Anti-inflammatory oleanane triterpenes from Tripterygium wilfordii cell suspension cultures by fungal elicitation.

Treatment of cell suspension cultures of Tripterygium wilfordii with an autoclaved Botrytis sp. homogenate rapidly increased the synthesis of a family of oleanane and friedelane triterpenes, including the antiinflammatory oleanane triterpene 3ß,22α-dihydroxyolean-12-en-29-oic acid. This compound exceeded 30 mg · l(-1) in 13 day elicitations with 12 l bioreactors, in contrast to control levels of less than 5 mg · l(-1). Cell cultures treated with the fungal elicitor provided higher triterpene yields in less time than cultures in a diterpene production medium or whole plants. Elicited production has been developed for commercial application in light of the successful treatment of rheumatoid arthritis with Tripterygium extracts.

Studies on New Components and Stereochemistry of Diterpenoids from Trypterygium wilfordii.

An extensive investigation of the petroleum ether extract of TRIPTERYGIUM WILFORDII plants have revealed the presence of four new compounds, triptonoterpene, neotriptonoterpene, triptonodiol, and neotriptonolide, in addition to the diterpenes isolated and characterized earlier. The structures of these novel natural products have been elucidated.

Biotransformation of Dehydroabietic, Abietic, and Isopimaric Acids by Mortierella isabellina Immobilized in Polyurethane Foam.

Polyurethane foam supports immobilization of Mortierella isabellina ATCC 38063, a zygomycete that hydroxylates and thereby detoxifies resin acids found in certain pulp mill effluents. The fungus becomes entrapped as it threads growing mycelia through the open foam matrix. The tenacity of binding and the amount of biomass bound depend on initial spore numbers and nutrient concentration. Optimal dehydroabietic acid transformation occurs with early-stationary-phase foam-bound mycelia suspended in buffer at pH 6.5 to 8.5 with aeration >0.1 liter liter min and near a temperature maximum of 33 degrees C. Neither a greater number of similarly loaded foam cubes nor a greater surface area for similar amounts of biomass increased transformation rates. Compared to free mycelial cultures, foam presence retards growth and decreases the rate of precursor transformation but does not alter the nature of the metabolites formed from dehydroabietic acid, abietic acid, or isopimaric acid. Diffusional barriers or binding of substrates in an inaccessible manner may cause the observed inhibitions. Overall, foam immobilization stabilizes enzymic activity and is a facile laboratory process with scaleup potential.

Biotransformation of dehydroabietic acid with resting cell suspensions and calcium alginate-immobilized cells of Mortierella isabellina.

Mortierella isabellina ATCC 38063 is a zygomycete capable of hydroxylating fish-toxic resin acids which occur in certain pulp mill effluents to nontoxic metabolites. Addition of dehydroabietic acid (1) (80 mg/liter) to a freshly inoculated culture of M. isabellina in dextrose-yeast extract broth resulted in precursor disappearance in 28 to 30 h. During growth phase, hydroxylation occurred at C-2, whereas hydroxylation at C-15 and C-16 commenced with onset of stationary phase. Alternatively, 1 added to stationary-phase culture (40 mg/liter) disappeared within 2 h and hydroxylation occurred concurrently at C-2, C-15, and C-16. Enzymatic activity of stationary-phase culture was totally cell associated and was present despite the absence of 1 during the preparatory growth phase. Resuspension of mature fungi as free mycelia or immobilized in calcium alginate beads did not diminish the effectiveness of the biotransformation, although two new metabolites, 15-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (5) and 16-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (7) were formed. Immobilized mycelia retained hydroxylase activity for greater than 110 days whether or not they were challenged with fresh 1 on a regular basis. In this respect they are more long-lived than resuspended free mycelia are.

Alkaloid production in Catharanthus roseus (L.) G. Don cell cultures. XIII. Effects of bioregulators on indole alkaloid biosynthesis.

A study on the effect of various bioregulators on the biosynthesis of ajmalicine (8) and catharanthine (9) in plant tissue cultures of Catharanthus roseus is described. It is shown that 1,1-dimethylpiperidine bromide (3) and 2-diethylaminoethyl-3,4-dimethylphenylether (7) are effective in increasing these alkaloid levels in the cell line PRL #200. Such studies may prove beneficial in larger scale experiments designed for the production of these alkaloids.

Cultivation of Tripterygium wilfordii Tissue Cultures for the Production of the Cytotoxic Diterpene Tripdiolide.

The production of the cytotoxic agent tripdiolide employing plant tissue cultures of TRIPTERYGIUM WILFORDII is demonstrated. Detailed investigations of a developed cell line of T. WILFORDII in terms of tripdiolide production versus variations in growth conditions, for example media composition, are presented. Also the age and size of the inoculum and the incubation period with respect to tripdiolide formation are evaluated.

Alkaloid production in Catharanthus roseus cell cultures : XII. Biosynthetic capacity of callus from original explants and regenerated shoots.

Callus derived from hypocotyls of periwinkle, Catharanthus roseus, responded to culture on nutrient media supplementedwith IAA, BA, and zeatin with shoot formation at low frequencies. However, shoot regenerating callus could be very successfully propagated and subcultured. Alkaloid profiles of callus derived from the original explants (hypocotyls) as well as callus derived from regenerated shoots were almost identical. Subcultures of old callus (initiated in 1978) failed completely to grow shoots. In programs for long-term preservation of alkaloid producing cell lines by regeneration and storage of shoots, selection for ability to form shoots would have to precede selection for alkaloid production.

Alkaloid Production in Catharanthus roseus cell cultures VIII.

A cell line of Catharanthus roseus (L.) G. Don coded PRL # 200, was characterized with respect to its biosynthetic capabilities for indolealkaloids, in particular catharanthine, in suspension cultures. Other alkaloids isolated are vallesiachotamine isomers, ajmalicine, hörhammericine, hörhammerinine, vindolinine, 19-epivindolinine and strictosidine lactam.

Alkaloid production in Catharanthus roseus (L.) G. Don. : VI. Variation in alkaloid spectra of cell lines derived from one single leaf.

Analysis of 76 cell clones derived from one leaf of a periwinkle plant (Catharanthus roseus (L.) G. Don) showed the occurrence of Corynanthe-, Strychnos-, and Aspidosperma-type alkaloids. The majority of clones (62%) displayed compounds of all three types. Variation of the alkaloid spectra of the cell clones was low when compared to that found previously with serially subcultured callus and cell suspensions derived from different plants.

Studies on the total synthesis of bisindole alkaloids in the vinblastine-vincristine series.

A series of investigations involving the total syntheses of a variety of indole and dihydroindole alkaloids in the Aspidosperma, Iboga and Vinca families are discussed. In this lecture particular emphasis is placed on studies relating to the synthesis of the clinically important bisindole or "dimeric" alkaloids of the vinblastine-vincristine family. Apart from the activity demonstrated by the natural alkaloids, it is shown that closely related synthetic derivatives can possess interesting anti-tumor activity as well.