Hystar 3

Development and optimization of the analytical method for the quantification of quinine was carried out by high-performance liquid chromatography (HPLC), using a Dionex UltiMate 3000 instrument (Thermo Scientific, Waltham, MA, USA), equipped with a photodiode array detector model DAD-3000 (RS), coupled to an Amazon Speed Ion-Trap mass spectrometer analyzer (AmaZon speed, Bruker, Billerica, MA, USA) with an electrospray ionization source (ESI). The system was controlled using Bruker Daltonics HyStar 3.2 Software with an Ethernet data interface. The separations were performed through a C18 reversed-phase column, 150 mm long, 4.6 mm internal diameter, and with a fixed phase of 2.6 μm particle size (AcuarateTM RP-MS column, Thermo Scientific, USA). The mobile phase consisted of solvent A (Water 99.9%/0.1% formic acid) and solvent B (MeOH), with a flow rate of 0.5 mL/min in a gradient mode. The elution gradient was conducted at a constant flow rate of 0.5 mL/min as follows: 0 min, 100% A; 2–15 min, 93% A; 35 min, 60% A; 45–47 min, 30% A; 57 min, initial conditions until 60 min as a re-equilibration step. The sample volume injected was 10 μL. Detection was performed at a wavelength of 330 and 280 nm, for quinine and caffeine (internal standard), respectively. Caffeine was selected as an internal standard due to its structural and chemical similarities to quinine. Importantly, caffeine elutes at a distinct retention time from quinine, effectively preventing peak overlap and ensuring accurate and precise quantification of quinine in the samples. The mass spectrometry conditions were set to operate in positive ionization mode, with the following parameters: ionization capillary voltage was maintained at 4.500 V; the mass range was specified as m/z 100–2000; the nebulizer pressure was adjusted to 26.0 psi; the nitrogen drying gas temperature was set at 200 °C; the flow rate of the dry nitrogen gas was 6.0 L/min; rolling averages were configured to 2 counts; and the number of averages was established at 2, with ion charge control (ICC) activated. Method optimization included the evaluation of linearity, precision, and accuracy. Five quinine standards (3, 25, 50, 75, 100 μg/mL) were prepared from a 2000 μg/mL standard solution. Linearity was assessed by constructing calibration curves, obtaining a determination coefficient (R²) of 0.9990, demonstrating the high precision and reliability of the method.

The UHPLC-MS analysis was conducted using a Bruker Daltonics MaXis Impact (Bruker GmbH, Bremen, Germany) system, which included a Thermo Scientific HPLC UltiMate 3000 setup featuring a Dionex Ultimate quaternary pump and ESI+-QTOF-MS detection. The analysis was performed on a C18 reverse-phase column (Kinetex, UPLC C18, 5 µm, 4.6 × 150 mm, Phenomenex, Torrance, CA, USA) at 25 °C with a flow rate of 0.8 mL/min. An injection volume of 25 microliters was used. The mobile phase consisted of a gradient of eluent A (water containing 0.1% formic acid) and eluent B (methanol/acetonitrile/isopropanol 1:1:1, containing 0.1% formic acid). The gradient profile was as follows: 70% A at min 0, 30% A at min 4, 0% A at min 7, 30% A at min 10, and returning to 70% A at min 13, followed by an additional 2 min with 70% A. The total run time was 15 min. All measurements were conducted in duplicate. If differences were found, a third run was conducted. Finally, the average matrix was considered.
The mass spectrometry parameters were configured for a mass range of 100 to 1000 Da. The nebulizing gas pressure was set to 2.8 bar, the drying gas flow was maintained at 12 L/min, and the drying gas temperature was set to 300 °C. Calibration with sodium formate was performed before each chromatographic run. Instrument control and data processing utilized specific software from Bruker Daltonics, including Chromeleon, TofControl 3.2, Hystar 3.2, and Data Analysis 4.2.

