Bioaccessibility and inhibitory effects on digestive ...

2 downloads 0 Views 952KB Size Report
Apr 28, 2018 - In this study, the aim was to determine the bioaccessibilities of carnosic acid in sage and rosemary and in vitro in- hibitory effects of these ...
International Journal of Biological Macromolecules 115 (2018) 933–939

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Bioaccessibility and inhibitory effects on digestive enzymes of carnosic acid in sage and rosemary Pınar Ercan ⁎, Sedef Nehir El Food Engineering Department, Engineering Faculty, Ege University, 35100 Bornova, Izmir, Turkey

a r t i c l e

i n f o

Article history: Received 1 December 2017 Received in revised form 24 April 2018 Accepted 25 April 2018 Available online 28 April 2018 Chemical compounds studied in this article: Carnosic acid (PubChem CID: 65126) 4-Nitrophenyl α-D-glucopyranoside (PNPG) (PubChem CID: 92969) Pefabloc® SC (PubChem CID: 186136) Phosphoric acid (PubChem CID:1004) Hexane (PubChem CID:8058) Methanol (PubChem CID: 887)

a b s t r a c t In this study, the aim was to determine the bioaccessibilities of carnosic acid in sage and rosemary and in vitro inhibitory effects of these samples on lipid and starch digestive enzymes by evaluating the lipase, α-amylase and αglucosidase enzyme inhibition activities. The content of carnosic acid in rosemary (18.72 ± 0.33 mg/g) was found to be higher than that content of that in sage (3.76 ± 0.13 mg/g) (p b 0.05). The carnosic acid bioaccessibilities were found as 45.10 ± 1.88% and 38.32 ± 0.21% in sage and rosemary, respectively. The tested sage and rosemary showed inhibitory activity against α-glucosidase (Concentration of inhibitor required to produce a 50% inhibition of the initial rate of reaction - IC50 88.49 ± 2.35, 76.80 ± 1.68 μg/mL, respectively), α-amylase (IC50 107.65 ± 12.64, 95.65 ± 2.73 μg/mL, respectively) and lipase (IC50 6.20 ± 0.63, 4.31 ± 0.62 μg/mL, respectively). Furthermore, to the best of our knowledge, this is the first work that carnosic acid standard equivalent inhibition capacities (CAEIC50) for these food samples were determined and these values were in agreement with the IC50 values. These results show that sage and rosemary are potent inhibitors of lipase, α-amylase and α-glucosidase digestive enzymes. © 2018 Elsevier B.V. All rights reserved.

Keywords: Carnosic acid α-Amylase α-Glucosidase Lipase Inhibitors In vitro bioaccessibility

1. Introduction Obesity incidence increased at an alarming rate and is becoming a worldwide health problem, with incalculable social costs, in recent years [1,2]. This disease has acquired epidemic proportions projected to reach 2.3 billion of overweight adults and 700 million obese adults, respectively by 2015. Although some anti-obesity drugs exist, they have more side effects than beneficial effects [1]. For this reason, a wide variety of natural materials have been explored for their obesity treatment potential [2]. There are varieties of bioactive components, such as saponins, polyphenols, caffeine, chitosan, conjugated linoleic acid, which can induce weight loss and prevent diet-induced obesity [2,3,4]. Carnosic acid is one of the examples of bioactive compounds found in nature and has anti-obesity activity [1,4]. ⁎ Corresponding author. E-mail addresses: [email protected], (P. Ercan), [email protected] (S.N. El).

https://doi.org/10.1016/j.ijbiomac.2018.04.139 0141-8130/© 2018 Elsevier B.V. All rights reserved.

