Sharifi-Rad et al. Cell. Mol. Biol.2016, 62 (6): 80-85 Cellular and Molecular ISSN: 1165-158X Biology doi: 10.14715/cmb/2016.62.6.15 Original Research Inhibitory activity on type 2 diabetes and hypertension key-enzymes, and antioxidant capacity of Veronica persica phenolic-rich extracts M. Sharifi-Rad1, G. S. Tayeboon2, J. Sharifi-Rad3, 4*, M. Iriti5, E. M. Varoni6, and S. Razazi7 1 Zabol University of Medical Sciences, Zabol 61663335, Iran 2 Department of Biology, Payamenoor University, PO BOX19395-3697, Tehran, Iran 3 Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol 61615-585, Iran 4 Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol 61615-585, Iran 5 Department of Agricultural and Environmental Sciences, Milan State University, via G. Celoria 2, Milan 20133, Italy 6 Department of Biomedical, Surgical and Dental Sciences, Milan State University, via Beldiletto 1/3, Milan 20142, Italy 7 Department of Chemistry, Sharif University of Technology, Tehran, Iran Abstract: Veronica genus (Plantaginaceae) is broadly distributed in different habitats. In this study, the inhibitory activity of free soluble and conjugated phenolic extracts of Veronica persica on key enzymes associated to type 2 diabetes (α-glucosidase and α-amylase) and hyper- tension (angiotensin I converting enzyme, ACE) was assessed, as well as their antioxidant power. Our results showed that both the extracts inhibited α-amylase, α-glucosidase and ACE in a dose-dependent manner. In particular, free phenolic extract significantly P( < 0.05) inhibited α-glucosidase (IC50 532.97 µg/mL), whereas conjugated phenolic extract significantly (P < 0.05) inhibited α-amylase (IC50 489.73 µg/mL) and ACE (290.06 µg/mL). The enzyme inhibitory activities of the extracts were not associated with their phenolic content. Anyway, the inhibition of α-amylase, α-glucosidase and ACE, along with the antioxidant capacity of the phenolic-rich extracts, could represent a putative mechanism through which V. persica exerts its antidiabetes and antihypertension effects. Key words: Plantaginaceae, α-amylase, α-glucosidase, angiotension I converting enzyme, traditional medicine, ethnopharmacology. Introduction phytochemicals, particularly phenolics, may play a pro- tective role both in diabetes and hypertension (11, 12). Type-2 diabetes mellitus accounts for over 90% of Nonetheless, chronic hyperglycemia in diabetes all instances of diabetes, both in developed and deve- triggers oxidative stress at tissue and organ levels (6), loping countries. About 382 million people are consi- which could be controlled by antioxidant agents. Inte- dered to be living with diabetes all over the world, with rest on natural, dietary antioxidants is currently growing an impressive prediction of 471 million people with the (13-16). Phenolic compounds represent a large group disease by the year 2035 (1). Postprandial hyperglyce- of bioactive phytochemicals which occur in almost all mia has been implicated in the development of insulin medicinal and food plants, because of their very high resistance (2), being one of the first markers of glucose ecological relevance in plant organisms (17). Their in homeostasis deregulation (3). In addition, hypertension, vitro antioxidant power, as well as other pharmacologi- cardiovascular disease and diabetic neuropathy are as- cal activities, have been extensively demonstrated since sociate with this status (4, 5). A therapeutic approach in years (18). the prevention/control of hyperglycemia is through the The genus Veronica is the most important genus delay of glucose absorption by inhibiting important en- belonging to the Plantaginaceae family, with about zymes involved in carbohydrate hydrolysis (α-amylase) 500 species widely diffused in boreal hemisphere and and glucose release from disaccharide (α-glucosidase). present in many parts of the austral hemisphere. This However, some drugs widely used (acarbose and migli- is indicative of high ecological adaptability and genetic tol) to reduce blood glucose level are not exempt from plasticity, with species growing in humid to dry envi- a number of pharmacological side effects (6, 7). There- ronments, from the sea level to alpine biotopes (19). In fore, there exists a continued necessity for alternative, traditional medicine, Veronica species are used as diure- natural α-amylase and α-glucosidase inhibitors with tic, wound healing, antirheumatic and anticancer agents high specificity and less adverse effects. (20-22). In addition, stems and leaves of some Veronica As previously introduced, long term diabetes is ge- species are edible, either raw or cooked (23). Despite nerally associated with hypertension (8), which, in turn, the use of Veronica species in traditional healing sys- represents risk factors for cardiovascular diseases and tems is extensively reported, there exists a paucity of is chronic renal failure (8, 9). Angiotensin-I converting enzyme (ACE) is a zinc metallopeptidase that converts Received March 1, 2016; Accepted May 22, 2016; Published May 30, 2016 angiotensin-I to angiotensin-II, a strong vasoconstric- * Corresponding author: Javad Sharifi-Rad, Department of Pharmacognosy, tor, and breaks down bradykinin, a vasodilator (10). Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran. Inhibition of ACE is considered an effective therapeutic Email: [email protected] approach in the control of hypertension both in diabetic and nondiabetic patients (10). In this context, dietary Copyright: © 2016 by the C.M.B. Association. All rights reserved. 80 M. Sharifi-Rad et al. 2016 | Volume 62 | Issue 6 Biological activities of Veronica persica phenolic-rich extracts. information documenting their pharmacological acti- solution by method of Oyaizu (26). A 3 mL aliquot was vities and mechanisms involved in the observed health mixed with 3 mL of 200 mM sodium phosphate buffer benefits. Veronica persica (common names: birdeye (pH 6.5) and 3 mL of 1% potassium ferricyanide. The speedwell, common field-speedwell, Persian speedwell, mixture was incubated at 45 °C for 25 min and then large field speedwell, bird's-eye or winter speedwell‎‎) is was added 3 mL of 10% trichloroacetic acid. This mix- a flowering plant native to Eurasia. To the best of our ture was centrifuged at 2600 rpm for 15 min. Five mL knowledge, no information is available on the possible of the supernatant were mixed with an equal volume of mechanism(s) of action of V. persica extracts in decrea- distilled water and 1 mL of 0.1% ferric chloride. The sing blood glycemia and management of hypertension. absorbance was measured at 700 nm (A700) in the spec- Hence, the present study aims primarily at investigating trophotometer-UV (Shimadzu A160) and ferric redu- the inhibitory activity of phenolic-rich extracts of V. cing power was subsequently calculated using ascorbic persica on key enzymes involved in diabetes and hyper- acid as standard. tension, i.e. α-amylase, α-glucosidase and ACE. Total antioxidant capacity Materials and Methods The total antioxidant capacity was by 2,2-azinobis(3- ethylbenzothiazoline-6-sulfonate radical (ABTS) Plant material scavenging ability of the extracts according to the Aerial parts (stems, leaves and flowers) of Veronica method described by Sharifi Rad et al. (27). The ABTS persica were collected at flowering stage, in November was generated by reacting ABTS aqueous solution (7 2014, from wild plants in the mountains of Meymand, mM) with K2S2O8 (2.45 mM, final concentration), in the Firuzabad County, Fars Province, Iran. The plant was dark, for 14 h and adjusting the absorbance at 734 nm taxonomically identified by a botanist and a specimen to 0.700 with ethanol. Then, 0.2 mL of appropriate dilu- was deposited at the Herbarium of Pharmacognosy, De- tion of the extracts were added to 2.0 mL ABTS solution partment of the Faculty of Pharmacy affiliated to Zabol and the absorbance was measured at 734 nm in the spec- University of Medical Sciences of Iran. The collected trophotometer-UV (Shimadzu A160) after 15 min. The plant materials were dried in the shade, powdered and trolox equivalent antioxidant capacity (TEAC) was sub- stored at 10 °C until required for analysis. sequently calculated using trolox (a synthetic antioxi- dant) as the standard. Free soluble phenolic extraction Free phenolic extraction was performed according to α-Amylase inhibitory activity a slight modified method described by Chu et al. (24). Phenolic extract dilutions (0-150 µL) and 500 µL Twenty grams of the powdered sample were extracted of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 with 80% acetone (1:5, w/v) and filtered (filter paper M NaCl) containing porcine pancreatic α-amylase (EC Whatman No. 2) under vacuum. The filtrate was then 3.2.1.1) (0.5 mg/mL) were incubated at 25 °C for 15 evaporated by a rotary evaporator under vacuum at min. Then, 500 µL of 1% starch solution in 0.02 M 40 °C until about 90% of the filtrate had been evapo- sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) rated and then lyophilized to obtain a dry extract. The were added to each tube. The reaction mixture was in- residues were kept for conjugated phenolic extraction, cubated at 25 °C for 15 min and stopped with 1.0 mL while extract was kept at -4 °C for next analyses. of dinitrosalicylic acid colour reagent. Afterwards, the mixture was incubated in a boiling distilled water bath Conjugated phenolic extraction for 5 min, and cooled to room temperature (24±1°C). The residue from free extraction was flushed with The reaction mixture was then diluted by adding 10 nitrogen and hydrolyzed with about 30 mL of 4 M mL of distilled water, and absorbance measured at 540 NaOH solution at room temperature (24 ± 1°C) for 1 nm (A540) by using spectrophotometer-UV (Shimadzu h with shaking. Afterward, the pH of the mixture was A160). The α-amylase inhibitory activity was expressed adjusted to pH 2 with concentrated HCl and the conju- as % inhibition (28).