 at 100 μg/ml against amelanotic melanoma cells. The most antiproliferative compounds against amelanotic melanoma cells were kaempferol-3-O-β-D-glucopyranoside and irisolidone with a percentage of inhibition of 100 and 96.6, respectively, and against large lung carcinoma cells with a percentage of inhibition of 82.1 and 84.6, respectively. Significant activity on the amelanotic melanoma cell line was also showed by irigenin-7-O-β-D-glucopyranoside, with a percentage of inhibition of 89.3. The compounds isovitexin and isoorientin-6-O’’-β-D-glucopyranoside showed a selective activity against amelanotic melanoma cells with a percentage of inhibition of 83.2 and 79.8, respectively.

A novel biflavone di-C-glucoside, 6,6″-di-C-β-D-glucopyranoside-methylene-(8,8″)- biapigenin (1), was isolated from the leaves of Jatropha curcas L. (Euphorbiaceae), together with six known compounds; apigenin 7-O-β-D-neohesperidoside (2), apigenin 7-O-β-Dgalactoside (3), orientin (4), vitexin (5), vicenin II (6), and apigenin (7). Their structures were determined on the basis of extensive chemical and spectroscopic analyses (UV, NMR and HRESI-MS). The immunomodulatory effect of an 80% aqueous methanol extract (AME) and compounds 1 - 5 (0.25 mg/kg body wt) to one-day-old specific pathogen-free (SPF) chicks was determined. Stimulation of both humoral and cell-mediated seroresponse was observed, especially those of AME and compound 1. Remarkable effective increases of the antibody titers, lymphocyte and macrophage cells, in blood were recorded. SPF chicks treated with the tested samples exhibited protection against Newcastle disease challenge virus after being vaccinated.

The known labdane-type diterpenoids anticopalic acid (1) and 3β-hydroxyanticopalic acid (2) were isolated from extracts of the aerial parts of Vitex hemsleyi Briq. (Labiatae). The acid 1 showed an antifeedant, dose-dependent activity against Spodoptera frugiperda (J. E. Smith) (Lepidoptera:Noctuidae). To our knowledge this is the first report on the antifeedant activity of a labdane-type diterpene against S. frugiperda.

The bioactivity of the flavonoids pinostrobin (1), pinocembrin (2), tectochrysin (3), galangin 3-methyl ether (4), and tiliroside (5) isolated from Lychnophora markgravii aerial parts was investigated in vitro against amastigote stages of Leishmania amazonensis. The compounds were isolated by several chromatographic techniques and their chemical structures were established by ESI-MS and NMR spectroscopic data. The flavonoids 1 and 3 were the most active compounds; they markedly reduced the viability of Leishmania amastigotes.

The phloroglucinol glucoside derivative [2,4-dihydroxy-6-(β-D-glucopyranosyloxy)phenyl]- butan-1-one (1), roseoside (2), and kaempferol-3-O-β-D-glucopyranoside (3) were isolated from the aerial parts of Conyza aegyptiaca (L.). To the best of our knowledge, this is the first isolation of compounds 1 - 3 from C. aegyptiaca. Their structures were determined by spectroscopic techniques including, IR, HR-EIMS, and extensive 500 MHz 1D- and 2D-NMR analyses ( 1 H, 13 C NMR, DEPT, 1 H- 1 H COSY, HMQC and HMBC experiments). The antioxidant activity of 1, using the DPPH assay, was investigated; in addition, 1 was investigated against different types of cell lines, including Hep-G2, HCT-116, and RAW 264.7 for its cytotoxic effects. Also, this is the first report on the activity of 1.

