Whole-Cell Mediated 11β-Hydroxylation on the Basic Limonoid Skeleton by Cunninghamella echinulata

Regio- and stereoselective 11β-hydroxylation was achieved on the basic limonoid skeleton through microbial transformation. Whole cells of Cunninghamella echinulata efficiently converted basic limonoids such as epoxyazadiradione, azadiradione, and gedunin to their 11β-hydroxy analogues as the sole metabolite. Fermentation conditions affecting the efficiency (96%) of biotransformation including substrate concentration, incubation period, pH, and temperature were optimized. The position and stereochemistry of hydroxyl functionality on the isolated metabolites were established through extensive spectroscopic and spectrometric studies (1D, 2D NMR, ESI-MS, and MS/MS)

Insecticidal Potential of Defense Metabolites from <i>Ocimum kilimandscharicum</i> against <i>Helicoverpa armigera</i>

<div><p>Genus <i>Ocimum</i> contains a reservoir of diverse secondary metabolites, which are known for their defense and medicinal value. However, the defense-related metabolites from this genus have not been studied in depth. To gain deeper insight into inducible defense metabolites, we examined the overall biochemical and metabolic changes in <i>Ocimum kilimandscharicum</i> that occurred in response to the feeding of <i>Helicoverpa armigera</i> larvae. Metabolic analysis revealed that the primary and secondary metabolism of local and systemic tissues in <i>O. kilimandscharicum</i> was severely affected following larval infestation. Moreover, levels of specific secondary metabolites like camphor, limonene and β-caryophyllene (known to be involved in defense) significantly increased in leaves upon insect attack. Choice assays conducted by exposing <i>H. armigera</i> larvae on <i>O. kilimandscharicum</i> and tomato leaves, demonstrated that <i>O. kilimandscharicum</i> significantly deters larval feeding. Further, when larvae were fed on <i>O. kilimandscharicum</i> leaves, average body weight decreased and mortality of the larvae increased. Larvae fed on artificial diet supplemented with <i>O. kilimandscharicum</i> leaf extract, camphor, limonene and β-caryophyllene showed growth retardation, increased mortality rates and pupal deformities. Digestive enzymes of <i>H. armigera -</i> namely, amylase, protease and lipase- showed variable patterns after feeding on <i>O. kilimandscharicum,</i> which implies striving of the larvae to attain required nutrition for growth, development and metamorphosis. Evidently, selected metabolites from <i>O. kilimandscharicum</i> possess significant insecticidal activity.</p></div

Digestive enzymes of <i>H. armigera</i> larvae fed on <i>O. kilimandscharicum</i> leaves.

<p>Changes in the levels of <b>A.</b> protease <b>B.</b> amylase <b>C.</b> lipase activity of <i>H. armigera</i> second-instar larvae fed on <i>O. kilimandscharicum</i> plants at 12 h, 24 h, day 3 and day 6. One way ANOVA followed by Tukey's multiple comparisons test suggested significant difference between the data at. <i>p</i><0.001 (indicated as ‘***’), <i>p</i><0.01 (indicated as ‘**’), <i>p</i><0.05 (indicated as ‘*’). Results are an average of three independent experiments conducted in duplicate. Error bars represent Mean ± SD.</p

Two way analysis of variance for growth inhibition and percentage mortality of <i>H. armigera</i> upon exposure to <i>O. kilimandscharicum</i> leaf extract and selected metabolites on various days.

<p>DF = Degrees of freedom, SS = Sum of squares, MS = Mean square, n = numerator, d = denominator, p = probability of significance, α = 0.05.</p

Metabolic changes in leaves of <i>O. kilimandscharicum</i> following <i>H. armigera</i> infestation.

<p>Heat map representing relative expression of a sub-set of volatiles elicited in leaf tissue during <i>O. kilimandscharicum</i>-<i>H. armigera</i> interaction; comparison between metabolite profiles of local (L) and systemic (S) leaf tissue in <i>O. kilimandscharicum</i>, 12 h and 24 h after feeding by <i>H. armigera</i>, and also on days 3 (D3) and 6 (D6), compared to control (C) plants.</p

Metabolic changes in stems and roots of <i>O. kilimandscharicum</i> following <i>H. armigera</i> infestation.

<p>Heat map representing relative expression of a sub-set of volatiles elicited in <b>A.</b> stems and <b>B.</b> roots during <i>O. kilimandscharicum</i>- <i>H. armigera</i> interaction at 12 h, 24 h, and on days 3 (D3) and 6 (D6) as compared to control (C) plants.</p

Two way analysis of variance for macromolecular content of <i>O. kilimandscharicum</i> leaves, stem and root on various days of <i>H. armigera</i> infestation.

<p>DF = Degrees of freedom, SS = Sum of squares, MS = Mean square, n = numerator, d = denominator, p = probability of significance, α = 0.05.</p

Two way analysis of variance for performance of <i>H. armigera</i> on various days feeding on tomato and <i>O. kilimandscharicum</i> leaves.

<p>DF = Degrees of freedom, SS = Sum of squares, MS = Mean square, n = numerator, d = denominator, p = probability of significance, α = 0.05.</p