Chemicals and reagents

The following adsorbents were used for purification: Diaion-HP20 (Mitsubishi, Japan) and silica gel 60 (0.04-0.063 mm) (Merck KGaA, Darmstadt, Germany). For the mobile phase, Dichloromethane, methanol, and ethyl acetate were obtained from VWR Chemicals (France).

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Plant material

The aerial parts of Allium saxatile Bieb. were collected in Javakheti, the region of Georgia. The plant was collected and identified by Dr. Tsiala Gviniashvili of the Institute of Botany at Ilia State University. The voucher specimen had been deposited at the same institute (# TBI1034088).

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Extraction and fractionation

The entire plant was dried and milled into particles of 1 mm. The powdered plant was extracted with 80% EtOH using an ultrasonic water bath at 50°C. The extract was dried with a rotary evaporator.

 

The dried extract of the plant was subjected to Diaion HP-20 column chromatography. The mobile phase was H2O-MeOH in gradient conditions and EtOAc to give four enriched fractions: A. saxatile F1, A. saxatile F2, A. saxatile F3, and A. saxatile F4.

 

Isolation

Isolation of compounds was carried out using column chromatography. The stationary phase was silica gel 60 (0.04-0.063 mm) in a dichloromethane/MeOH/water (24:14:3 V/V/V) system.

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Nuclear Magnetic Resonance (NMR)

The ¹H and ¹³C NMR spectra of the obtained compounds were recorded using a Bruker AVANCE NEO 500 MHz NMR spectrometer equipped with a cryoprobe, where deuterated methanol was used as the deuterated solvent.

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Nuclear Magnetic Resonance (NMR)

The ¹H and ¹³C NMR spectra of the obtained compounds were recorded using a Bruker AVANCE NEO 500 MHz NMR spectrometer equipped with a cryoprobe, where deuterated methanol was used as the deuterated solvent.

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Mass spectrometry

Samples for mass spectrometry were prepared in acetonitrile at a concentration of 100 μg/mL. The experiments were performed on an Acquity ultra-high-performance liquid chromatography (Waters) system equipped with an ACQUITY UPLC BEH C18 column (2.1 mm x 50 mm, 1.7 μm particle size; Waters). The UPLC is coupled to a Synapt XS HDMS Q-TOF mass spectrometer with an ESI source (Waters).  The mobile phase consisted of a mixture of water and acetonitrile, each containing 0.1% formic acid. The LC was performed at a flow rate of 0.4 mL/min, with the following gradient conditions: 5% acetonitrile from 0 to 1 minute, 5–100% acetonitrile from 1 to 28 minutes, and 100% acetonitrile from 28 to 30 minutes. The samples were analyzed using a UPLC-HDMSe workflow with a mass range from 50 to 2000 Da and a collision energy ramp from 20 to 70 V. Data analysis was performed using MassLynx V4.2.

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Chromatographic instrument and conditions

An Agilent Technologies Model 1260 Infinity liquid chromatography system was used for HPLC analyses, which was equipped with a vacuum degasser, a binary pump, an autosampler, and a photodiode array detector (DAD). The system was operated using ChemStation software. The separation was achieved using an Eclipse plus C18 (4.6 x 250 mm; 5 µm) column, with a mobile phase composed of water and acetonitrile (77:23, v/v) at a flow rate of 0.8 mL/min. The injection volume was 10 μL, and all separations were performed at 25°C. UV spectra were recorded in the detection range of 200-400 nm for all peaks, with quantification carried out at a single wavelength of 320 nm for astragalin.

 

Preparation of standard solution

The standard solution of astragalin was prepared in 50% methanol to a final concentration of 1.0 mg/mL. A series of working solutions of astragalin (n=5) was ready to obtain various concentration levels (0.06-1.0 mg/mL). The appropriate volume of astragalin solution was transferred into a 10.0 mL volumetric flask, and the volume was adjusted to 10.0 mL with the mobile phase. All prepared standard solutions were filtered through a 0.45 μm membrane filter (Millipore, ref HVPL04700) before HPLC analyses.

 

Preparation of sample solution

10.0 mg of the crude extract of A. saxatile was weighed and dissolved in 50% methanol to make a final volume of 100.0 mL in a 100 mL volumetric flask. Then, 2 mL of this solution was filtered through a 0.45 µm Millipore filter into an HPLC vial.

 

Validation and assay

The HPLC method developed for analyzing the crude extract of A. saxatile was validated in accordance with the ICH guidelines. The linearity of the method was determined for astragalin using five different concentrations, and calibration curves were constructed. The concentration range was 0.06 to 1.0 mg/mL for astragalin. Calibration curves were constructed by plotting peak areas against concentrations, and the linearity was assessed using least squares regression to calculate the slope, y-intercept, and coefficient of correlation (R2). The limits of detection and quantification were determined based on signal-to-noise ratios of 3:1 and 10:1, respectively. The precision of the method was evaluated for both intra-day and inter-day variability, and the accuracy of the method was assessed using a recovery test. Intra-day precision was calculated from the analysis of six sample solutions prepared independently on the same day. Inter-day precision was evaluated by repeating the same procedure on three other days; the standard deviation and the RSD (Relative Standard Deviation) values were calculated for each day. The recovery test is a method used to evaluate the accuracy of an analytical method. In this case, known quantities of astragalin standard solutions were added to the real samples (crude extract of A. saxatile) at three different concentration levels: 50%, 100%, and 150%. The spiked samples were then analyzed in triplicate using the HPLC method previously described. The recovery was calculated as the ratio of the measured concentration of the spiked sample to the expected concentration, expressed as a percentage of the predicted concentration.