The protein digest was analyzed by the nano-HPLC apparatus Proxeon Easy-nLC (Proxeon, Odense, Denmark) coupled by a nanoelectrosprayer to ultrahigh-resolution quadrupole-time of flight mass spectrometer MaXis Q-TOF (Bruker Daltonics, Bremen, Germany). The software packages used for controlling the instruments were HyStar 3.2 and micrOTOF-control 3.0, and ProteinScape 3.0 and DataAnalysis 4.0 for data collection and manipulation (Bruker Daltonics).
The samples (5 μL) were injected into precolumn (trap column) NS-MP-10 Biosphere C18 (particle size: 5 μm, pore size: 12 nm, length: 20 mm, inner diameter: 100 μm) and column NS-AC-12dp3-C18 Biosphere C18 (particle size: 3 μm, pore size: 12 nm, length: 200 mm, inner diameter: 75 μm) both prepared by NanoSeparations (Nieuwkoop, Holland).
The separation was done by a linear gradient between water (phase A) and acetonitrile (phase B) both containing 0.1% (v/v) formic acid. The elution started by mobile phase consists with 5% B, next 5 min followed by a gradient elution to 7% B, after by gradient elution 30% B at 180 min. Next 10 min the column was eluted by a gradient to 50% B, and the last 10 min eluted by a gradient to 100% B. Finally, the column was washed with 100% B for 20 min. The column was equilibrated between runs by 5% B for 10 min. The temperature of separation was an ambient temperature (25 °C) when the flow rate was 0.20 μL/min.
On-line nano-electrospray ionization (easy nano-ESI) was used in positive mode. The ESI voltage was set to +4.5 kV, scan time 3 Hz. The drying gas was nitrogen: flow rate 4 L/min and temperature 180 °C; The nebulizer pressure was set as 100 kPa. The masses were scanned in the range from 50 to 2200 m/z. As the internal mass lock was used a monocharged ion of C₂₄H₁₉F₃₆N₃O₆P₃ (m/z 1221.9906). To enable an accurate molecular mass determination the mass spectra corresponding to each signal from the total ion current chromatogram were averaged.

Chemicals Porcine pancreas α-amylase type VI (EC 3.2.1.1), α-glucosidase type I from Baker’s Yeast (EC 3.2.1.20), 3,5-dinitrosalicylic acid (DNS), p-nitrophenyl-α-D-glucopyranose (PNPG), maltose and acarbose were obtained from Sigma-Aldrich (Paris, France). Soluble starch, sodium dihydrogenphosphate (NaH₂PO₄), sodium potassium tartrate and sodium chloride were purchased from Merck. Analytical grade solvents for extraction and HPLC grade solvents for chromatography were from Scharlau (Barcelona, Spain). HPLC grade water was obtained by an EASY-pure II (Barnstead, Dubuque IA, USA) water purification system. Deuterated solvents were purchased from Armar Chemicals (Döttingen, Switzerland).
General Analytical HPLC separations were carried out on a system consisting of a 1100 series binary high-pressure mixing pump with degasser module, column oven and a 1100 series PDA detector (all Agilent, Waldbronn, Germany). A Gilson 215 liquid handler with a Gilson 819 injection module and 50 μL loop was used as autosampler. The HPLC was coupled to an Esquire 3000 Plus ion trap mass spectrometer equipped with an electrospray (ESI) interface (Bruker Daltonics, Bremen, Germany). Data acquisition and processing was performed using HyStar 3.0 software (Bruker Daltonics). Semi-preparative HPLC separations were carried out on an Agilent 1100 series HPLC system consisting of a 1100 series quaternary low-pressure mixing pump with degasser module, column oven, a 1100 series PDA detector, and an autosampler with a 1000 μL loop. The preparative HPLC system consisted of a Shimadzu SCL-10VP controller and binary pump (LC-8A), a UV–vis SPD-M10A VP detector and Class-VP 6.12 as software. NMR spectra were recorded on an Avance III spectrometer operating at 500 MHz and 125 MHz for ¹H and ¹³C, respectively (Bruker Biospin, Fällanden, Switzerland). A 1 mm TXI probe was used, and data processing was performed with Topspin 2.1 (Bruker). Absorbance of enzyme-assay reaction mixture was measured by BioTek microplate reader (XS2).