Eleven strains of endophytic fungi which habitat in an endangered, Chinese endemic medicinal plant, Dysosma pleiantha (Hance) Woodson, were isolated and tested for their cytotoxic activity using the brine shrimp lethality bioassay. Six isolates were found to exhibit some cytotoxic activity. Extracts of F1273, F1276, and F1280, which were identified as Trichoderma citrinoviride, Chaetomium globosum and Ascomycete sp., in particular, showed most potent activity with LC 50 values of 4.86, 7.71, and 14.88 μg/ml, respectively. These results indicate that endophytic fungi of Dysosma pleiantha could be a promising source for antitumour agents.

In our ongoing screening culture fluid extracts of Gloeoporus (Caloporus) dichrous strain 83065 inhibited the germination of Magnaporthe grisea and Fusarium graminearum spores. While isolating the active metabolites two new caloporosides, caloporoside G and caloporoside H, in addition to the known caloporoside derivatives F-16438G, caloporoside A, and 2-hydroxy-6-(16-hydroxyheptadecyl)benzoic acid were obtained.

Gene expression in the yeast retrotransposon Ty2 is regulated at transcriptional and translational levels. In this study, we have shown that the transcription of Ty2 is partially dependent on the membrane-bound glucose sensors Gpr1p and Mth1p in Saccharomyces cerevisiae. Transcription of Ty2 decreased approx. 3-fold in the gpr1, mth1 yeast mutant. Moreover, our results revealed that the transcription of Ty2 fluctuates during the growth stages of S. cerevisae. Both transcription and the frameshift rate of Ty2 rapidly dropped when the stationary stage yeast cells were inoculated into fresh medium. There was an instant activation of Ty2 transcription and a high level expression during the entire logarithmic stage of yeast growth. However, the transcription of Ty2 decreased 2-fold when the yeast cultures entered the stationary stage. The frameshift rate in Ty2 also varied depending on the growth conditions. The highest frameshift level was observed during the mid-logarithmic stage. It decreased up to 2-fold during the stationary stage. Furthermore, we have found that the frameshift rate of Ty2 diminished at least 5-fold in slowly growing yeasts. These results indicate that the transcription and the frameshift efficiency are coordinately regulated in the retrotransposon Ty2 depending on the growth conditions of S. cerevisiae.

In vitro shoot and callus cultures of the endemic South-African shrubs: Cyclopia intermedia E. Mey., Cyclopia subternata Vogel, and Cyclopia genistoides (L.) Vent. (Fabaceae) were established and examined for the presence of polyphenolic compounds. The xanthones mangiferin and isomangiferin, as well as the flavanones hesperidin and eriocitrin were identified by LC-ESI-MS and LC-DAD, and analyzed quantitatively by HPLC. The respective intact plants were analyzed for comparison. From all in vitro cultures, the highest levels of mangiferin (1.55%) and isomangiferin (0.56%) were recorded in C. subternata microshoots, compared to 1.31% and 0.49% found in the intact plant. Callus cultures of all species synthesized only trace amounts of mangiferin and isomangiferin. Hesperidin and eriocitrin contents were significanly lower in all in vitro cultures, in comparison to the respective intact plants. Among the obtained in vitro biomasses, the highest hesperidin content was recorded in C. intermedia (0.9%) and C. subternata (0.87%) microshoots, whereas C. subternata callus was characterized by the best growth parameters and highest hesperidin content (0.69%) from all examined Cyclopia calli.

Three-month-old seedlings of Lycoris chinensis were treated with biotic and abiotic elicitors: yeast elicitor (YE), methyl jasmonate (MJ), salicylic acid (SA), and sodium nitroprusside as NO donator (NO). We have shown that the addition of MJ and NO promotes the accumulation of galanthamine in these seedlings. The effect of these elicitors on the growth of the seedlings, as well as on the amount of the alkaloids accumulated in the seedlings was studied. The results showed that, in general, high doses of MJ and SA had a negative effect on the growth of the seedlings, while appropriate doses of NO and YE had a positive effect on the growth of the seedlings. It was remarkable that the addition of MJ, NO, and YE can promote galanthamine accumulation in seedlings. The accumulation was higher in treatments at higher concentrations of NO (100 μM), where the release of galanthamine was 1.72-fold higher than that of the control at the 10 th day of culture. The highest values of lycorine were obtained in seedlings treated with YE at a concentration of 0.01 g/l and by the 10 th day of culture; the level was 1.38 times of the control.

