Multiple pharmacological approaches on hydroalcoholic extracts from different parts of Cynoglossum creticum Mill. (Boraginaceae)

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Multiple pharmacological approaches on

hydroalcoholic extracts from different parts of

Cynoglossum creticum

Mill. (Boraginaceae)

Luigi Menghini, Claudio Ferrante, Gokhan Zengin, Mohamad Fawzi

Mahomoodally, Lidia Leporini, Marcello Locatelli, Francesco Cacciagrano,

Lucia Recinella, Annalisa Chiavaroli, Sheila Leone, Luigi Brunetti & Giustino

Orlando

To cite this article: Luigi Menghini, Claudio Ferrante, Gokhan Zengin, Mohamad Fawzi Mahomoodally, Lidia Leporini, Marcello Locatelli, Francesco Cacciagrano, Lucia Recinella, Annalisa Chiavaroli, Sheila Leone, Luigi Brunetti & Giustino Orlando (2019) Multiple

pharmacological approaches on hydroalcoholic extracts from different parts of Cynoglossum

creticum Mill. (Boraginaceae), Plant Biosystems - An International Journal Dealing with all Aspects

of Plant Biology, 153:5, 633-639, DOI: 10.1080/11263504.2018.1527790

To link to this article: https://doi.org/10.1080/11263504.2018.1527790

Published online: 08 Nov 2018. _{Submit your article to this journal}

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Multiple pharmacological approaches on hydroalcoholic extracts from different

parts of Cynoglossum creticum Mill. (Boraginaceae)

Luigi Menghinia, Claudio Ferrantea, Gokhan Zenginb, Mohamad Fawzi Mahomoodallyc, Lidia Leporinia, Marcello Locatellia , Francesco Cacciagranoa, Lucia Recinellaa, Annalisa Chiavarolia, Sheila Leonea, Luigi Brunettiaand Giustino Orlandoa

a

Department of Pharmacy, University“G. D’Annunzio” of Chieti-Pescara, Chieti, Italy;bDepartment of Biology, Selcuk University, Science Faculty, Konya, Turkey;cDepartment of Health Sciences, Faculty of Science, University of Mauritius, R
eduit, Mauritius

ABSTRACT

Cynoglossum creticum Mill (Boraginaceae) is used traditionally as a remedy to manage several human ailments. In this context, the present study aimed to perform multiple pharmacological investigations on the hydroalcoholic extracts prepared from Cynoglossum roots and aerial parts (leaves and flowers). We evaluated the antioxidant and enzyme inhibitory (against cholinesterases,a-glucosidase, a-amylase, lipase and tyrosinase) activity of the extracts. The protective effect(s) of the extracts on cardiomyocyte C2C12 and intestinal HCT116 cell lines challenged with hydrogen peroxide (H2O2) was studied. We found that the aerial parts harbored the highest amount of phenolic compounds. Generally, aerial parts showed significant antioxidant and enzyme inhibitory effects. Leaves exhibited the best lipase inhibitory activity (173.15 mgOE/g extract), followed by flowers and roots. The root and aerial extracts were equally able to blunt intracellular H2O2 induced reactive oxygen species production from both C2C12 and HCT116 cell lines. Both cells lines could be treated with scalar concentrations of root and flower extracts in the range 50–300 lg/mL without interferences on cell viability. In conclusion, the present study showed protective effects exerted by Cynoglossum extracts, which could serve as a foun-dation for the development of pharmaceuticals and nutraceuticals derived from Cynoglossum.

