LIPID PEROXIDATION INHIBITION STUDY OF FLOWER EXTRACT AND TWO COUMARINS ISOLATED FROM DAPHNE MEZEREUM L.

The medicinal importance of the genus Daphne L. is related to the richness in the expansive range of different classes of natural products and bioactive phytochemicals, such as coumarins, flavonoids, lignans and different classes of terpenes. The current study reports on the lipid peroxidation effect of diethyl-ether macerate of Daphne mezereum L. flowers and of two coumarins we have isolated from the aqueous subfraction of the crude diethyl-ether extract. All three tested samples, D. mezereum flowers extract (IC 50 = 25.1 ± 2.9 mM) and isolated coumarins: umbelliferone (IC 50 = 7.1 ± 2.6 mM) and herniarin (IC 50 = 19.0 ± 1.3 mM), exhibited notable antioxidative potential in lipid peroxidation assay. None of the samples, however, had an inhibitory effect as pronounced as standardly applied antioxidants Trolox (IC 50 = 22 ± 6 μM), caffeic acid (IC 50 = 15 ± 3 μM) and quercetin (IC 50 = 23 ± 6 μM). Taken altogether, the results of our studies bring forward new data regarding the antioxidant activities of D. mezereum species. Acta Medica Medianae 2024;63(1):39-46.


Introduction
Named after the water nymph in Greek myth that was turned into a laurel tree, encompassing 95 species of flowering shrubs, Daphne L. is the most diverse genus of Thymelaeaceae family.The genus is native to certain regions of sub-tropical Asia, but is also distributed in Europe and north Africa.Until now, in Europe's Flora, the presence of 17 species of this genus has been identified [1].Daphne mezereum L. is among plants that grows mostly in Europe and Asia and one of seven Daphne species native to Serbia [2].The decorative, strongly scented flowers are produced in early spring on the bare stems before the leaves appear.The fruit is a bright red berry, very poisonous for humans, but despite striking toxicity, because of its desirable horticultural characteristics, D. mezereum become one of the most popular perennial flowering shrubs [3].
As was confirmed in earlier studies, Daphne plants are rich source of pharmacologically important molecules, indicating broad potential use in medicine [4][5][6][7][8][9][10][11][12].The genus is having a long history in traditional medicine as a remedy for the treatment of rheumatism, ulcers, treatments for aches, inflammation, and abortificient [13,14].Previous phytochemical studies shows that plants of the genus contain a large number of classes of bioactive secondary metabolites, dominated by A M M P a p e r A c c e p t e d coumarins, flavonoids, lignans, diterpenes and steroids [5,6,9,11,12].
Despite numerous data on the compositional analysis of Daphne species from Serbia (Daphne alpina subsp.alpina [24], Daphne cneorum L. [25], Daphne blagayana L. Freyer [26,27] and Daphne malyana Blečić [28]), with antioxidant capacity determined and correlated to coumarins, phenolic acids and flavonoids, similar investigation was never published for D. mezereum (except the congress announcement we have reported in 2022 [29]).Therefore, the aim of our study was to provide information on antioxidant activities of extract obtained from flowers by maceration with diethyl-ether.Along with this investigation we are reporting data on two simple coumarins we have isolated from D. mezereum whose antioxidant properties we also tested in lipid peroxidation assay.

Chemicals
All of the starting materials, standards and solvents were of analytical reagent grade, obtained from commercial sources and used without further purification (unless specified otherwise, all chemicals were purchased from Merck (Darmstadt, Germany)

Isolation of simple coumarins
Dry DE extract was sub-fractioned according to the modified procedures given in Komissarenko et al., 1994 [30] and in Ness et al., 1996 [31] with hot water, filtrated, and re-extracted with chloroform.Upon drying, chloroform extract was evaporated to give 1.