In order to find the minimal structural requirements to maintain microtubule binding, 12 taxol analogues have been docked to the taxol binding site of tubulin. By comparing the interactions of each analogue with β-tubulin, the structure-activity relationships are summarized as follow: C-2 benzoyl and taxane ring systems are the essential groups for microtubule binding, the improvements of bioactivity and bioavailability are dependent on the substituents at positions C-1, C-4, C-7, C-9, C-10, and C-14, whereas the C-13 side chain mainly provides a specific binding.

Salvia miltiorrhiza is an annual plant growing in China, Mongolia, Korea and some other Asian countries. The extract from S. miltiorrhiza roots has been used for supporting healthy cardiovascular and circulatory systems during the last decade. The active constituents of S. miltiorrhiza from different areas vary significantly, and the wild resources are overexploited. To adapt the demand for active constituents of S. miltiorrhiza against cardiovascular-related diseases, alternative materials need to be developed. The aim of the present work was to investigate the possibility of S. miltiorrhiza hairy roots as the alternative materials. The results showed that S. miltiorrhiza hairy roots are genetically stable. The contents of salvianolic acid B and tanshinone IIA, two main active constituents in hairy roots, determined by the assessment of combining fl ow cytometry and phytochemical analysis, are comparable to or significantly lower than in wild plant roots. The extract from S. miltiorrhiza hairy roots also had similar protection activity for hypoxia and reoxygenation injury in rat cardiac myocytes like that from wild plant roots. S. miltiorrhiza hairy roots may be alternative materials to obtain the drug or healthy food for cardiovascular-related diseases.

Betacyanins and anthocyanins, two main red fl ower pigments, never occur together in the same plant. Although the anthocyanin biosynthetic pathway has been well analyzed, the biosynthetic genes and the regulatory mechanism of the betacyanin biosynthesis are still obscure. We cloned two cDNAs of DOPA dioxygenase from Phytolacca americana, PaDOD1 and PaDOD2, that may be involved in the betalain biosynthesis. The deduced amino acid sequence of PaDOD1 and PaDOD2 showed approximately 80% homology to each other. The promoter regions of PaDOD1 and PaDOD2 were isolated by inverse PCR and analyzed using PLACE database. Some putative MYB, bHLH, and environmental stress-responsive transcription factor binding sites were detected in the PaDOD1 and PaDOD2 promoter regions. Expression patterns of PaDOD1 and PaDOD2 in suspension cultures of P. americana were investigated by semiquantitative RT-PCR. The transcripts of PaDODs were found in both betacyanin-producing red cells and non-betacyanin-producing white cells, suggesting that not only the expression of DOD, but also the supplementation of DOPA might be a regulatory step for the betalain biosynthesis in P. americana.

A random genomic library of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) was constructed and viral factors were identified by screening the regulator(s) for helicase gene expression. DNAs of 238 library plasmids were used to co-transfect with the reporter plasmid, pHp510-luc, in which the luciferase (luc) gene was driven by the baculovirus helicase promoter. Results showed that eight plasmids of the library strengthened the luciferase activity more than 1000-fold. Sequence analyses revealed that all of the eight plasmids contained an intactie-1 coding region. To confirm the reliability of the screening library, pHp510-luc was co-transfected with the cloned early gene which revealed that the BmNPV IE-1 was the only early factor that could stimulate the helicase promoter. The function analyses suggested that genome-wide screening factors through the library are powerful means to investigate the transcriptional regulation of dsDNA viruses.