ARTICLE HISTORY Received 20 February 2018 Accepted 19 September 2018 KEYWORDS Cynoglossum; HPLC-PDA; antioxidant; enzyme inhibition; cell assays; natural agents; cardiomyocyte

1. Introduction

Medicinal plants have long been used and appraised as therapeutic agents due to the presence of bioactive com-pounds, such as phenolics and terpernoids (Zhang and Ma

2018). Such natural products possess great structural and chemical diversity that cannot be compared to any synthetic libraries of small molecules. Drug discovery from natural products has continued to inspire novel discoveries in chem-istry, biology and medicine for both communicable and

non-communicable diseases (Newman and Cragg 2012; Shen

2015). Several reports tend to suggest that herbal medicine and related natural products are gaining much momentum for the treatment and prophylaxis of a panoply of human diseases. Phytochemicals harnessed from traditionally used medicinal plants have been proposed to being evolutionarily optimized as drug-like molecules and remain the best sour-ces of pharmacophores and drug leads to address health challenges (Shen 2015). Several studies have proved that plants are excellent sources of active phytochemicals which can serve as template molecules in the prevention of differ-ent diseases. One such class of bioactive compounds wide-spread in the plant kingdom are the phenolic compounds, known to be responsible for the therapeutic potential of plant species (Mollica et al.2018; Zengin et al.2018a,2018b). Recent studies have focused on the emerging role of

phenolic compounds in the management of several diseases (Zengin et al.2017a,2017b).

The Boraginaceae family includes a wide group of plants (about 2000 species) with potential antimicrobial/antiviral, antitumor and anti-inflammatory activities, possibly related to multiple biologically active molecules such as naphthaqui-nones, flavonoids, terpenoids and phenols (Helmst€adter

2016). Cynoglossum creticum Mill belongs to the

Boraginaceae family and is native, common, and widely dis-tributed in all Italian regions (Selvi and Sutor
y 2012). It is traditionally used as a remedy for cold head and other inflammatory diseases of the upper respiratory tract, such as cough, laryngitis, throat inflammation, against purulent boils, and running sores (Palmese et al. 2001; Helmst€adter 2016). This species has not been investigated pharmacologically but has been reported to contain pyrrolizidine alkaloids (Asibal et al. 1989; El-Shazly et al. 1996). The phytochemical profile has been recently described (Dresler et al. 2017), reporting significant amounts of allantoin, phenolic acids and rutin. Nonetheless, a rigorous pharmacological investigation is still lacking.

With the aim to provide a scientific rational substantiating its medicinal use, the present work was designed to perform multiple in vitro tests on hydroalcoholic extracts from Cynoglossum roots and aerial parts (leaves and flowers). In

CONTACTGokhan Zengin gokhanzengin@selcuk.edu.tr Department of Biology, Selcuk University, Science Faculty, Konya, Turkey.

ß 2018 Societ
a Botanica Italiana

2019, VOL. 153, NO. 5, 633_–639

https://doi.org/10.1080/11263504.2018.1527790

this context, we evaluated acetylcholinesterase (AChE), butyr-ylcholinesterase (BChE),a-glucosidase and a-amylase activity of extracts prepared from different parts. Additionally, we investigated the protective effects of the extracts on cardio-myocyte C2C12 and intestinal HCT116 cell lines challenged with an oxidative stress stimulus (hydrogen peroxide). Finally, in order to identify the putative mechanism, we performed a comparative analytical evaluation on the extracts for the identification and quantification of active phytochemicals. It is anticipated that results gathered herein will open new ave-nues for potential applications in the management of inflam-mation and oxidative stress-related pathologies.

2. Materials and methods 2.1. Plant material and extraction

A representative sampling of wild population of C. creticum was done from plants at full blooming status growing in Chieti (GPS: N 42
2205.71200; E 14
9011.40200). Plant organs were manually divided in roots and aerial parts. After dehy-dration in a ventilated oven in the dark at 40
C, until con-stant weight, plants were grinded and stored in vacuum bags, in the dark until extraction.

Extraction was performed with a mixture of ethanol/water 50/50, at room temperature in a sonicator bath. In order to obtain an exhaustive extraction, each sample was subjected to three extraction cycles with new solvents (Brockmeyer et al. 2015; Menghini et al. 2018). The final plant material/ solvent ratio was 1/25 (w/v). Extracts were freshly prepared just before use.

