ABSTRACT
An open reading frame (ORF) of isopentenyl-diphosphate delta isomerase gene (FuIPI) was cloned from Fritillaria unibracteata Hsiao et K. C. Hsia. (F. unibracteata). Furthermore, the bioinformatics and functional analyses of FuIPI were performed in this study. The result showed that, the ORF of FuIPI gene was 825 bp, encoding a polypeptide of 274 amino acids in length, with a relative molecular mass of about 31 kD and a theoretical isoelectric point of 5.61. Sequence analysis showed that FuIPI contained conserved structural domains and key residues involved in the catalyzing process. The phylogenetic analysis exhibited that FuIPI was closely related to IPIs of Dendrobium officinale and Musa acuminate. Real-time PCR analysis showed that FuIPI was distributed in different tissues of F. unibracteata, but had the highest transcriptional level in leaves, followed by stems, bulbs, and flowers. Furthermore, the FuIPI protein was successfully expressed in Escherichia coli BL21(DE3). The purified FuIPI protein successfully catalyzed the conversion from isopentenyl diphosphate (IPP) to dimethylallyl pyrophosphate (DMAPP). The above results provided a theoretical basis for further investigation of the molecular role of FuIPI in the biosynthesis of alkaloids.
ABSTRACT
OBJECTIVE:To investigate antitussive,antiasthmatic,phlegm-resolving and anti-inflammatory effects of Fritillaria cirrhosa and Fritillaria unibracteata. METHODS:Mice and guinea pig were randomly divided into blank control group (distilled water),ethanol extract groups,water extraction groups and powder groups of F. cirrhosa and F. unibracteata(1.13 g/kg for mice, 0.42 g/kg for guinea pig,calculated by crude drug),positive control group,with 10 mice(guinea pig)in each group. Each group was given relevant medicine intragastrically once a day for consecutive 5 d. Relative indicators were detected 30 min after last medication. The ammonia water induced cough method was used to investigate antitussive effect (dextromethorphan hydrobromide as positive drug,0.013 g/kg)by determining coughing latent period and coughing times within 3 min. After inducing asthma with 2％ histamine phosphate for 15 s, antiasthmatic effect (aminophylline as positive drug, 0.033 g/kg) was investigated by determining coughing latent period and the number of guinea pig with convulsive fall. The phenol red injection method was used to investigate phlegm-resolving effect (ambroxol as positive drug,21.304 g/kg) by determining the content of phenol red in the trachea of mice in phenol red expectoration test. The anti-inflammatory effect(dexamethasone acetate as positive drug,9.225×10-4 g/kg)was investigated by determining ear swelling degree and inhibition rate of ear swelling mice in xylene-induced inflammation test. RESULTS:Compared with blank control group, coughing latent period and cough induction latent period prolonged significantly in ethanol extract of F. cirrhosa and F. unibracteata groups,F. cirrhosa and F. unibracteata powder groups,positive control group, and coughing time decreased significantly within 3 min. The number of mice with convulsive fall decreased significantly in F. cirrhosa powder group, F. unibracteata ethanol group,F. unibracteata powder group and positive control group. The content of phenol red in the trachea of mice increased significantly,while ear swelling and inhibitory rate of ear swelling decreased significantly,with statistical significance (P<0.05 or P<0.01). There was no statistical significance in each index of mice between powder group and positive control group (P>0.05). Compared with corresponding groups of F. cirrhosa,coughing latent period of mice prolonged significantly in F. unibracteata ethanol extract group (P<0.05), other indexes had no statistical significance(P>0.05). CONCLUSIONS:The antitussive,antiasthmatic,phlegm-resolving and anti-inflammatory effects of F. cirrhosa are similar to or worse than those of F. unibracteata.
ABSTRACT
To explore the resource of endophytic actinomycete in Fritillaria unibracteata, and alleviate the shortage of F. unibracteata resource, using F. unibracteata as experimental materials which growth in the western Sichuan plateau and cut its healthy bulb. Pure culture, insert, TLC and Oxford cup were applied to observe the mycelial morphology, research the ability of producing alkaloid and its antibacterial activity. Totally, 14 endophytic actinomycete strains were isolated by using Gao culture media. Based on the color reaction, 5 typical strains were selected for producing alkaloid. Through the TLC technique, all strains produced 2 obvious alkaloids spots. Antibacterial activity determination showed that the antimicrobial effects of 2 strains is prominent, the diameter up to 11 mm.16S rRNA gene sequence comparison analysis showed that 5 strains belonging to the Streptomyces. The alkaloids produced by endophytic actinomycetes are not related to F. unibracteata, but its fermentation liquid has antibacterial effect, it is worthy of further study.