Plant materialThe aerial parts of S. chloroleuca Rech. f. & Aell. were collected from Shahrestanak, Tehran province of Iran, in June 2008 at an altitude of 2300 m. The plant was botanically identified by Dr. Ali Sonboli of Biology Department of Medicinal Plants and Drug Research Institute, Shahid Beheshti University, Tehran, Iran. Voucher specimen (MPH 845) has been deposited at the herbarium of Medicinal Plants and Drugs, Research Institute, Shahid Beheshti University, Tehran, Iran.
Extraction and isolation Dried leaf material (100 g) was ground with a ZM 1 ultra-centrifugal mill (Retsch, Haan, Germany) equipped with a 0.75 mm Conidur ring sieve, and extracted by successive percolation with n-hexane, ethylacetate and methanol (2 L each). After evaporation to dryness under reduced pressure, 20 g of methanol extract was obtained. The extract was suspended in distilled water and loaded onto a Diaion HP-20 column (5 × 40 cm) i.d. After washing with water, the column was eluted with methanol (3 L), to provide a fraction enriched in phenolic compounds (8.1 g). This fraction was subjected to column chromatography over sephadex LH-20 (2×50 cm) i.d, eluted with methanol. After screening by TLC the obtained fractions with similar compositions were pooled, to yield 5 combined fractions (F1-F5). These main fractions were assayed for their α-amylase and α-glucosidase inhibition activities. The most active fractions were separated by preparative HPLC (SunFire C₁₈, 5 μm, 150 × 30 mm i.d., Waters) with 10-100 % of methanol in water (both containing 0.1 % formic acid), over 40 min at a flow rate of 20 mL/min, and injection volume of 200 µL. Collected peaks from preparative HPLC were evaporated and subjected to semi-preparative HPLC (SunFire C₁₈, 5 μm, 150 × 10 mm i.d., Waters) with 10-100 % methanol in water (both containing 0.1 % formic acid) over 40 min, at a flow rate of 4 mL/min. Several injections yielded compounds 1 (8 mg), 2 (5 mg) from F3 and 3 (6 mg) from F4. The n-hexane extract was separated on silica gel using n-hexane-ethylacetate mixtures as eluent. Fractions obtained with 40% ethylacetate (250 mg) were purified by semi-preparative HPLC, and yielded the known compound salvigenin (4 (link)) (20 mg). The detailed purification process of active components (1 (link)-4 (link)) was performed by the flowchart scheme described in Figure 1.
Luteolin 7-O-glucoside (1)¹H NMR (500 MHz, DMSO-d₆) δ 3.16-3.46 (m, sugar-H), 3.69(d, J = 11.0 Hz, H-5″), 5.02 (d, J = 7.4 Hz, H-1″), 6.41 (d, J = 2.0 Hz, H-6), 6.67 (s, H-3), 6.74 (d, J = 2.0 Hz, H-8), 6.87 (d, J = 8.3 Hz, H-5′), 7.37-7.40 (m, H-2′,6′). UV λ_max 254 nm, 350 nm. MS (m/z) 447.1 [M-H]^-.
Luteolin 7-O-glucuronide (2)¹H NMR (500 MHz, DMSO-d₆) δ 3.28-3.51 (m, sugar-H), 3.98 (d, J = 9.3 Hz, H-5″), 5.23 (d, J = 7.2 Hz, H-1″), 6.45 (d, J = 2.0 Hz, H-6), 6.70 (s, H-3), 6.79 (d, J = 2.0 Hz, H-8), 6.91 (d, J = 8.5 Hz, H-5′), 7.40-7.45 (br s, H-2′, 6′). UV λ_max 254 nm, 350 nm. MS (m/z) 461.1 [M-H]^-.