Carnosic acid is a phenolic diterpene with a formula C20H28O4, which is considered to be the most important antioxidant constituent of sage and rosemary. Carnosic acid also has anti-inflammatory, antiplatelet, antibacterial, anticancer and anti-adipogenic properties enabling weight loss [5,6]. The objectives of this study were to determine the carnosic acid content of sage and rosemary and its in vitro bioaccessibilities of sage and rosemary and in vitro bioaccessibilities of carnosic acid in sage and rosemary and to evaluate the inhibitory effects of sage and rosemary on lipid and carbohydrate digestive enzymes (lipase, α-amylase, and αglucosidase). In this study, a standardized static in vitro digestion method, which is recently published by the COST FA1005 Action INFOGEST, an international network joined by N200 scientists from 32 countries working in the field of digestion, was applied to analyze the in vitro bioaccessibilities of the carnosic acid in samples. They concluded that this protocol should be tested by different research groups for a variety of application on different food samples [7,8].

934

P. Ercan, S.N. El / International Journal of Biological Macromolecules 115 (2018) 933–939

2. Materials and methods 2.1. Chemicals Carnosic acid (91209), 4-nitrophenyl α-D-glucopyranoside (PNPG) (N0877), 4-methylumbelliferyl oleate (4MUO) (75164), α-amylase (A1031), pepsin (P7012), pancreatin (P1750), lipase (L3126), Pefabloc® SC (76307), phosphoric acid (79606), hexane (34859) were purchased from Sigma (St. Louis, MO, US). Methanol (1586018) was purchased from Merck. All reagents were analytical grade. 2.2. Samples Dried leaves of sage (Salvia fruticosa Mill.) and dried rosemary (Rosmarinus officinalis L.) were purchased at different times from three different markets in Izmir, Turkey. Dried leaves of sage (10 g) were infused in 250 mL of boiling water for 10 min and filtered. 2.3. In vitro digestion Samples were subjected to in vitro digestion according to the procedure recently described by Minekus et al. [8]. In this consensus protocol, within the COST FA1005 INFOGEST Network, the practical static digestion method is based on human gastrointestinal physiologically relevant conditions [7]. 2.3.1. Simulated digestion fluids Simulated digestion fluids were made up of the corresponding electrolyte stock solutions, enzymes, CaCl2 and water. The electrolyte stock solutions were 1.25 × concentrated i.e. 4 parts of electrolyte stock solution + 1 part of water gives the correct ionic composition in the simulated digestion fluids. The volumes of Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) were calculated for a final volume of 500 mL for each simulated fluid, according to Table 1 [8]. 2.3.2. Digestion procedure 2.3.2.1. Oral phase: final ratio of food to SSF of 50:50 (w/v). The 5 g of solid or 5 mL of liquid food were mixed with 4 mL of SSF electrolyte stock solution and minced together. Next, 25 μL of 0.3 M CaCl2 and 975 μL of water were added and thoroughly mixed. The sample was incubated for 2 min at 37 °C at 150 rpm in an orbital shaker (Stuart S 1500, UK) [8]. In our study, salivary α-amylase was not added because the aim of this study included determination of α-amylase inhibition activity of samples in the intestinal phase.

2.3.2.2. Gastric phase: final ratio of food to SGF of 50:50 (v/v). Oral bolus was mixed with 8 mL of SGF electrolyte stock solution and 5 μL of 0.3 M CaCl2. The pH was adjusted to 3.0 with 3 M HCl. A 1 mL of porcine pepsin stock solution (2000 U/mL in the final mixture) was added and the volume was adjusted to 10 mL with water. The sample was incubated for 2 h at 37 °C at 150 rpm [8]. 2.3.2.3. Intestinal phase: final ratio of gastric chyme to SIF of 50:50 (v/v). Gastric chyme was mixed with 11 mL of SIF electrolyte stock solution and pH was adjusted to 7.0 with 1 M NaOH. A 40 μL of 0.3 M CaCl2 and 2.5 mL of fresh bile (160 mM in fresh bile) were added to the mixture. The activity of pancreatin solution in SIF was adjusted to reach 100 U/mL of trypsin activity in the final mixture and additional pancreatic lipase (to obtain 2000 U/mL in the final mixture) was added to the mixture according to the calculated lipase activity of the pancreatin. Final volume was made up with water to 40 mL. The sample was incubated for 2 h at 37 °C at 150 rpm. After 2 h, 8 μL of 500 mM Pefabloc was added to the samples to stop the enzyme reaction. The samples were stored at −18 °C under nitrogen until analysis [8]. 2.3.3. Carnosic acid bioaccessibility Carnosic acid bioaccesibility was calculated as percentage of carnosic acid content of in vitro digested sample (S) and carnosic acid content of sample before in vitro digestion (C). %Bioaccesibility ¼ ðS=CÞ  100

