Direct q system

The cell-free synthesized FLG was isolated by immobilized-metal affinity chromatography (IMAC). The nitrilotriacetic acid (NTA) agarose matrices (Qiagen, Germantown, MD) were charged with Ni²⁺ and then washed with deionized water (Direct-Q system, Millipore, Burlington, MA) 10 times. All buffers contain the same composition of 50 mM of monosodium phosphate and 300 mM of sodium chloride with the different imidazole concentrations for each step of purification (10 mM for binding, 20 mM for wash, and 250 mM for elution). The CFPS reaction was transferred to a centrifugal filter unit [Amicon Ultra, MWCO (10 kDa)] and centrifuged at 14,000 × g RCF for 15 min. The filtrate was discarded, and an equal volume of lysis buffer containing protease inhibitors (Thermo Fisher, Waltham, MA) was added to the concentrate and subjected to repeated centrifuge to remove salts and other components in CFPS that possibly interfere with the histidine binding. The tagged proteins were then loaded onto the Ni-NTA matrices and immobilized, washed twice, and eluted by adding an elution buffer containing protease inhibitors. The imidazole in elute was removed by buffer exchange to Tris buffer (pH 8.0) containing protease inhibitors. The purified FLG was stored at −20°C until use.

Fresh flowers of P. suffruticosa Andr. were collected at full bloom stage in April, from the National Flower Garden (137 m altitude, 34°39″ N latitude and 112°27″ E longitude), Luoyang, China. Altogether eight cultivars were collected, namely ‘JYH’ (‘JuanYeHong’), ‘JXQ’ (‘JinXiuQiu’), ‘SHT’ (‘ShanHuTai’), ‘FDB’ (‘FengDanBai’), ‘HH’ (‘HuHong’), ‘YH’ (‘YaoHuang’), ‘BXT’ (‘BaiXueTa’) and ‘WLPS’ (‘WuLongPengSheng’). The plant materials were identified by Professor Huanling Zhang, Peony Institute of Luoyang. Voucher specimens are deposited at the Herbarium of Department of Pharmaceutical Sciences, Henan University of Science and Technology. The water contents of fresh flowers were determined by distillation with toluene [39 ]. Analytical standards of 2-phenylethanol, 1,3,5-trimethoxybenzene, geraniol and citronellol were supplied by Aladdin Chemistry Co., Ltd. (Shanghai, China). Mixed standard of n-alkanes C7-C30 (1000 μg/mL for each alkane component in hexane) from Supelco (Bellefonte, PA, USA) was used as received. Deionized water was prepared with Millipore Direct-Q system. Other reagents were of analytical grade.

Standard compound naringenin were purchased from Sigma-Aldrich (St. Louis, MO, United States), and formononetin, pseudobaptigenin, onogenin, sativanone, medicarpin, and maackiain were purified from hydrolyzed extracts of Ononis spinosa root in our laboratory. The isoflavone glucoside standards (formononetin-, pseudobaptigenin-, onogenin-, sativanone-, maackiain-, and medicarpin glucoside) were isolated in our laboratory, too (Gampe et al., 2020 (link)). High-performance liquid chromatography (HPLC) and Mass spectrometry (MS)-grade methanol and acetonitrile were purchased from Fischer Scientific Co. (Fair Lawn, NJ, United States); LiChropur formic- and acetic acid were obtained from Merck (Darmstadt, Germany). Purified water was prepared using a Millipore Direct-Q system (Millipore Corp., Bedford, MA, United States).

Lippia sidoides EO was purchased from PRONAT (Produtos Naturais do Nordeste LTDA, Horizonte, Ceará, Brazil). The constituents of the NLCs were Gelucire 50/13 (Gattefossé, Saint-Priest, France), oleic acid (LabSynth, Diadema, São Paulo, Brazil), Labrasol (Gattefossé, Saint-Priest, France), Span^® 80 (Sigma-Aldrich, Darmstadt, Germany), Tween^® 80 (LabSynth, Diadema, São Paulo, Brazil), and Poloxamer 188 (Kolliphor P 188 Micro, kindly donated by BASF, Ludwigshafen, Germany). Mixtures of whey protein (Arla Foods Ingredients S.A., Buenos Aires, Argentina), arabic gum (Nexira, São Paulo, Brazil), and colloidal silicon dioxide (Aerosil 200, Evonik Degussa, Essen, Germany) were used as spray-drying carriers. ultrapure water (Direct Q System, Millipore, Merck KGaA, Darmstadt, Germany) was used in the preparation of NLCs. Other chemicals and solvents used were of pharmaceutical or of high performance liquid chromatography (HPLC) grade.

Methanol ≥99.9% was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and acetone was from Carlo Erba Reagents (Val de Reuil CEDEX, France). EM, 2,3-Butanedione 97% (DA), 2,3-Pentanedione 97% (AP), N,OBis(trimethylsilyl)trifluorocetamide (BSTFA) 1%–TMCS 99%, and 3-methoxyphenethyl alcohol (internal standard—ISTD) were purchased from Sigma-Aldrich (Steinheim, Germany). The reagent o-Phenylenediamine 98% was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Nicotine, propylene glycol (PG), and vegetable glycol (VG) were provided by NOBACCO (Koropi, Greece). Ultrapure water was produced by a Millipore Direct-Q System (Molsheim, France).

Glycerol (Sigma-Aldrich, Burlington, MA, USA) and polyethylene glycols (TatChemProduct, Kazan, Russia) of various molecular mass 300 Da (PEG 300), 6000 Da (PEG 6000), and 40,000 Da (PEG 40,000) were chosen as crowding agents. For experiments with crowding systems the Glycerol/water and PEG/water solutions were prepared with agitation by magnetic stirrer. Water solutions were prepared using mQ-water (Direct-Q System, Millipore, Andover, MA, USA). The concentrated stoke solutions of lyophilized BSA (50 mg/mL) and α-casein (50 mg/mL) (Sigma-Aldrich, Burlington, MA, USA) were prepared in 100% D₂O. Aliquots of 120 μL protein stock solution were poured into crowding agent–water (H₂O) mixtures (480 μL) to attain total volume 600 μL and protein concentration 10 mg/mL. For all protein solutions, the pH was adjusted by microliter amounts of NaOH and HCl to a physiological value 7.4. Such a pH value was used to prevent aggregation of proteins [61 (link),104 (link)]. All experiments were conducted at 298 K.