Advanced Research on Biological Properties-A Study on the Activity of the Apis mellifera Antioxidant System and the Crystallographic and Spectroscopic Properties of 7-Diethylamino-4-hydroxycoumarin

Abstract

The search for substances that increase the immunity of bees is becoming a necessity in the era of various environmental threats and the declining immunocompetence of these insects. Therefore, we tested the biological and physicochemical properties of 7-diethylamino-4-hydroxycoumarin (7DOC). In a cage test, two groups of bees were created: a control group fed with sugar syrup and an experimental group fed with sugar syrup with the addition of 7DOC. In each group, the longevity of the bees was determined and the protein concentrations and antioxidant activities in the bees' hemolymph were determined. The bees fed with 7DOC lived 2.7 times longer than those in the control group. The protein concentrations and activities of SOD, CAT, GPx and GST, as well as the TAC levels, were significantly higher in the hemolymph of the supplemented workers. To confirm these potent biological properties of 7DOC, the UV-Vis spectra, emission and excitation of fluorescence, synchronous spectra and finally the fluorescence lifetimes of this compound were measured using the time-correlated single photon counting method, in various environments differing in polarity and in the environment applied in bee research. This compound was shown to be sensitive to changes in solvent polarity. The spectroscopic assays were complemented with crystallographic tests of the obtained monocrystals of the aforementioned compounds, which attested to the aggregation effects observed in the spectra measurements for the selected coumarin. The research results confirm that this compound has the potential to be implemented in apiary management, which will be our application goal, but further research into apiary conditions is required.

Keywords: X-ray crystallography; antioxidants; coumarin derivatives; honeybee; molecular spectroscopy.

Scheme 1

Structure of 7-diethylamino-4-hydroxycoumarin (7DOC).

Figure 1

Percentage of live bees supplemented with 7DOC and in the non-supplemented control group.

Figure 2

Protein concentrations (mg/mL) in the workers’ hemolymph along with the aging processes, with or without 7DOC supplementation (two-way ANOVA; group: F_(1,268) = 311,55, p = 0.0000, se ± 0.018843 (control), se ± 0.062063 (7DOC); days of supplementation: F_(4,264) = 2360,1; p = 0.0000, se ± 0.014635 (1st day), se ± 0.010349 (7–35 day); group × days of supplementation: F_(4,264) = 656,64; p = 0.0000, se ± 0.014635; lowercase letters (a, b)—statistically significant differences between groups on a given day of the bees’ life).

Figure 3

Antioxidant activities in workers’ hemolymph along with the aging processes, after supplementation with 7DOC. Lowercase letters (a, b)—statistically significant differences between groups on a given day of the bees’ life.

Figure 4

Total antioxidant capacity (TAC) levels in workers’ hemolymph along with the aging processes, after 7DOC supplementation (two-way ANOVA; group: F_(1,268) = 776.42, p = 0.0000, se ± 0.244479 (control), se ± 0.428825 (7DOC; days of supplementation: F_(4,264) = 1937.7; p = 0.0000, se ± 0.120715 (1st day), se ± 0.085359 (7–35 days); group × days of supplementation: F_(4,264) = 473.85, p = 0.0000, se ± 0.120715; lowercase letters (a, b)—statistically significant differences between groups on a given day of the bees’ life).

Figure 5

Atom labeling and Anisotropic Displacement Parameters plotted at 50% level of probability for 7-dietyloamino4-hydroxycoumarin molecule. Ethyl groups C12-C13, C10-C11 are at 77.58° and 85.50° angle, respectively, to the mean plane created by aromatic rings.

Figure 6

A 7DOC asymmetric unit with intermolecular interactions. All contacts (hydrogen bonds and π-π stacking) are with molecules of oppositely positioned ethyl groups.

Figure 7

Packing of crystals along different crystallographic directions (I)—a, (II)—b, (III)—c.

Figure 8

Panel (A): Electron absorption spectra for 7DOC in some solvents and in the water/sugar mixture. Panels (B–D): Fluorescence emission spectra corresponding to absorption spectra from Panel (A) at excitation with an appropriate wavelength.

Figure 9

Panel (A): Fluorescence excitation spectra corresponding to the spectra from Figure 8A; Panel (B): Selected RLS spectra also corresponding to the ones displayed in Figure 8A.

Figure 10

An example of the course of fluorescence anisotropy in methanol (black squares) and in the water/sugar mixture (red dots).

Figure 11

The fluorescence decay of 7DOC depends on the solvent. The top panel shows the decay of 7DOC fluorescence emission (dotted curves) and respective best exponential fits (continuous lines). The bottom panel shows residual distributions for the presented fits.