2.4. Carnosic acid content Carnosic acid content was measured by High-Performance Liquid Chromatograph (HPLC) method according to Yan et al.' study [6] with some modifications. The sample was vortexed for 60 s and spiked with 100 μL 2.5 mol/L hydrochloric acid and vortexed for 120 s. The mixture was extracted with 4 mL of n-hexane–isopropyl alcohol (9:1, v/v) by shaking for 5 min, centrifuged at 6000 rpm for 10 min at 0 °C and the supernatant was then transferred to another tube and evaporated to dryness under a gentle stream of nitrogen at room temperature. The residue was reconstituted with 200 μL of methanol–phosphoric acid (95:5, v/v) and filtered through a 0.22 μm filter prior to HPLC analysis. Primary stock solution of carnosic acid was prepared in methanol– phosphoric acid (95:5, v/v) at concentrations of 530.0 μg/mL and this primary stock solution was stored at 4 °C until used. The analytical HPLC system consisted of a Agilent Technologies 1200 series HPLC equipped with a Diode Array Detector and a Agilent PLRP-S column (300 A, 8 μm, 150 × 4.6 mm). A mobile phase composed of acetonitrile–0.1% phosphoric acid solution (55:45, v/v) was used

Table 1 Composition of stock solutions of simulated digestion fluids. SSF (pH 7) Salt solution added

KCl KH2PO4 NaHCO3 NaCl MgCl2(H2O)

SGF (pH 3)

SIF (pH 7)

Stock conc.

mL of stock added to prepare 0.4 L

Final salt conc. in SSF

mL of stock added to prepare 0.4 L

Final salt conc. in SGF

mL of stock added to prepare 0.4 L

Final salt conc. in SIF

mol/L

mL

mmol/L

mL

mmol/L

mL

mmol/L

0.5 0.5 1 2 0.15

15.1 3.7 6.8 – 0.5

15.1 3.7 13.6 – 0.15

6.9 0.9 12.5 11.8 0.4

6.9 0.9 25 47.2 0.12

6.8 0.8 42.5 9.6 1.1

6.8 0.8 85 38.4 0.33

0.5 0.3 6

0.06 – 0.09

0.06 1.5 1.1

0.5 – 1.3

0.5 0.15 15.6

– – 0.7

– 0.6 8.4

6

(NH4)2CO3 CaCl2(H2O)2 HCl

SSF, Simulated Salivary Fluid; SGF, Simulated Gastric Fluid; SIF, Simulated Intestinal Fluid. The concentrations correspond to 400 mL and final volume was up to 500 mL after addition of the enzymes, bile and CaCl2(H2O)2 during in vitro digestion. The necessary volume of CaCl2(H2O)2 was added to the final mixture of the digestion medium to prevent precipitation.

P. Ercan, S.N. El / International Journal of Biological Macromolecules 115 (2018) 933–939

935

reaction vials and 20 μL of α-glucosidase solution was added to each vial, followed by incubation at 37 °C for 10 min. Then, 40 μL of PNPG solution was added to initiate the digestion. After 15 min, 10 μL of 0.1 M EDTA and 190 μL of 1 M Na2CO3 were added for reaction termination. An aliquot of 200 μL was withdrawn from each vial and added into separate wells of a microplate reader (Thermo Scientific Varioskan Flash, Finland). Absorbance at 400 nm was measured. A control vial was prepared by replacing the inhibitor solution with phosphate buffer. The entire experiment was repeated by substituting the activeα-glucosidase with inactive α-glucosidase treated at 100 °C for 10 min.

