2-Methoxybenzoic acid → Salicylic Acid

Temperature

-80 °C

Salicylic acid was extracted and analyzed by HPLC using a modification of the methods described in Meuwly et al., 1993. For salicylic acid analysis, 0.3 to 0.5 gFW leaf tissue from E. orontii-infected leaves (7 dpi) was frozen in glass tubes with liquid nitrogen and either used directly or stored at −80° C. The frozen tissue was ground in liquid nitrogen to a fine powder using a chilled glass rod. Three ml of 90% methanol and 250 ng o-anisic acid (internal standard) were added to each sample. Samples were vortexed, sonicated for 20 minutes, and centrifuged for 20 minutes at 3000 rpm in a table-top centrifuge. The supernatant was transferred to a new tube, and the pellet was re-extracted with 2 ml of 90% methanol. The two supernatants were combined, divided into two portions of equal volumes (for total SA and free SA measurements), vacuum dried, and frozen at −80° C. For total SA, 500 μl β-glucosidase (80 U/ml in 100 mM sodium acetate, pH 5.2; Sigma, St. Louis, Mo.) was added to each sample. The samples were sonicated for 5 minutes, vortexed, covered with foil, and incubated for 90 minutes at 37° C. For both total and free SA samples, 2.5 ml 5% trichloroacetic acid was added, and the samples were vortexed, sonicated for 5 minutes, and centrifuged at 3000 rpm for 15 minutes. The supernatant was extracted twice with 2.5 ml of a 1:1 (v/v) mixture of ethyl acetate:cyclopentane. The organic phases were combined, vacuum dried, and frozen at −80° C. Just prior to loading samples on the HPLC, each was resuspended in 250 μl of 20% methanol, vortexed, sonicated for 5 minutes, and filtered through a 0.22 μm nylon filter.

1. extractionSalicylic acid was extracted
2. customorontii-infected leaves (7 dpi)
3. temperaturewas frozen in glass tubes with liquid nitrogen
4. customstored at −80° C
5. customsonicated for 20 minutes
6. customThe supernatant was transferred to a new tube
7. extractionthe pellet was re-extracted with 2 ml of 90% methanol
8. customdried
9. additionFor total SA, 500 μl β-glucosidase (80 U/ml in 100 mM sodium acetate, pH 5.2; Sigma, St. Louis, Mo.) was added to each sample
10. customThe samples were sonicated for 5 minutes
11. waitincubated for 90 minutes at 37° C
12. additionFor both total and free SA samples, 2.5 ml 5% trichloroacetic acid was added
13. customsonicated for 5 minutes
14. waitcentrifuged at 3000 rpm for 15 minutes
15. extractionThe supernatant was extracted twice with 2.5 ml of a 1:1 (v/v) mixture of ethyl acetate
16. customvacuum dried
17. temperaturefrozen at −80° C
18. customsonicated for 5 minutes
19. filtrationfiltered through a 0.22 μm nylon
20. filtrationfilter

ORD record

ord-2a124ba7a30241d8aa00be3649b08b3c

Source dataset

uspto-grants-2006_07

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07070772B2