Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation.

Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control various pathways involving such metabolic reactions in the plant. As a representative experimental validation of this notion, WSCYP93Id was heterologouly expressed in Escherichia coli and catalytic capabilities of the recombinant WSCYP93Id protein were evaluated using withanolides as substrates. Optimized assays with some major withanolides (withanone, withaferin A and withanolide A) involving spectrophotometric as well as high-pressure liquid chromatography (HPLC)-based evaluation (product detection) of the reactions showed conversion of withaferin A to a hydroxylated product. The genes belonging to other CYP group are possibly involved in some specialised synthesis such as that of brassinosteroids.

Zantholic acid, a new monoterpenoid from Zanthoxylum zanthoxyloides.

From the extract of Zanthoxylum zanthoxyloides fruits, a new menthane monoterpenoid, (4R,7R)-8-carboxy-7,9-dihydroxy-trans-menthane, tentatively, named as zantholic acid along with several known compounds was isolated. Its structure was determined using spectroscopic methods. The cytotoxic activity of zantholic acid against a panel of cancer cell lines was evaluated using the MTT assay. The results show that zantholic acid possesses a selective cytotoxic activity towards breast cancer cell lines.

Antibacterial, cytotoxic activities and chemical composition of fruits of two Cameroonian Zanthoxylum species.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum leprieurii (Syn. Fagara leprieurii) is a plant extensively used by traditional healers in Cameroon for the treatment of stomach disorders, gonorrhea, intestinal parasites and sterility. The infusion from the fruit is taken in Cameroon for the treatment of sickle cell anemia, as well. Similarly, the roots of Zanthoxylum zanthoxyloides (Syn. Fagara zanthoxyloides) are also used for wound dressing, sickle cell anemia and as pain reliever, in addition to the uses mentioned for Zanthoxylum leprieurii. AIM OF THE STUDY: To scientifically validate their traditional use, the in vitro antimicrobial and anticancer assays were performed. In addition, chemical compositions of their fruit extracts and essential oils were also studied. MATERIAL AND METHODS: The chemical compositions of their essential oils and methanol extracts were studied. GC and GC-MS were used for analyzing the essential oils while various chromatographic methods were used to isolate the pure compounds from methanol extracts. MTT assay was used for the cytotoxic studies of methanol extracts, their fractions and some pure compounds. Antimicrobial activity of methanol extracts, some isolated compounds as well as essential oils was tested against a panel of human pathogenic bacteria by diffusion and dilution methods. RESULTS: The principal constituents of the essential oil of Zanthoxylum zanthoxyloides were identified as citronellol and geraniol whereas Zanthoxylum leprieurii was rich in E-ß-ocimene. Furthermore, we report for the first time, the presence of 7,8-dimethoxycoumarin and sinapic acid in the methanol extract of Zanthoxylum leprieurii. In vitro bioassays reveal that the methanol extracts, essential oil and some of the isolated compounds from fruits of both the plants exhibit moderate anticancer and antimicrobial activities. CONCLUSION: Altogether, the present findings justify the traditional use of Zanthoxylum zanthoxyloides and Zanthoxylum leprieurii in the treatment of stomach disorders and cancer disease. The antimicrobial properties in the essential oil of Zanthoxylum zanthoxyloides are of additional significance.

An alkenol and C-methylated flavones from Callistemon lanceolatus leaves.

Callistemon lanceolatus leaves after extraction with a combination of organic solvents have afforded 3-methyltetradec-2-en-7-ol and the rare C-methylated flavones: 5-hydroxy-7,4'-dimethoxy-6,8-dimethylflavone and 5-hydroxy-7,4'-dimethoxy-6-methylflavone. Structures have been established by spectroscopic methods. Isolation of C-methylated flavones from C. lanceolatus supported the view that they are characteristic to Myrtaceae.

Crystal structure of artemisinic acid: a possible biogenetic precursor of antimalarial artemisinin from Artemisia annua.

Artemisinic acid [1], a possible biogenetic precursor of the antimalarial artemisinin [2], was isolated from the hexane extract of Artemisia annua. X-ray crystallography of the dimer of artemisinic acid shows that the cyclization during intermolecular hydrogen bonding occurs by the opposite orientation of the alpha, beta-methylene group in each molecule. Complete spectroscopic data of 1 are also given.

Volatile Constituents of Dracocephalum nutans.

The essential oil of DRACOCEPHALUM NUTANS L. (Lamiaceae) has been investigated for the first time. The GC-MS analysis of the oil showed the presence of about 25 monoterpenoids including pinocamphone (56.4%), beta-pinene (12.7%), isopinocamphone (4.3%), alpha-phellandrene (4.6%), and isopinocampheol (3.7%).

The Essential Oil of Perilla ocimoides: A Rich Source of Rosefuran.

The essential oil of a strain of PERILLA OCIMOIDES from Bangladesh, cultivated at Lucknow, after GC-MS investigations afforded rosefuran ( 1) as the major constituent (58%) along with 17 others including beta-caryophyllene, perilla ketone ( 2), perillene, alpha-cedrene, germacrene-D, and isobis-abolene.

New Prenylflavanones and Chalcones from Helichrysum rugulosum.

Both the aerial parts and the roots of HELICHRYSUM RUGULOSUM afforded a complex mixture of prenylated flavanones and chalcones. The structures were elucidated by spectroscopic methods.

Weitere Phloroglucin- und alpha-Pyron-Derivate aus Helichrysum-Arten.

The investigation of three South African HELICHRYSUM species afforded a tremetone derivative, where the acetyl group is methylated, four phloroglucin derivatives, two of them being chromones, four alpha-pyrone derivatives and a dihydro chalcone also derived from phloroglucine. The structures were elucidated by sepectroscopic methods.