Agrobacterium Enhances Xanthone Production in Hypericum Perforatum Cell Suspensions

Supplementary material

Agrobacterium enhances xanthone production in Hypericum perforatum cell suspensions

Plant Growth Regulation

Oliver Tusevskia, Jasmina Petreska Stanoevab, Marina Stefovab, Sonja Gadzovska Simica*

aDepartment of Plant Physiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, P.O. Box 162, 1000 Skopje, Macedonia.

bDepartment of Analytical Chemistry, Institute of Chemistry, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, P.O.Box 162, 1000 Skopje, Macedonia.

*Corresponding author. Tel: +389 2 3249 801; +389 78 22 00 65; Fax: +389 2 3228 141.

E-mailaddress: (Sonja Gadzovska Simic)

Material and methods

Quantification of secondary metabolites and antioxidant activity

Total phenolic (TP) content

An aliquot of the diluted extract (100 μL) was mixed with 500 μL Folin–Ciocalteu reagent (1:9, v/v) and 400 μL 0.7 M Na2CO3. Samples were incubated for 5 min at 50ºC and then cooled for 5 min at room temperature. Absorbance was measured spectrophotometrically at 765 nm. The standard of gallic acid was prepared in the same manner and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (mg GAE·g-1 DW).

Total flavonoid (TF) content

A 100 μL aliquot of appropriately diluted extract was mixed with 400 μL distilled water, then 30 μL 5% NaNO2 was added, and allowed to react for 5 min. Following this, 30 μL 10% AlCl3 was added and the mixture stood for further 5 min. Finally, to the reaction mixture 200 μL of 1 M NaOH and 240 μL distilled water were added, and the absorbance at 510 nm was obtained against blank prepared similarly, by replacing extract with distilled water. The TF content was calculated from a calibration curve using catechin as a standard. The results were expressed as milligrams of catechin equivalents (CE) per gram of dry weight (mg CE·g-1 DW).

Total flavanol (TFL) content

An aliquot of the diluted methanolic extract (40 μL) was mixed with 200 μL 0.1% 4-(dimethylamino)-cinnamaldehyde (DMACA). A solution of DMACA was prepared in a mixture of 1M HCl with CH3OH at a 1:9 (v/v) ratio. The samples were incubated for 10 min. at room temperature and absorbance was measured at 640 nm. The concentration of TFL was calculated using catechin as a standard. The results were expressed as milligrams of catechin equivalents (CE) per gram of dry weight (mg CE·g-1 DW).

DPPH radical scavenging activity

In the presence of an antioxidant, the purple colour of DPPH• decays, and the change of absorbance can be followed at 517 nm. The reaction mixture consisted of 10 μL extract and 290 μL 0.25 mM DPPH methanolic solution. A control sample was included, in which extract was replaced by methanol. The reaction for scavenging DPPH• was carried out at room temperature in the dark for 10 min, and then the reduction in absorbance was recorded at 517 nm against methanol blank. Ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and catechin were used as positive controls. The radical scavenging activity was calculated as a percentage of DPPH• discolouration.

Supplementary Fig. S1. Linear correlations measured between the DPPH radical scavenging activity and total phenolics (a), flavonoids (b) and flavanols (c) in H. perforatum cell cultures.