Acetonitrile

The samples of the worker bees and the brood were lyophilized using a Labconco Freezone 2.5 freeze dryer (Labconco, USA) (pressure: 0.024 mbar; temperature: 50 °C, time: 168 h).
Analytical portions of 5 g of the lyophilized animals or honey were shaken with 10 mL of acetonitrile (Chempur, Poland). Then, a mixture of salts containing 4 g of anhydrous magnesium sulfate (VI) (Chempur, Poland), 1 g of sodium chloride (Chempur, Poland), 1 g of trisodium citrate (Chempur, Poland), and 0.5 g of sesquihydrate disodium hydrogen citrate (Chempur, Poland) was added. The contents were shaken for 2 min and centrifuged for 5 min at 4500 rpm at 21 °C. Six millilitres of the acetonitrile phase was transferred to a polypropylene test tube that contained 150 mg of PSA (primary secondary amine) (Agilent, USA) and 900 mg of anhydrous sodium sulfate(VI) (Chempur, Poland). The extract was vigorously shaken for 2 min and centrifuged for 5 min as described above. Four millilitres of the obtained extract was taken and transferred to a glass tube, evaporated to dryness on a Heidolph Efficient Labware 4000 rotary evaporator (Heidolph, Germany), and then dissolved in 4 mL of petroleum ether (Chempur, Poland).

TPTZ reagent was purchased from Fluka Analytical (Buchs, Switzerland), formic acid from Fisher Scientific (Waltham, MA, USA), DPPH^* and ABTS^*+ reagents, as well as pectinase enzyme from Rhizopus sp. were supplied by Sigma-Aldrich Company (Saint Louis, MO, USA). Acetonitrile, methanol, Folin-Ciocalteau reagent, and acetate buffer (pH 3.6) were purchased from Chempur (Piekary Śląskie, Poland), and sodium carbonate from POCH (Gliwice, Poland).

Deionized water, purified with the Direct-QUV (Millipore, France) system, was used for all aqueous solutions. Acetonitrile and methanol of HPLC grade were purchased from Chempur (Piekary Śląskie, Poland). Phosphoric acid was purchased from Sigma-Aldrich (Switzerland); sodium hydroxide, from P.P.H. Stanlab (Lublin, Poland). Disodium hydrogen phosphate and monosodium phosphate were purchased from POCH S.A (Gliwice, Poland).
The pterin standards L-biopterin, D-neopterin, pterin, pterin 6-carboxylic acid, isoxanthopterin and xanthopterin were purchased from Dr B. Schircks Laboratories (Jona, Switzerland).

Acetone (HPLC grade) came from Sigma-Aldrich (Poznań, Poland); Acetonitrile (HPLC grade) came from Chempur (Piekary Śląskie, Poland); Carotenoids, vitamin C, chlorophylls, and phenolics standards (HPLC grade 99.5–99.9% pure) came from Fluka and Sigma-Aldrich (Poznań, Poland): β-carotene, caffeic acid, chlorogenic acid, chlorophyll a, chlorophyll b, dehydroascorbic acid, ferulic acid, gallic acid, kaempferol-3-O-glucoside, lutein, l-ascorbic acid, p-coumaric acid, quercetin, quercetin-3-O-rutinoside; Ethyl acetate came from Sigma Aldrich (Poznań, Poland); Magnesium carbonate (ultrapure) came from Chempur (Piekary Śląskie, Poland); Meta-phosphoric acid came from Sigma-Aldrich (Poznań, Poland); Methanol (HPLC grade) came from Chempur (Piekary Śląskie, Poland); Ortho-phosphoric acid came from Chempur (Piekary Śląskie, Poland).