2.2. Chemicals

Chemical standards: gallic acid, catechin, chlorogenic acid, 4-hydroxybenzoic acid, vanillic acid, epicatechin, syringic acid, 3-hydroxybenzoic acid, 3-hydroxy-4-methoxybenzaldehyde, p-coumaric acid, rutin, sinapinic acid, t-ferulic acid, naringin, 2,3-dimethoxybenzoic acid, benzoic acid, o-coumaric acid, quercetin, 8-cinnamoyl harpagide (harpagoside), t-cinnamic acid, naringenin and carvacrol (>98%) were purchased from Sigma-Aldrich (Milan, Italy). Solvents (acetonitrile (HPLC-grade), methanol (HPLC-(HPLC-grade), acetic acid (99%)) were obtained from Carlo Erba Reagents (Milan, Italy). Centrifuge model 5804 (Eppendorf, Hamburg, Germany), vortex (VELP Scientifica Srl, Usmate, Italy), and ultrasound bath (Falc

Instruments, Treviglio, Italy) were used as

add-itional equipment.

2.3. HPLC analysis

HPLC-PDA analyses were performed by a validated method as reported in the literature (Locatelli et al., 2017; Sobolev

et al. 2018) using an HPLC Waters liquid chromatograph

(model 600 solvent pump, 2996 PDA). The mobile phase was directly degassed on-line using a Biotech 4CH DEGASI Compact (Uppsala, Sweden). Empower v.2 Software (Waters Spa, Milford, MA) was used to collect and analyze data. The

analyses were carried out using gradient elution mode on a C18 reversed-phase column (Prodigy ODS(3), 4.6 150 mm,

5 mm; Phemomenex, Torrance, CA), thermostated at 30
C

(±1
C). The gradient elution was achieved by a solution of water-acetonitrile (93:7 ratio, with 3% of acetic acid) as initial conditions. The complete separation was achieved in 60 min. The gradient elution program used for the analyses is shown below.

The stock solutions of 20 standards were made at concen-tration of 1 mg/mL in a final volume of 10 mL of methanol. Working solutions of mixed standards at the concentrations of 0.25, 0.5, 1, 2.5, 5, 10 and 20lg/mL were made by dilution of stock solution in volumetric flasks with the mobile phase. Then the standards were injected into the HPLC-UV/Vis sys-tem. Calibration curves were obtained at corresponding max-imum detection wavelength for each phenolics, using a series of standard solutions over the concentration range from the LOQ of each analyte to 50lg/mL (upper limit of quantifi-cation). All calibration curves were linear over the concentra-tion range tested with the determinaconcentra-tion coefficients 0.9361. The limits of detection (LOD, S/N¼ 3) were 0.075 lg/mL for each analyte. The LOQ (S/N¼ 10) was 0.25 lg/mL for each analyte at corresponding maximum wavelength. The precision (RSD%) and the trueness of the HPLC determination was in the range ±15% for the studied phenolics at three quality control levels within the linearity range.

2.4. In vitro antioxidant assays

2.4.1. Anti-oxidant properties, total phenolic and total fla-vonoid content

Antioxidant assays (DPPH (1-diphenyl-2-picrylhydrazyl), ABTS (20-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid) radical scavenging, CUPRAC (cupric reducing antioxidant capacity), FRAP (ferric reducing antioxidant power), phosphomolybde-num and metal chelating) were performed as reported in our previous publications (Grochowski et al. 2017; Mocan et al.

2018). Antioxidant capacities were expressed as equivalents of trolox (mmol TEs/g extract). The total phenolic content was also determined by the Folin-Ciocalteu method and expressed as gallic acid equivalents (mg GAEs/g extract), whereas total flavonoids content was determined using AlCl3

method and expressed as rutin equivalents (mg REs/g extract). The possible inhibitory effects of the studied extracts against cholinesterases (by Ellman’s method), tyro-sinase, a-amylase and a-glucosidase were evaluated using

standard in vitro bio-assays according to (Grochowski

et al.2017). TIME FLOW %A %B 0 1 mL/min 93 7 0.1 93 7 30 72 28 38 75 25 45 2 98 47 2 98 48 93 7 58 93 7 634 L. MENGHINI ET AL.