Diosmetin 7-O-glucuronide (3)¹H NMR (500 MHz, DMSO-d₆) δ 3.33-3.45 (m, sugar-H), 3.90 (s, OMe-4′), 4.02 (d, J = 9.6 Hz, H-5″), 5.25 (d, J = 7.3 Hz, H-1″), 6.47 (d, J = 2.0 Hz, H-6), 6.86 (d, J = 2.0 Hz, H-8), 6.93 (s, H-3), 6.95 (d, J = 8.3 Hz, H-5′), 7.55-7.40 (m, H-2′, 6′). UV λ_max 268 nm, 345 nm. MS (m/z) 475.1 [M-H]^-.
Salvigenin (4)¹H NMR (500 MHz, CDCl₃) δ 3.89 (s, OMe-4′), 3.92 (s, OMe-7), 3.96 (s, OMe-6), 6.54 (s, H-8), 6.58 (s, H-3), 7.02 (d, J = 9.0 Hz, H-3′, 5′), 7.84 (d, J = 9.0 Hz, H-2′, 6′). UV λ_max 274 nm, 330 nm. MS (m/z) 329.1 [M+H]⁺.
α-Amylase inhibition assayα-Amylase inhibition activity was assessed by a previously reported procedure with some modifications (17 ). The assay system, which was carried out in 96-well plates, comprised the following components in a total volume of 250 µL: 100 mM sodium phosphate (pH 6.8), 17 mM NaCl, 1.5 mg Soluble starch, 50 µL of inhibitor solution in DMSO at various concentrations (for pure compounds 12.5, 25, 50, 100 and 150 µM), and 10 µL of enzyme solution (25 unit/mL). After incubation at 37 °C for 30 min, the reaction was stopped by addition of 20 µL NaOH (2N) and 20 µL color reagent (4.4 µM of 3,5-dinitrosalisylic acid, 106 µM of potassium sodium tartarate tetrahydrate and 40 µM of NaOH) followed by a 20 min incubation at 100 °C water bath. α-Amylase activity was determined by measuring the absorbance of the mixture, due to the maltose generated at 540 nm. Individual blanks were prepared to correct for the blank ground absorbance, where the enzyme was replaced with buffer as follows:
Corrected absorbance of test sample = Absorbance of sample –absorbance of blank
From the net absorbance obtained, the % (w/v) of maltose generated was calculated from the equation obtained from the maltose standard calibration curve (0–0.1%, w/v, maltose).
Control incubations, representing 100% enzyme activity, were conducted in the same manner replacing the plant extract with DMSO. The percentage of α-amylase inhibition was calculated by the following equations:
$\mathit{\%\; reaction}=\frac{\mathit{mean\; of\; maltose\; in\; sample}}{\mathit{mean\; of\; malt}\mathit{ose\; in\; control}}\times 100$
% α-amylase inhibition activity= 100 - % reaction
α-Glucosidase inhibition assayThe α-glucosidase inhibition was measured according to an earlier reported bioassay method (18 ). The mixture contained 20 µL α-glucosidase (0.5 unit/mL), 120 µL of 0.1 M phosphate buffer (pH 6.9) and 10 µL of test sample at varying concentrations (for pure compounds 5, 10, 15, 30 and 50 µM). The mixed solution was incubated in 96-well plates at 37 °C for 15 min. After preincubation, the enzymatic reaction was initiated by adding 20 µL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9), and the reaction mixture was incubated for another 15 min at 37 °C. The reaction was stopped by adding 80 µL of 0.2 M sodium carbonate solution and then the absorbance was measured by microplate reader at 405 nm. The reaction system without plant extracts was used as control and the system without α-glucosidase was used as blank for correcting the background absorbance. The inhibitory rate of sample on α-glucosidase was calculated by the following formula:
$\mathbf{\%\; Inhibition}=\left(\frac{\mathit{control\; absorbance}-\mathit{sample\; absorbance}}{\mathit{control\; absorbance}}\right)\times 100$
Statistical AnalysisStatistical analyses were done using GraphPad Prism version 5.00 for Windows. Differences were evaluated by one-way analysis of variance (ANOVA) test completed by Tukey’s multicomparison test. Statistical significance was declared at a p<0.05. All assays were performed at least in triplicate and the results were expressed as mean ± standard deviation (SD). IC₅₀ values were determined by plotting a percent inhibition versus concentration curve for all assays.