DNA extraction from food is always problematic especially from highly processed samples which contain only trace amounts of severely degraded DNA fragments. In this work, to extract trace amounts of small DNA fragments of the traditional Chinese medicine (TCM) colla corii asini derived from highly processed Equus asinus skin, three strategies were compared for its authentication. With some optimizations, the modified QIAquick spin column method achieved higher DNA yield and purity in comparison with the “SDS/proteinase K” method and the “Wizard magnetic DNA purification system for food” method. Further studies showed that at least 0.4 g colla corii asini was needed to obtain enough DNA extracts for PCR-based detection by the method and only amplicons of less than 100 bp could be generated from the DNA extracts which confirmed the efficiency of the method in small DNA fragment extraction. The DNA obtained by this method was suitable to be used in PCR-based authentications.

(Z)-5-dodecen-1-ol (Z5 - 12:OH), (Z)-5-dodecenyl acetate (Z5 - 12:Ac), and (Z)-5-tetradecenyl acetate (Z5 - 14:Ac) were found in the extracts of the female sex pheromone gland of the carpenterworm moth Holcocerus artemisiae Chou et Hua, a pest of Artemisia filifolia. The average amounts of Z5 - 12:OH, Z5 - 12:Ac, and Z5 - 14:Ac in a single sex pheromone gland of a calling moth were (7.14 ± 0.73) ng, (54.20 ± 0.34) ng, and (38.70 ± 0.46) ng, respectively. Electroantennography (EAG) of these compounds and their analogues demonstrated that Z5 - 12:Ac excitated the largest male EAG response, followed by Z5 - 14:Ac. Traps baited with rubber septa impregnated with Z5 - 12:Ac (500 μg/septum) and Z5 - 14:Ac (300 μg/septum) were more effective than traps with other baits or virgin females. Addition of Z5 - 12:OH to rubber septa did not enhance the trap catches, but (E,Z)-3,5-dodecadienyl acetate (E3,Z5 - 12:Ac) enhanced the trap catch.

Glucan preparations, primarily modifi ed water-soluble glucans, are involved in the activation of the body’s natural defense mechanisms and in the acceleration of the skin’s wound-healing processes. Pleuran, an insoluble β-D-glucan in hydrogel form, offers a natural alternative to more common chemically derivated soluble β-D-glucans. Pleuran was applied to human keratinocyte primary cultures, and after 24 h of incubation the release of matrix metalloproteinase 9 (MMP-9) and metalloproteinase 2 (MMP-2) by stimulated keratinocytes was detected using gelatine zymography. There was a concentration-dependent increase in pro-MMP-9 release after treatment with pleuran over the concentration range of 2 to 200 μg/ml, but pro-MMP-2 was detected at a constant level. Moreover, the active forms of both MMPs were not detectable, indicating that in vitro autoactivation of these zymogens did not occur. The results indicate that pleuran is a potent keratinocyte stimulator of pro- MMP-9 release, which implies its application in dermatological therapies

DNA repair is critical for successful chemo- and radiotherapy of human tumours, because their genotoxic sensitivity may vary in different types of cancer cells. In this study we have compared DNA damage and the efficiency of its repair after genotoxic treatment with hydrogen peroxide, cisplatin and γ-radiation of head and neck squamous cell carcinoma (HNSCC). Lymphocytes and tissue cells from biopsies of 37 cancer patients and 35 healthy donors as well as the HTB-43 larynx cancer cell line were employed. The cell sensitivity to genotoxic treatment was estimated by the MTT survival assay. The extent of DNA damage and efficiency of its repair was examined by the alkaline comet assay. Among the examined treatments, we found that HNSCC cells were the most sensitive to γ-radiation and displayed impaired DNA repair. In particular, DNA damage was repaired less effectively in cells from HNSCC metastasis than healthy controls. In conclusion, our results suggest that the different genotoxic sensitivity of HNSCC cells may depend on their DNA repair capacity what in turn may be connected with the effectiveness of head and neck cancer therapy.