Fig. 1. α-Glucosidase inhibition - concentration graph of samples.

throughout the analysis at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. The column was maintained at 25 °C. The wavelength used for identification and quantification of the carnosic acid was 210 nm. The retention time of carnosic acid was found as 10.54 ± 0.01 min. Carnosic acid contents of samples were calculated with using calibration curves (y = 58.972x + 80.837, R2 = 0.9984) (Fig. 5). 2.5. Enzyme inhibition assays 2.5.1. Sample preparation For the extraction of samples which were used for enzyme inhibition assays, sample of 0.5 g to 5 g were weighted. Then, 10 mL of phosphate buffer (50 mM, pH 6.9) containing 6.85 mM NaCl, was added to samples and vortexed at 3000/min for 1.5 min. After applying a 107 W ultrasonic probe to vortexed samples for 1 min, the samples were centrifuged at 8500 ×g for 15 min. The supernatant was filtered through blue ribbon filter paper. The pellet was retreated with the same procedures and centrifuged. The filtered supernatants were combined and used for enzyme inhibition assays. The extracts of samples were diluted with phosphate buffer to obtain samples at varying concentrations. 2.5.2. α-Glucosidase inhibition assay α-Glucosidase inhibition was measured by a spectrophotometric method according to Koh et al. [9] with some modifications. Reaction substrate 4-nitrophenyl α-D-glucopyranoside (PNPG) (30 mM) and αglucosidase (AGH) (25 mg/mL) solutions were prepared in phosphate buffer saline (PBS), respectively. After vortexing for 10 min, the αglucosidase mixture was centrifuged at 4 °C 10000 ×g for 30 min. The clear supernatant was subsequently utilized in the assay. Briefly, 340 μL of inhibitors of different concentrations were pipetted into separate

Fig. 2. α-Amylase inhibition - concentration graph of samples.

2.5.3. α-Amylase inhibition assay α-Amylase inhibition was measured by a spectrophotometric method according to Koh et al. [9] and Yang et al. [10] with some modifications. First, 820 μL samples of inhibitor of different concentrations were pipetted into separate reaction vials and 100 μL of α-amylase enzyme solution was added into each vial, and incubated at 37 °C for 10 min. Next, 80 μL of potato starch solution (1%) was pipetted into each reaction vial. After 12 min incubation in a 37 °C water bath, 500 μL of HCl (10%) was added to each vial for reaction termination. Next, 150 μL of iodine solution (0.0025 M I2/0.0065 M KI) and 500 μL of distilled water were added. Upon the addition of 500 μL of deionized water, an aliquot of 200 μL from each reaction vial was pipetted into separate wells of a microplate reader (Thermo Scientific Varioskan Flash, Finland). Absorbance at 620 nm was measured. A control vial was prepared by replacing the inhibitor solution with phosphate buffer. The entire experiment was repeated by substituting the active αamylase enzyme with denatured α-amylase enzyme solution treated at 100 °C for 10 min. 2.5.4. Pancreatic lipase inhibition assay Pancreatic lipase activity was measured according to Sugiyama et al. [11] using 4-methylumbelliferyl oleate (4MUO) as the substrate. Twenty-five microliters of the sample solution dissolved in water and 25 μL of the pancreatic lipase solution (1 mg/mL) were mixed in the well of a microplate reader. Fifty microliters of 4MUO solution (0.1 mM) dissolved in Dulbecco's phosphate buffered saline (9.6 g/L) was then added to initiate the enzyme reaction. After incubation at 23 °C for 20 min, 100 μL of 0.1 M sodium citrate (pH 4.2) was added to stop the reaction. The amount of 4-methylumbelliferone released by lipase was measured using a fluorescence microplate reader at an excitation wavelength of 320 nm and an emission wavelength of 450 nm. 2.5.5. Determination of inhibition parameters The α-glycosidase, α-amylase, lipase enzyme inhibition assays were performed for each extracts of samples at varying concentrations as

Fig. 3. Lipase inhibition - concentration graph of samples.