Tryptic digests were also analyzed on an additional liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) system. In detail, LC-QTOF-tandem MS analysis on a nano reverse phase (RP) column was performed on a maXis HD Q-TOF mass spectrometer equipped with a CaptiveSpray nanoBooster source (both Bruker) coupled to a Ultimate 3000 nano ultra-performance liquid chromatography system (Thermo Scientific, Breda, The Netherlands). The mass spectrometer and the LC were controlled by Hystar 3.2 (Bruker).
One microliter of the tryptic digest (1:7 dilution, dissolved in water) was loaded onto a C18 μ-pre column (PepMap100; 300 μm × 5 mm, 5 μm, 100 Å, Thermo Scientific) with 10 μL/min of 99% water/ 1% ACN/ 0.05% TFA for 5 min. (Glyco-) peptides were separated on a C18 analytical column (Acclaim PepMap RSLC; 75 μm × 15 cm, 2 μm, 100 Å, Thermo Scientific, Breda) and elution was performed at a flow rate of 700 nL/min with buffer A [water containing 0.1% FA (v/v)] and buffer B [80% acetonitrile/20% water containing 0.1% FA (v/v)]. A linear gradient of 3%–40% buffer B in 15 min was applied followed by column washing and reconditioning.
The CaptiveSpray nanoBooster was operated with acetonitrile-enriched gas (0.2 bar) and 3 L/min dry gas at 150 °C and a capillary voltage of 1200 V. MS spectra were acquired within a mass range of m/z 50–2800. As before, basic stepping mode was applied for the tandem MS collision energy (100%–50%) each 80% and 20% of the time, respectively, and collision energies were set as a linear curve in a m/z dependent manner ranging from 55 eV at m/z 700 to 124 eV at m/z 1800, for all charge states. In this setup, product-ion spectra were generated from the three most abundant precursors in a range of m/z 550–2800 with an isolation width of 8–10 Da depending on m/z values. MS was performed at a spectra rate of 1 Hz, tandem MS at 0.5 to 2 Hz dependent upon precursor intensity.

The HPLC system (1100/1200 series, Agilent, Waldbronn, Germany) consisted of a binary pump (G1312A), an autosampler (G1329B), and a DAD-detector (G1316A). It was coupled to a HCT Ultra Ion Trap mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray ionization source (ESI). The anthocyanins and copigments were separated on a Luna C18(2) 3 μ column (150 × 2.0 mm, Phenomenex (Torrance, CA, USA)) using water/acetonitrile/formic acid (95/3/2; v/v/v) (eluent A) and water/acetonitrile/formic acid (48/50/2; v/v/v) (eluent B) at a flow rate of 200 μL/min. Gradient elution was performed, starting with 6% eluent B and rising to 35% over 30 min. The level of eluent B was then set to 40% until minute 35, and then 90% until minute 45. This level was maintained for 5 minutes before being reduced to 30% until minute 55. Finally, the initial conditions (6% eluent B) were restored until minute 70. The ESI source was operated in positive mode (anthocyanins), negative mode (copigments) and alternating mode (+/−3000 V), using nitrogen as the nebulizer (50 psi) and drying gas (10 L/min, 365 °C). The sample extracts were dissolved in 2 mL of eluent A. Aliquots of 5 μL of each sample were analyzed by the HPLC-PDA-ESI-MS/MS method described above using the Bruker Hystar 3.2, Bruker ESICompass 1.3 for HCT/Esquire, and Data Analysis Version 3.0 software packages (Bruker Daltonics, Bremen, Germany).