The following were used: cyclopentanone (Sigma-Aldrich, St. Louis, MI, USA, ≥99%), cyclohexanone (Sigma-Aldrich, ≥99%), cycloheptanone (Sigma-Aldrich, ≥99%), cyclooctanone (Sigma-Aldrich, ≥98%), cyclododecanone (Merck, Rahway, NJ USA, ≥98%), 2-methylcyclohexanone (Aldrich, St. Louis, MI, USA, ≥98%), 3-methylcyclohexanone (Aldrich, ≥97%), 4-methylcyclohexanone (Aldrich, ≥99%), cyclohexanol (Sigma-Aldrich, >99%), cyclohexane (Sigma-Aldrich, >99%), manganese(II) acetylacetonate (Sigma-Aldrich, 99%), cobalt(II) acetylacetonate (Sigma-Aldrich, 97%), manganese(II) acetate tetrahydrate (Sigma-Aldrich, 99.99%), cobalt(II) acetate tetrahydrate (Sigma-Aldrich, 99.99%), manganase(II) nitrate tetrahydrate (Sigma-Aldrich, ≥97%), cobalt(II) nitrate hexahydrate (Sigma-Aldrich, >99%), isopentyl nitrite (Sigma-Aldrich, 96%), tert-butyl nitrite (Sigma-Aldrich, 90%), N-hydroxyphthalimide (Acros, Waltham, MA, USA, 98%), methyl ethyl ketone (Sigma-Aldrich, 99.7%), nitric acid (Sigma-Aldrich, 60%), pentyl nitrite (Sigma-Aldrich, 98%), acetic acid (Chempur, Karlsruhe, Germany, 99.5%), acetonitrile (Chempur, 99.5%), benzonitrile (Sigma-Aldrich, 99%), sulfuric acid (Chempur, 98%), and methanol (Chempur, 99.8%).

Raw cilostazol produced by Glenmark (Mumbai, India) was kindly gifted by Przedsiębiorstwo Farmaceutyczne Lek-Am Sp. z o.o. Vivapharm HPMC E6 (Hypromellose 2910) was donated by JRS Pharma (Rettenmaier Polska Sp. z o.o., Warsaw, Poland). Other materials were purchased commercially. P.a. grade glacial acetic acid, acetonitrile, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and methanol were obtained from Chempur (Piekary Śląskie, Poland) and poly(ethylene glycol) average MW 400 g/mol (PEG400) was obtained from Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA). Poly(vinyl alcohol) MW 9-10 kDa 80% hydrolyzed (PVA 9–10 kDa), poly(vinyl alcohol) average MW 30–70 kDa 87–90% hydrolyzed (PVA 30–70 kDa), Kolliphor P188 (poloxamer 188, PX188) and Kolliphor P407 (poloxamer 407, PX407) were purchased from Sigma Aldrich (Merck Life Science Sp.z.o.o., Poznan, Poland). Hydroxypropyl methylcellulose HPMC E50 and dioctyl sulfosuccinate sodium salt 96% (DOSS) were obtained from Alfa Aesar (Ward Hill, MA, USA). Hydroxypropyl methylcellulose 86 kDa HPMC K4M, polyvinylpyrrolidone average MW 50 kDa (PVP K30), hydroxypropyl cellulose average MW 100 kDa (HPC) were obtained from Acros Organics and sodium dodecyl sulfate Ph. Eur. grade (SDS) was purchased from Pol-Aura (Dywity, Poland).

Cyclohexanone (Grupa Azoty S.A., Puławy, Poland, ≥99.9%), cyclohexane (Grupa Azoty S.A., Puławy, Poland, ≥99,9%), cyclohexanol (Grupa Azoty S.A., Puławy, Poland, ≥99.9%), manganese(II) acetylacetonate (Sigma-Aldrich, 99%), cobalt(II) acetylacetonate (Sigma-Aldrich, 97%), sulfuric acid (Chempur, 95%), methanol (Chempur, 99.8%), acetic acid (Chempur, 99.5%), acetonitrile (Chempur, 99.5%), benzonitrile (Chempur 99.5%), N-hydroxyphthalimide (Sigma-Aldrich, 97%).