Lipid peroxidation inhibition by thiobarbituric acid-malondialdehyde assay
Lipid peroxidation (LP) is a free radical mediated chain reaction that once initiated results in oxidative degradation of polyunsaturated lipids.
The final product of lipid peroxidation is malondialdehyde (MDA), a short-chain aldehyde, which is a biochemical marker of cell membrane oxidative damage [33].
Lipid peroxidation and the LP inhibition in the presence of the tested compounds, was measured by thiobarbituric acid-malondialdehyde (TBA-MDA) assay according to the procedure given in Lazarević et al., 2020 [34].

A M M P a p e r A c c e p t e d
The composition of the extract was analyzed and confirmed with HPLC-DAD, by the comparison of the retention time and UV spectrum with the coumarin standards, and by recording 1 H and 13 C NMR for the isolated compounds.The obtained spectral data have confirmed the identity of the isolated coumarins.These data coincide well with the previous reports [35,36] and fully assigned 1 H and 13 C spectra are presented in Figure 2.

Conclusion
e r A c c e p t e d of the solution was transferred into a 5-mm Wilmad, 528-TR-7 NMR tube.NMR analysis1 H NMR and13 C NMR spectra were recorded on a Bruker AVANCE DPX300 spectrometer at 300 MHz and 75 MHz, respectively.All NMR spectra were recorded at 298 K in DMSO-d6 (isotopic enrichment 99.95%) solution.Chemical shifts (δ) are given as parts per million (ppm), relative to tetramethylsilane (TMS) as internal standard with multiplicity reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet; coupling constants (J) are shown in hertz (Hz); number and assignment of protons.The experimental error in the measured 1H-1H coupling constants was ± 0.5 Hz.HPLC analysisHPLC analysis was performed using the Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump and a diode array detector.The test sample solutions were prepared in methanol diluted up to 100 ppm.The analyses were carried out on a reverse phase Purospher STAR RP-18e column (125 mm x 3 mm, 3.0 μm, Merck, KGaA, 64271 Darmtstadt, Germany) by maintaining column temperature at 30 °C.Mobile phase A was trifluoroacetic acid (0.1%) solution which was prepared by dissolving 1.0 mL of trifluoroacetic acid in 1000 mL of water, and methanol was used as mobile phase B. The injection volume was 10 μL and the flow A M M P a p e r A c c e p t e d rate was 0.5 mL/min.The wavelength was fixed at 254 nm.For crude DE extract (A) the data were acquired by using gradient elution system: 0-10min, 50% A, increasing the ratio of phase B to 90% and decreasing phase A to 10%;10-12 min, holding 10% A and 90% B phase; 12-13 min decreasing of B phase to 50% and increasing A phase to 50%; 13-15 min, holding 50% A and 50% B phase.For subfractionated extract (B) and coumarins (C and D) isolated therefrom, data were acquired by using gradient elution system: 0-18 min, 80% A, increasing the ratio of phase B to 90% and decreasing phase A to 10%;18-20 min, holding 10% A and 90% B phase; 20-21 min decreasing of B phase to 20 % and increasing A phase to 80%; 21-22 min, holding 20% A and 80% B phase.

Table 1 .
Lipid peroxidation inhibition effects of three tested samples (IC50 values given in mM) and of selected antioxidants (IC50 values given in μM) shown that D. mezereum DE extract, by inhibiting the LP process, have antioxidant properties and that this effect can be partially attributed to the presence of simple coumarins A M M P a p e r A c c e p t e d

Funding
Epidemiological studies have correlated the ingestion of coumarin based compounds in the diet with beneficial health effect mainly due to their antioxidant activity.Exhibiting antioxidant activity by inhibiting lipid peroxidation, studied coumarins are representing such compounds.Further phytochemical and pharmacological evaluations are needed before shedding further light on the potential application of D. mezereum.The work was funded by the Ministry of Science and Technological Development of Serbia, Project 451-03-47/2023-01/200113 and Faculty of Medicine, University of Niš Internal project No. 40.tumor associated antigen expression in human melanoma cells.Anticancer Res 1986; 6(4):765-74.e r A c c e p t e d

Table 1 .
The obtained results indicated that all samples [D.mezereum