Extraction, capsule preparation, and analysis
The celery seed extraction was performed using 80% ethanol (Merck,
Germany). The NBP purchased from Langchem, Inc. (Shanghai, China) was
used for the standardization of the celery extract. The celery seeds
were purchased from Imam Pharmacy (Mashhad, Iran). Herbarium of the
School of Pharmacy certified their identity (voucher number:
293-0107-18). Briefly, the extraction process was done as follows. An
amount of 800 g celery seeds were powdered, suspended in 2400 mL
ethanol-water (80/20, v/v), and shaken for 1 hour in the darkness at
room temperature. After filtration, the remaining suspended wet powder
was collected and the abovementioned step was repeated two more times to
complete the extraction process. Finally, the collected liquid was
filtered again by a Buchner filtration set to create a cleaner extract
with higher quality. The extract was sprayed onto the mixture of
AEROSIL® (colloidal silicon dioxide) and maltodextrin
in a fluid bed processor at the bed temperature of 35±5 °C. In the next
step, the wet granules were dried in the fluid bed processor instrument
to decrease the moisture. Finally, the dried granules were powdered and
filled into the capsules. Each patient received four celery seeds (1.34
g extract per day) or placebo capsules per day. An Acme 9000 system
(Young Lin, South Korea) consisting of an SP930D solvent delivery
module, SDV50A solvent mixing vacuum degasser, column oven CTS30, UV730
dual-wavelength UV/VIS detector, and ODSA C18 (4.6 mm×150 mm, 5-µm)
column was applied to the chromatographic determination of NBP. The data
analysis was carried out in Autochro-3000. The column temperature, flow
rate, injection volume, and UV detector were 50°C, 1 mL/min, 20 µL, and
230 nm, respectively. Moreover, the gradient method was used in which
the mobile-phase composition was 20% HPLC-grade methanol in water and
changed to 80% during 20 min. A concentration of 100 µg/mL from the
capsule content was prepared in HPLC-grade methanol and injected into
the HPLC. The concentration of NBP was measured based on the comparison
of the area under the curve with the NBP standard solution.