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In Vitro Comparative Assessment of the Inhibitory Effects of Single and Combined Spices Against Glucose-Synthesizing Enzymes

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In Vitro Comparative Assessment of the Inhibitory Effects of Single and Combined Spices Against Glucose-Synthesizing Enzymes Adeyeoluwa et al Tropical Journal of Pharmaceutical Research June 2020; 19 (6): 1209-1214 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v19i6.14 Original Research Article In vitro comparative assessment of the inhibitory effects of single and combined spices against glucose-synthesizing enzymes Temitayo Esther Adeyeoluwa, Fatai Oladunni Balogun, Anofi Omotayo Tom Ashafa* Phytomedicine and Phytopharmacology Research Group, Department of Plant Sciences, University of the Free State, QwaQwa Campus, Private Bag x13, Phuthaditjhaba 9866, QwaQwa, Free State, South Africa *For correspondence: Email: [email protected] Sent for review: 15 November 2019 Revised accepted: 23 May 2020 Abstract Purpose: To assess the individual and synergistic inhibitory activities of five (5) tropical spices, viz, Zingiber officinale Rosc., Xylopia aethiopica (Dun.) A. Rich, Piper guineense Schum & Thonn, Myristica fragrans Houtt. and Allium sativum L. against glucose hydrolysing enzymes. Methods: In vitro assessment of the alpha-amylase and alpha-glucosidase inhibitory activities of cold water, hot water, ethanol and aqueous-ethanol extracts of each of the five spices and their combination was investigated using enzymatic protocols. Acarbose was used as the standard. Results: Only four extracts showed strong inhibition against alpha-glucosidase and weak inhibition against alpha-amylase. Going by their half-maximal inhibitory concentrations (IC50), the combined spices exhibited the best concurrent inhibitory activity against both hydrolysing enzymes (665.83 ± 27.50 µg/mL for alpha-glucosidase) and (1166.86 ± 14.70 µg/mL for alpha-amylase). Conclusion: The findings from this study demonstrate the enhanced antihyperglycemic potentials of the combined spices compared to the individual spices for the management of diabetes. Keywords: Hyperglycemia, Diabetes, Alpha-amylase, Alpha-glucosidase, Zingiber officinale, Xylopia aethiopica, Piper guineense, Myristica fragrans, Allium sativum, Spices This is an Open Access article that uses a fund-ing model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION million people are living with type-2 diabetes in South Africa and there are over 425 million cases Diabetes continues to mount danger globally as worldwide. Undoubtedly, these figures are a result of an increase in the intake of refined expected to significantly increase by the year food and an unhealthy dietary lifestyle. Type-2 2045 particularly amongst the low and average diabetes, also known as insulin-resistant income earners, while more than 50% cases of diabetes, accounts for more than 90% of diabetes mellitus go undiagnosed globally [3]. diabetes cases worldwide [1]. As stated by the International Diabetes Federation [2], over 1.8 -------------------------------------------------------------------------------------------------------------------------------Trop J Pharm Res, June 2020;---------------------- 19(6): 1209 © 2020 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License Adeyeoluwa et al Adverse effects from conventionally used the University of the Free State QwaQwa antidiabetic drugs such as sulfonylurea, incretin, Campus herbarium were AdeMed/01/2018/QHB and thiazolidinedione inhibitors [4] continue to (Ginger), AdeMed/02/2018/QHB (Ethiopian create pressure for the discovery of natural pepper), AdeMed/03/2018/QHB (Black pepper), substances. These natural products have AdeMed/04/2018/QHB (Nutmeg), AdeMed/05/ become popular remedial and health-enhancing 2018/QHB (Garlic). The spices were separately agents because of their known possession of dried, ground and stored in sealed glass bottles secondary metabolites [5]. Health benefits at 4oC until they were to be used. derived from spices are due to these secondary metabolites such as phenolic compounds which Extraction procedure have been reported to be potent anti- hyperglycemic and anti-diabetic agents [6]. Five grams of each powdered spice were extracted separately with 250 mL each of cold Ginger has been reported to be effective and water, hot water (boiled at 90oC), ethanol and safe in the management of hyperglycemia, aqueous-ethanol for 2 days