936

P. Ercan, S.N. El / International Journal of Biological Macromolecules 115 (2018) 933–939

Table 2 Carnosic acid contents and bioaccessibilities of samples. Samples

Carnosic acid content before in vitro digestion (mg/g)

Carnosic acid content after in vitro digestion (mg/g)

Bioaccesibility (%)

Sage Rosemary

3.76 ± 0.13a,B 18.72 ± 0.33a,A

1.69 ± 0.05b,B 7.17 ± 0.13b,A

45.10 ± 1.88A 38.32 ± 0.21B

Different capital letters within the same column shows statistically significant difference. Different lower case letters within the same rows shows statistically significant difference (p b 0.05). (n = 6).

described below. Percentage of enzyme inhibition was calculated with the following equation. Inhibitionð%Þ ¼ 100     ðAcontrol −Acontrolblank Þ− Asample −Asampleblank =ðAcontrol −Acontrolblank Þ

where Acontrol, Acontrolblank, Asample, and Asampleblank refer to the absorbance value of reaction vial containing active enzyme and buffer, inactive enzyme and buffer, active enzyme and inhibitor, and inactive enzyme and inhibitor, respectively. Substrate was present in all of these vials. The assay was performed in triplicate, and a curve of percentage inhibition against inhibitor concentration was plotted with the averaged values (Figs. 1–3). IC50 (IC: inhibition capacity; IC50: the content of the sample/or carnosic acid (in μg/mL), which causing 50% inhibition of the digestive enzyme) of each inhibitor was determined by interpolation from the curve with using GraphPad Prism program (Table 3). Moreover, the enzyme inhibition assays were performed for the samples at the concentration of 1 mg/mL after in vitro stomach digestion to observe the effect of samples on α-glycosidase, α-amylase, lipase enzymes before intestinal phase (Table 4). The enzyme inhibition percentage triggered by carnosic acid was used as a standard to determine and compared with food samples. It was found that sage and rosemary samples at 1 mg/mL concentration had 3.76 and 18.72 μg/mL of carnosic acid, respectively. Therefore, enzyme inhibition percentages with carnosic acid were determined at the concentrations of 3.76 and 18.72 μg/mL (Table 5). Moreover, carnosic acid equivalent inhibition capacity (CEIC) was calculated for each sample as described below. A curve of percentage inhibition for samples (used in different μM) and the curves for carnosic acid (used in different μM) were fitted to exponential equations. Then, the slope of the equations, which is equal to derivative of the equation at 50% inhibition of each inhibitor were used to find carnosic acid equivalent inhibition capacity (CAEIC50, mM/g) as shown in the below equation (Table 6). If Equation of sample y ¼ a ln ðxÞ þ b Equation of carnosic acid y ¼ A ln ðxÞ þ B V ¼ Volume of sample ðmLÞ CAEIC ¼ a=ðA  VÞ

2.6. Statistical analysis All experiments were performed in triplicate and parallel. Six values for each sample were averaged (n = 6). IC50 values were calculated Table 3 IC50 values of samples for α-glucosidase, α-amylase, lipase. Samples

IC50 values (μg/mL) α-Glucosidase

Sage Rosemary

A,b

88.49 ± 2.35 76.80 ± 1.68B,b

α-Amylase

Lipase A,a

107.65 ± 12.64 95.65 ± 2.73A,a

6.20 ± 0.63A,c 4.31 ± 0.62B,c

Different capital letters within the same column shows statistically significant difference. Different lower case letters within the same rows shows statistically significant difference (p b 0.05). (n = 6).