2.4.2. Pharmacological studies

In vitro studies. C2C12 and HCT116 cells were cultured in DMEM (Euroclone) supplemented with 10% (v:v) heat-inacti-vated fetal bovine serum and 1.2% (v:v) penicillin G/strepto-mycin in 75 cm2 tissue culture flask (n¼ 5 individual culture flasks for each condition). The cultured cells were maintained in humidified incubator with 5% CO2 at 37
C. For cell

differ-entiation, C2C12 and HCT116 cell suspensions at a density of 1 106 cells/mL were treated with various doses (10, 50 and 100 ng/mL) of phorbol myristate acetate (PMA, Fluka) for 24 or 48 h (induction phase). Thereafter, the PMA-treated cells were washed twice with ice-cold pH 7.4 phosphate buffer solu-tion (PBS) to remove PMA and non-adherent cells, whereas the adherent cells were further maintained for 48 h (recovery phase). Morphology of cells was examined under an inverted phase-contrast microscope. To assess the basal cytotoxicity of extracts, a viability test was performed on 96-microwell plates, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium brom-ide (MTT) test. Cells were incubated with extracts (ranging con-centration 10–100 lg/mL) for 24 h. Ten microliters of MTT (5 mg/mL) were added to each well and incubated for 3 h. The formazan dye formed was extracted with dimethyl sulfoxide and absorbance recorded as previously described (Menghini et al.2011). Possible effect(s) on cell viability was evaluated in comparison to untreated control group.

ROS generation: ROS generation was assessed using a ROS-sensitive fluorescence indicator, 20,70 -dichlorodihydrofluor-escein diacetate (DCFH-DA). When DCFH-DA is introduced into viable cells, it penetrates the cell and becomes deacetylated by intracellular esterases to form 20,70 -dichlorodihydrofluores-cein (DCFH), which can react quantitatively with ROS within the cell, and be converted to 20,70-dichlorofluorescein (DCF), which is detected by a fluorescence spectrophotometer. To determine intracellular effects on ROS production, cells were seeded in a black 96-well plate (1.5 104 cells/well) in

medium containing 25 mg/mL extracts. Immediately after

seeding, the cells were stimulated for 1 h with H2O2 (1 mM).

After the cells were incubated with DCFH-DA (20lM) for

30 min, the fluorescence intensity was measured at an excita-tion wavelength of 485 nm and an emission wavelength of 530 nm, using a fluorescence microplate reader.

2.5. Statistical analysis

For antioxidant and enzyme inhibitory results, one-way ana-lysis of variance (ANOVA) (with Tukey’s assay) was employed to detect differences among the extracts (p< .05). This statis-tical analysis was calculated by SPSS v. 17.0 program. For cell assays, statistical analysis was performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San

Diego, CA). Means ± S.E.M were determined for each experi-mental group and analyzed by one-way (ANOVA) followed by Newman-Keuls comparison multiple test. Statistical signifi-cance was set at p< .05.

3. Results and discussion 3.1. Bioactive compounds

Phenolic compounds are potential sources of bioactive agents to design functional products, with excellent bio-logical effects such as antioxidant, antimicrobial, and anti-cancer (Gonc¸alves and Romano 2017). In this respect, we investigated the pharmacological potential and phytochem-ical profile of Cynoglossum. As reported inTable 1, the high-est total amount of bioactive components was obtained in leaves, followed by flowers and roots. Particularly, we observed higher concentrations of catechin, epicatechin, chlorogenic, vanillic, 3-OH benzoic, sinapinic, t-ferulic, ben-zoic and o-coumaric acids in aerial parts fraction, compared to the root extract. The root extracts contained a higher con-centration of syringic acid, 2,3-diMeO benzoic acid, naringin, naringenin and above all of quercetin, as compared to the aerial parts (Table 2). These results are consistent with previ-ous phytochemical analyses performed on the methanolic extracts of Cynoglossum (Dresler et al.2017).