and filtered using obesity, inflammation and oxidative stress [4]. Whatman No. 1 filter paper. Filtrates from the Additionally, a potential glucose-lowering effect ethanol extraction were concentrated under a of Ethiopian pepper on acute hyperlipidemic rats stream of cold air while those from the water and while inhibiting the exponential weight gain of the aqueous-ethanol extractions were evaporated at animals has been reported [7]. Piperine, the 60oC with the use of a hot water bath. An principal alkaloid in black pepper was been appropriate quantity of each dried extract was re- reported to be an effective anti-hyperglycemic dissolved in distilled water (water extracts) or agent at sub-acute doses [8]. Nutmeg has been dimethyl sulfoxide (DMSO) (ethanol and used traditionally in the management of diabetes aqueous-ethanol extracts) to attain a stock in India and other countries [9] while information solution of 1 g/L. on the antioxidative and anti-inflammatory actions of garlic in addition to its traditional use in Evaluation of hypoglycemic activity controlling or managing diabetes over several generations have been reported [10]. Alpha-amylase inhibitory assay The scientific basis for the combination of these Fifty microliters of extract or acarbose (control) at spices in traditional formulations as well as a concentrations of 1.000, 0.750, 0.500, 0.250 and comparative evaluation of the combination of the 0.125 g/L was added to buffer (sodium five spices to each spice was investigated in this phosphate, 0.02M SPB), (pH 6.9), into which α- study. amylase (0.5 g/L) had been included, and incubated for 10 min at 25oC. Afterwards, 50 µL EXPERIMENTAL of starch (one per cent) in SPB solution was introduced into each tube and the mixture re- Chemicals incubated at 25oC for 10 min. The reaction process was terminated with 100 µL DNS colour Acarbose, sodium phosphate buffer, potassium reagent. The tubes were further suspended in phosphate buffer, porcine pancreatic alpha- 100oC water bath for 10 min, cooled and the amylase, starch, 3,5 dinitrosalicylic acid (DNS), strength lessened with distilled water (15 mL). alpha-glucosidase from Saccharomyces The absorbance was read at 540 nm with a WPA cerevisiae, p-nitrophenylglucopyranoside (p- Biowave II spectrophotometer (Biochrom, NPG), sodium carbonate (Na2CO3), and solvents Cambridge, UK) [11]. were obtained from Sigma-Aldrich (South Africa). The extracts’ inhibiting potential (H) evaluated Plant materials adopting Eq 1. Ginger (Zingiber officinale Rosc.), Ethiopian H(%) = {(Ac-Ae)/Ac} x 100 …………… (1) pepper (Xylopia aethiopica (Dun.) A. Rich, black pepper (Piper guineense Schumach. & Thonn), where Ac and Ae represent the absorbance nutmeg (Myristica fragrans Houtt.) and garlic values of control (water or DMSO) and extract, (Allium sativum L.) were purchased in August respectively. 2018 from local herb sellers at Ibadan, Nigeria. The spices were identified and authenticated by Alpha-glucosidase inhibitory assay Prof AOT Ashafa of the Department of Plant Sciences, University of the Free state (UFS). In labelled test tubes, 50 µL of varied Voucher specimens of the spices deposited at concentrations (0.125-1.000 g/L) of the extracts Trop J Pharm Res, June 2020; 19(6): 1210 Adeyeoluwa et al or control (acarbose) was combined with 100 µL version 5.01 for Windows, San Diego, California, of 0.02 M phosphate buffer (PB) (pH 6.9) into USA. which 1.0 U/mL of the α-glucosidase solution had been included. Fifty microliters of 5mM p-NPG RESULTS solution in 0.02 M PB (pH 6.9) was added and o incubated for 30 min at 37 C. The reaction was The Aq.ethanol extract of nutmeg (IC50: 270.75 ± stopped with 2 mL of 0.1M sodium carbonate 9.40 µg/mL) and the cold water extract of garlic and its absorbance read at 405 nm using a (IC50: 351.88±11.10 µg/mL) depicted (p<0.05) WPA Biowave II spectrophotometer [11]. higher α-amylase inhibitory activity when The extracts’ inhibitory potential (H) was compared with the other samples. There was an determined using Eq 1 above. insignificant statistical distinction (p>0.05) between acarbose (IC50: 487.47 ± 12.40 µg/mL) Half maximal concentration (IC50) was obtained and the Aq.ethanol extracts of Ethiopian pepper, from the linear equation as in Eq 2. black pepper, garlic and mixed spices(Table 1). y = mx+c ……………. (2) Aqueous ethanol extract of garlic (IC50: 364.71±21.40 µg/mL) and nutmeg (IC50: 428.28 where (value is 50) is the percent
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