Intestinal acetone powder

All plant samples were tentatively identified to species level by comparison with the reference materials at the herbarium of Queen Sirikit Botanic Garden, Chiang Mai, Thailand, and deposited as voucher specimens (PSRU-RUT001-008) in the collection of Faculty of Science and Technology, Pibulsongkram Rajabhat University (PSRU), Phitsanulok Province, Thailand.
Gallic acid was purchased from Merck (Germany) and folin-ciocalteu reagent was bought from BDH Chemicals Ltd. (Poole, England). The 2,2′-Diphenyl-1-picrylhydrazyl radical (DPPH) and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Fluka (Steinheim, Germany). 2, 2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was derived from SIGMA (Oakville, ON, Canada). Intestinal acetone powder and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle medium and fetal bovine serum were purchased from Invitrogen Corp. (Grand Island, NY, USA). All the solvents and other chemicals were an analytical grade (A.R.).

Intestinal Acetone powder from a rat source of α-glucosidase (EC 3.2.1.20), α-amylase from porcine pancreas type VI-B (EC 3.2.1.1), pancreatin from porcine pancreas, pepsin from the gastric mucosa of pigs, bile from bovine and ovine, TRIS-HCl, acarbose, caffeic acid, chlorogenic acid, (+)-catechin, cinchonine, (+)-catechin, (−)-epicatechin, kaempferol 3-glucoside, naringin, sodium chloride, maltose, formic acid, methanol, and acetonitrile were obtained from Sigma Aldrich (Steinheim, Germany). Acetone, ethanol, ethyl acetate, hydrochloric acid, sodium hydroxide, sodium bicarbonate, iodine, potassium iodide, disodium phosphate, and monosodium phosphate were purchased from Chempur (Piekary Śląskie, Poland). Potato starch and calcium chloride were purchased from POCH (Gliwice, Poland) and glucose test from Biomaxima SA (Lublin, Poland). Quercetin 3-glucoside, quercetin 3-rutinoside, quercetin 3-rhamnoside, and isorhamnetin 3-glucoside were obtained from Extrasynthese (Lyon, France). Procyanidin C1, procyanidin B1, procyanidin B2, neochlorogenic acid, cryptochlorogenic acid, and 3,5-dicaffeoylquinic acid were purchased from PhytoLab (Vestenbergsgreuth, Germany). Kaempferol was purchased from ICN Biomedicals (Costa Mesa, CA, USA). Ultrapurity water was prepared in the laboratory using a Simplicity Water Purification System (Millipore, Marlborough, MA, USA).

One hundred mg of intestinal acetone powder (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 1 mL of 0.1 M maleate buffer (pH 6) and homogenized for 6 min [39 (link)]. This was centrifuged at 3000× g for 30 min, a supernatant was taken, diluted with buffer solution at 1:2 (v/v), and used as an α-glucosidase enzyme solution. This enzyme solution (20 mL), each filtrate (20 mL), and 2% sucrose in maleate buffer (20 mL) were mixed and pre-incubated at 378 °C for 1 h. This was heated in a water bath at 100 °C for 10 min to stop the enzyme reaction, and then 20 mL was taken, mixed with a color reagent (3 mL; Glucose CII-Test Wako, Wako Pure Chemical Industries), and incubated at 378 °C for 5 min. Its absorbance was measured at 505 nm with UV visible spectrophotometer (T60UV, PG instruments Ltd., Lutterworth, UK). In addition, the control group replaced the sample solution with a buffer solution and proceeded in the same manner as described above, and the inhibition rate for the experimental group was calculated.