3.2. Antioxidant and protective effects

The phytochemical differences between aerial parts and root extracts tend to reflect the observed biological activities in

Table 1. Total phenolic, flavonoid and phenolic acid contents of the samples.

Extracts Total phenolic content (mgGAE/g extract) Total flavonoid content (mgRE/g extract) Total phenolic acid content (mgCE/g extract) Flowers 18.46 ± 0.35b _{4.39 ± 0.15}b _{4.83 ± 0.36}b

Leaves 45.35 ± 1.52a 21.77 ± 0.32a 11.74 ± 0.75a Roots 13.11 ± 0.16c _{0.68 ± 0.05}c _{3.56 ± 0.27}c

Values expressed are means ± S.D. of three parallel measurements. GAE: Gallic acid equivalent; RE: Rutin equivalent; CE: Caffeic acid equivalent. Data marked with different letters (a, b, c) within the same column indicate statistically significant differences in the samples (p<.05).

Table 2. Phenolic components in Cynoglossum extracts (4% w/v).

Phenolic compounds Roots Leaves Flowers

Gallic acid nd nd nd

Catechin 13.43 ± 0.25 6.38 ± 1.04 209.26 ± 7.50 Chlorogenic acid 3.91 ± 0.33 7.47±.0.46 15.83 ± 0.87 p-OH benzoic acid BLD nd nd Vanillic acid 0.50 ± 0.03 nd 1.14 ± 0.05 Epicatechin nd 4.60 ± 1.79 0.34 ± 0.05 Syringic acid 0.68 ± 0.13 nd nd 3-OH benzoic acid 1.09 ± 0.35 nd 2.32 ± 0.21 3-OH-4-MeO benzaldehyde 0.29 ± 0.07 BLD 0.22 ± 0.01 p-Coumaric acid BLD nd nd Rutin BLD 0.27 ± 0.01 1.35 ± 0.04 Sinapinic acid 0.20 ± 0.01 nd 0.98 ± 0.01 t-Ferulic acid 2.19±.07 nd 3.38 ± 0.18 Naringin 0.77 ± 0.42 nd nd 2,3-diMeO benzoic acid 1.15 ± 0.27 nd nd Benzoic acid 3.91 ± 0.21 nd 6.25 ± 0.13 o-Coumaric acid 14.59 ± 0.03 nd 79.31 ± 1.05 Quercetin 48.84 ± 0.03 nd 14.45 ± 0.04 harpagoside Nd nd nd t-Cinnamic acid nd nd nd Naringenin 2.19 ± 0.01 nd 0.66 ± 0.02 Carvacrol nd 0.33 ± 0.03 nd Total (lg/mL) 93.72 ± 1.12 18.88 ± 3.52 335.49 ± 9.56 nd: not detected.

vitro. Both aerial parts and root extracts displayed consider-able antioxidant activity. On the other hand, aerial parts extracts were found to be more effective, as showed by DPPH, ABTS, CUPRAC, FRAP and metal chelating activity bio-assays (Table 3). Apparently, these results could be related to the antioxidant properties of phenolic compounds (Huyut et al. 2017) and this approach was supported by several researchers, who reported a linear correlation between

phen-olic and antioxidant properties (Colak et al. 2017;

Limmongkon et al.2018).

The protective effects of the extracts were also investi-gated on selected C2C12 and HCT116 cell lines. First of all, we identified a concentration range of tolerability through MTT viability test. The results of viability tests indicated that both cells lines could be treated with scalar concentrations of root and flower extracts in the range 50–300 lg/mL with-out interferences on cell viability (Figures 1 and 2). According to the results from the MTT tests, we performed further experiments to evaluate the potential antioxidant

activity of the extracts (50–300 lg/mL) on C2C12 and

HCT116 cells challenged with a standard oxidative stimulus constituted by hydrogen peroxide (1 mM). The results indi-cated that aerial parts and root extracts were equally able to blunt ROS production induced by hydrogen peroxide, des-pite harboring higher phenolic compounds in the aerial parts, as compared to the roots (Figures 3 and 4). By con-trast, we observed that aerial parts extract were less potent compared to roots in blunting hydrogen peroxide-induced ROS production, in HCT116 cell line. This discrepancy could be related to several reasons. On one side, tumoral HCT116 cell line is characterized by a low grade of differentiation (Liu et al. 2017), and a constitutive activity of NF-jB pathway,

