Dear Radia,

For the best method to prepare pine needles from antioxidant enzyme assays, please read the following text:

In this study, we evaluated the antioxidant activity of pine needle extracts prepared with hot water, ethanol, hexane, hot water-hexane (HWH), and hot water-ethanol (HWE), using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical method. The hot water extract possessed superior antioxidant activity than the other extracts. We also compared the antioxidant activity of pine needle extracts through ROS inhibition activity in a cellular system using MC3T3 E-1 cells. The hot water extract exhibited the lowest ROS production. The pattern of HPLC analysis of each extract indicated that the hot water extract contained the highest proanthocyanidin level. The pine needle hot-water extract was then isolated and fractionated with Sephadex LH-20 column chromatography to determine the major contributor to its antioxidant activity. The No.7 and 12 fractions had high antioxidant activities, that is, the highest contents of proanthocyanidins and catechins, respectively. These results indicate that the antioxidant activity of procyanidins from the hot water extract of pine needles is positively related to not only polymeric proanthocyanidins but also to monomeric catechins. Moreover, the antioxidant activity of the pine needle hot water extract was similar to well-known antioxidants, such as vitamin C. This suggests that pine needle proanthocyanidins and catechins might be of interest for use as alternative antioxidants.

Preparation of pine needle extracts

Pine needles were ground mechanically into a powder form using a mortar and pestle. They were extracted in a manner similar to previous studies [19]. One kilogram of the pine needle powder was extracted with 2L of distilled water, ethanol, and hexane at 80℃ for 12 h to obtain the hot water, hexane, and ethanol extracts, respectively. The water-insoluble fraction was collected and then extracted with ethanol (HWE) and hexane (HWH), respectively. After 12h, each extract was filtered with Whatman filter paper, the solvents were then evaporated under reduced pressure, and the solid residue was lyophilized. The extracts were stored at -20℃ until used

this text was taken from a publication entitled "Antioxidant activity and analysis of proanthocyanidins from pine (Pinus densiflora) needles "  by Park et al. published in Nutr Res Pract. 2011 Aug; 5(4): 281–287.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180677/

Hoping this will be helpful,

Rafik

Dear Rafik

Thanks alot for your quick and complete answer, this was very helpful.

Kind regards,

Radia