Porcine pancreatic α amylase

α-Amylase inhibitory effect of COS extract was assessed using the pre-incubation method as described by Geethalakshmi et al. [32 ] from the method adapted from Bernfeld [33 (link)]. Porcine pancreatic α-amylase (Sigma) in ice-cold distilled water (5 unit/ml solution) and potato starch (1 % w/v) in 20 mM phosphate buffer (pH 6.9) with 6.7 mM sodium chloride were used. COS extract (40 μl) was mixed with 40 μl α-amylase and 80 μl of 20 mM phosphate buffered saline (pH 6.9) and pre-incubated for 15 min at 37 °C. Final concentration of COS extract used was 1 to 6.5 mg/ml. Starch (40 μl) was added after the pre-incubation and the reaction mixtures were incubated for 15 min at 37 °C. Dinitrosalicylic acid colour reagent was added (100 μl) to the tubes and incubated at 85 °C for 15 min. Distilled water (900 μl) was added to the tubes and the absorbance was measured at 540 nm. Appropriate blanks and controls were carried out. Acarbose (Sigma) was used as the standard inhibitor.

Gallic acid, rat intestinal acetone powder, porcine pancreatic α-amylase, 4-methylumbelliferone, glucose oxidase kits and 3,5-dinitrosalicylic acid p-nitrophenylbutylrate (p-NPB), oleic acid, phosphatidylcholine, glycodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, porcine cholesterol esterase, porcine pancreatic lipase were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cholesterol test kits were purchased from HUMAN GmbH Co. (Wiesbaden, Germany). Total bile acid kit was purchased from Bio-Quant Co. (San Diego, CA, USA). All other chemical reagents used in this study were of analytical grade.

Squid pens, crab shells and shrimp shells were obtained from Shin-Ma Frozen Food Co. (I-Lan, Taiwan) [45 (link)]. Shrimp head powder (SHP) was acquired from Fwu-Sow Industry (Taichun, Taiwan). Demineralized shrimp shell powder (deSSP) and demineralized crab shell powder (deCSP) were prepared according to the previously described methods [45 (link)]. Acarbose, p-nitrophenyl glucopyranoside and enzymes (yeast α-glucosidase, rat α-glucosidase, porcine pancreatic α-amylase, B. subtilis α-amylase, lysozyme, cellulase, bromelain and papain) were obtained from Sigma Chemical Co., St. Louis, MO, USA. The Macro-Prep High S column was obtained from BioRad (Hercules, CA, USA). All other reagents used were of the highest grade available.

This assay was used to assess the samples α‐amylase inhibitory properties. The squash sample extracts (1 mg/ml) diluted by 10 degrees (50 μl) were mixed with 500 μl of 0.02 mol/L sodium phosphate buffer (pH 6.9 with 0.006 mol/L NaCl) and 100 μl of 0.5 mg/ml porcine pancreatic α‐amylase (9000‐90‐2) (Sigma Aldrich Co., Ltd. #product A3176) and incubated at room temperature for 10 min. Then, 500 μl of 1% starch solution (9005‐25‐8) was added to the reaction mixture. The reaction mixture was incubated for 10 min at room temperature, and 1.0 ml of DNSA dinitrosalicylic acid (609‐99‐4) was added. The reaction was ceased by incubating in a boiling water bath for 5 min and cooled to room temperature. Then, diluted by adding 10 ml of distilled water, and absorbance was measured at 540 nm using Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific Instruments Co., Ltd.). The reference sample included all other reagents mentioned and the enzyme except the test sample. Assays were performed with a range of squash phenol and protein contents.
The α‐amylase inhibitory activity was expressed as percentage inhibition (Worthington, Weddell, & Neilson, 1993). $\text{Inhibition}\left(\%\right)=\left(\frac{{\text{Abs}}_{\text{ref}}-{\text{Abs}}_{\text{sample}}}{{\text{Abs}}_{\text{ref}}}\right)\times 100$
${\text{Abs}}_{\text{ref}}=\text{Absorbance of the reference}$
${\text{Abs}}_{\text{sample}}=\text{Absorbance of the test samples}$

All the catechin standards, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), 2,4,6‐tripyridyl‐s‐triazine (TPTZ), 2,2’‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt (ABTS), porcine pancreatic α‐Amylase (EC 3.2.1.1), and α‐Glucosidase (EC 3.2.1.20) were obtained from Sigma Chemical Corporation(Missouri, USA). Acarbose was purchased from Tokyo Chemical Industry Corporation. (Tokyo, Japan). Other chemicals and reagents used in the experiments were analytical grade or chromatographic grade.

d-(+)-Glucose, d-allose, d-galactose, Ag₂CO₃, L-cysteine methyl ester hydrochloride, phenyl isothiocyanate, pyridine, iPrOH, MTBE, porcine pancreatic α-amylase, acarbose, dinitrosalicylic acid, ACN and MeOH HPLC grade, and n-hexane, CHCl₃, MeOH, ACN, (CH₃)₂CO and EtOAc technical grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nano-pure water HPLC grade (18.2 MΩcm) was obtained from a NANO pure purification system (Barnstead/Thermolyne, Dubuque, IA, USA). Silica gel 60 F254 TLC plates, HCl and H₃PO₄ were purchased from EMD Millipore, Inc. (Darmstadt, Germany). Silica gel 60 Å 40-63 µm and AcOH glacial were purchased from VWR International LLC (West Chester, PA, Switzerland), while RP-C₁₈ Cosmosil 140 and formic acid were purchased from Nacalai Tesque, Inc., (Kyoto, Japan) and Alfa Aesar (Ward Hill, MA, USA), respectively. MeOH and pyridine NMR (perdeuterated) solvents were purchased from Cambridge Isotope Laboratories (Andover, MA, USA).

Acarbose, ranirestat, porcine pancreatic α-amylase, rat intestine acetone powder, aldose reductase, extra pure starch, dinitrosalicylic acid (DNS), p-nitrophenyl glucopyranoside (pNPG) and 2-chloro-4-nitrophenyl α-d-maltotrioside were obtained from Sigma-Aldrich, St. Louis, MO, USA. All other chemicals and reagents used are of analytical grade. Carpobrotus edulis leaves collected from the Agricultural Research Council—Vegetables, Industrial and Medicinal Plants campus in Pretoria, South Africa with voucher specimen Mulaudzi RB# 200 deposited in Bews Herbarium, University of KwaZulu-Natal, as described by Mulaudzi et al. [14 (link)] were lyophilised and ground into fine powders using a rotor mill (Fritsh Pulverisette 14, Labotec, Midrand, South Africa).