deeply involved in inflammation and oxidative stress (Elkady et al. 2016). Additionally, in HCT116 oroxylin, a flavonoid compound, is able to modulate Nrf-2 signaling, which could exert protective effects via the down-regulation of multiple inflammatory pathways, including NF-jB (Hu et al.2012). On the other hand, roots extract displayed higher content of quercetin (3.38-fold higher than in aerial parts), whose pro-tective effects could be due to the stimulation of Nrf-2 activ-ity (Weng et al.2017).

3.3. Enzyme inhibition

Plant secondary metabolites have been previously described as potential sources of novel enzyme inhibitors (Zengin et al.,2017a,2017b). Phenolic compounds, besides its role as antioxidant-antiradical activity have been described as poten-tial enzyme inhibitors (G€ulc¸in et al. 2016; Cespedes et al.

2017). In this context, we evaluated the effects of

Cynoglossum roots and aerial parts extracts on multiple enzymes such as AChE, BChE, a-glucosidase, a-amylase and lipase which are involved in Alzheimer’s disease (AD), type 2 diabetes (T2D) and obesity (Picot et al. 2017; Chen et al.

2018), respectively. AD and T2D are chronic diseases that often occur together in aged individuals. AD is characterized by neurodegeneration associated with progressive behav-ioral, cognitive, and memory function impairment (Avila et al. 2017). On the other hand, T2D is a chronic metabolic disease characterized by peripheral insulin resistance associ-ated with pancreatic b-cell and insulin deficit (Gonzalez-Franquesa and Patti 2017). Epidemiological studies revealed close relationships between AD and T2D onset (Vieira et al.

2017), consistently with the possible involvement of common

Table 3. Antioxidant properties of the samples.

Extracts

Phosphomolybdenum (mmol TE/g extract)

DPPH (mg TE/ g extract) ABTS (mg TE/ g extract) CUPRAC (mg TE/ g extract) FRAP (mg TE/ g extract) Metal chelating activity (mg EDTAE/ g extract) Flowers 0.47 ± 0.02c _{30.32 ± 0.52}b _{49.54 ± 1.01}b _{97.26 ± 1.42}b _{64.70 ± 1.86}b _{4.61 ± 0.22}a Leaves 1.00 ± 0.05a 69.28 ± 0.57a 108.26 ± 1.90a 221.46 ± 7.00a 149.88 ± 0.68a 3.37 ± 1.06b Roots 0.56 ± 0.06b _{21.74 ± 0.61}c _{34.31 ± 0.55}c _{71.46 ± 0.46}c _{43.91 ± 0.89}c _{1.78 ± 0.08}c

Values expressed are means ± S.D. of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA equivalent. Data marked with different letters (a, b, c) within the same column indicate statistically significant differences in the samples (p<.05).

Figure 1. Effect of hydroalcoholic Cynoglossum extract (50–300 lg/mL) on C2C12 cell viability. Data are expressed as means ± SEM. 636 L. MENGHINI ET AL.

physiopathological mechanisms, including inflammation, insulin resistance, and oxidative stress (Bozluolcay et al.2016; Arnold et al.2018; Folch et al. 2018). In agreement with the observed antioxidant effects, extracts from aerial parts, with the highest phenolic content (Dresler et al.2017), turned out to be most effective in inhibiting cholinesterases, a-amylase

and a-glucosidase activity. Particularly, the reported enzyme inhibitory activity of aerial parts could be related, at least partially, with the higher content of catechin, and chloro-genic acid, compared to roots (Zengin et al. 2017a, 2017b). Additionally, the majora-glucosidase activity inhibition, com-pared with a-amylase, is consistent with previous studies

Figure 2. Effect of hydroalcoholic Cynoglossum extract (50–300 lg/mL) on HCT116 cell viability. Data are expressed as means ± SEM.

Figure 3. Effect of hydroalcoholic Cynoglossum extract (50–300 lg/mL) on hydrogen peroxide-induced ROS production from C2C12 cells. Data are expressed as means ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc test. (ANOVA, p< .0001; post hoc p < .001 vs. CTR group.

Figure 4.Effect of hydroalcoholic Cynoglossum extract (50–300 lg/mL) on hydrogen peroxide-induced ROS production from HCT116 cells. Data are expressed as means ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc test. (ANOVA, p< .0001; post hoc p < .001 vs. CTR group.

(Llorent-Mart
ınez et al. 2017) thus further supporting a major

selectivity of phenolic compounds toward a-glucosidase,

rather than a-amylase. On the other hand, phenolic

com-pounds seem to bind in a similar way with both the enzymes

through the formation of non-covalent interactions

(Martinez-Gonzalez et al.2017).

Obesity is a major problem worldwide and effective man-agement strategies are need to decrease its prevalence (Suh et al. 2018). Pancreatic lipase inhibition is a good way to control body weight in obesity. From this point, anti-lipase compounds are considered as effective sources of anti-obes-ity drugs (Buchholz and Melzig2015). To this effect, the anti-lipase activity of Cynoglossum extracts was investigated. As can be seen in Table 4, the leaves exhibited the best lipase inhibitory activity with 173.15 mg OE/g extract, followed by flowers and roots. Tyrosinase is a main catalyst in the synthe-sis of melanin and thus the inhibition of tyrosinase is

consid-ered as an effective strategy for controlling

hyperpigmentation problems (Kim and Uyama 2005). In the present study, observed tyrosinase inhibitory effects can be ranked as leaves> flowers > roots. Taken together these results could be attributed to the presence of phenolic com-pounds. With regard to this fact, researchers have reported that phenolic compounds possess strong inhibitory potential on both pancreatic lipase and tyrosinase (Hsu and Yen2008; Lee et al. 2016). To the best of our knowledge, no study has provided evidence of the enzyme inhibitory potential of Cynoglossum extracts.

4. Conclusion

The present study has been designed to investigate the

chemical characterization and biological properties of

Cynoglossum extracts. The studied extracts were found to be rich in phenolic components such as catechin and chloro-genic acid. The extracts exhibited considerable antioxidant effects as observed from the in vitro and cell assays. In add-ition, the extracts showed promising inhibitory potential against key enzymes linked to major human pathologies. It is anticipated that the present study will provide a stimulus for further studies on this species. Taken together, C. creticum can be considered as a potential source of natural bioactive compounds that could be explored in the design of novel phyto-pharmaceuticals and dietary supplements.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

Marcello Locatelli http://orcid.org/0000-0002-0840-825X

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Table 4. Enzyme inhibitory effects of the samples.

Extracts AChE inhibition (mg GALAE/g extract) BChE inhibition (mg GALAE/g extract) Tyrosinase inhibition (mg KAE/g extract) Amylase inhibition (mmol ACAE/g extract)

Glucosidase inhibition (mmol ACAE/g extract)

Lipase inhibition (mg OE/g extract) Flowers 2.51 ± 0.01 3.18 ± 0.15c 60.47 ± 1.47b 0.30 ± 0.01b 4.57 ± 0.27b 79.73 ± 1.39b Leaves Na 6.90 ± 0.09a _{86.78 ± 0.08}a _{0.77 ± 0.01}a _{5.97 ± 0.32}a _{173.15 ± 3.72}a

Roots Na 3.44 ± 0.06b 38.96 ± 1.27c 0.26 ± 0.03b 4.49 ± 0.09b 79.50 ± 3.29b Values expressed are means ± S.D. of three parallel measurements. GALAE: Galatamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; OE:

Orlistat equivalent; na: not active. Data marked with different letters (a, b, c) within the same column indicate statistically significant differences in the sam-ples (p<.05).

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