with GraphPad Prism Version 5.01 (GraphPad Software, Inc., San Diego, CA). The MINITAB Statistical Software package (Version 17; State College, PA) was used for all statistical analyses. The two samples t-test was performed for investigating statistically significant differences within two samples. The statistical difference for more than two samples was determined by using one-way ANOVA Tukey comparison test. A p value of b0.05 was considered to be significant. 3. Results and discussion 3.1. Carnosic acid content and its bioaccessibility The total carnosic acid contents of sage (Salvia fruticosa Mill.) and rosemary (Rosmarinus officinalis L.) are shown in Table 2. The content of carnosic acid in rosemary (18.72 ± 0.33 mg/g) was found to be higher than sage (3.76 ± 0.13 mg/g) (p b 0.05). In the study of Luis & Johnson, the carnosic acid content was found as 12.18 ± 0.609 mg/g in rosemary (Rosmarinus officinalis L. plants- cultivar Sissinghurst English) [12]. Moreno et al. found the carnosic acid content of rosemary as 29.3 ± 2.9 g/100 g in leaves and 13.6 ± 1.3 g/100 g in flowers [13]. Peng et al. stated that the carnosic acid contents were 31.3, 30.4, 13.6 and 24.7 mg/g for the rosemary leaves which were extracted by supercritical fluid extraction at 60 °C, supercritical fluid extraction at 80 °C, ethanolic and acetonic, respectively [14]. Carnosic acid content of seven different brand sage was found between 2.99 ± 0.01–7.16 ± 0.16 mg/g in the study of Berker et al. [15]. Our results were found close to the results reported in the literature. The bioaccesibilities of carnosic acid in sage and rosemary were also determined in this study by an in vitro method. The simulated gastric/ small intestinal digestion models are being extensively used at present because they are rapid, safe and do not have the ethical restrictions of in vivo methods. In vitro methods either simulate the digestion and absorption processes (for bioavailability) or only the digestion process (for bioaccessibility), and the response measured is the concentration of a nutrient and other dietary bioactive compound in some kind of final extract [8,16]. In our study, carnosic acid bioaccessibilities were found as 45.10 ± 1.88 and 38.32 ± 0.21% in sage and rosemary, respectively (Table 2). Overall, little is known about the bioavailability of carnosic acid. Therefore, more research about the absorption, distribution and elimination of carnosic acid and its metabolites is necessary to fully understand their actions, in vivo [17]. Yan et al. [6] stated that the absorption of carnosic acid was slow (Tmax = 125.6 ± 118.4 min) after i.g. administration (90 mg/kg) to rats. However, the maximum plasma concentration was high and retained for a long time. The absolute bioavailability of carnosic acid was found high as 65.09 ± 1.422%, which would be a useful feature in future applications [6]. In the study of Doolaege et al. [17], the absorption, distribution and elimination of carnosic acid, the main antioxidant found in rosemary was studied, in vivo, in rats. For that purpose, carnosic acid was administrated in a single dose, intravenously (20.5 ± 4.2 mg/kg) and orally (64.3 ± 5.8 mg/kg), to four and nine rats, respectively. The bioavailability of carnosic acid, after 360 min, was 40.1%. Traces of carnosic acid were found in the rats' intestinal content, liver and muscle tissue of abdomen and legs. The recovery of carnosic acid in the feces, 24 h after oral administration, was 15.6 ± 8.2%. Carnosic acid is absorbed into the

P. Ercan, S.N. El / International Journal of Biological Macromolecules 115 (2018) 933–939

937

Table 4 Enzyme inhibitions of samples before and after in vitro stomach digestion. Samples (1 mg/mL concentration)

α-Glucosidase inhibition (%)

α-Amylase inhibition (%)

Lipase inhibition (%)

Sage

90.31 ± 1.38B,a 80.72 ± 1.16D,a 93.12 ± 0.38A,a 84.71 ± 0.86C,a

70.56 ± 0.83B,c 58.34 ± 0.67D,c 75.84 ± 0.38A,c 68.82 ± 0.86C,c

88.15 ± 0.51B,b 78.32 ± 0.59D,b 90.26 ± 0.66A,b 79.94 ± 0.66C,b

Rosemary

Before in vitro After in vitro Before in vitro After in vitro

Different capital letters within the same column shows statistically significant difference. Different lower case letters within the same rows shows statistically significant difference (p b 0.05). (n = 6).

bloodstream after oral administration in rats and is therefore bioavailable. It was found that carnosic acid in vivo is present in its free form and that its main elimination route is the fecal route [17].

3.2. Inhibitory activities of sage and rosemary against digestive enzymes Digestive enzyme inhibition by the samples was determined by calculating the IC50 with the lower numbers indicating the higher quality of enzymatic inhibition (Table 3). The IC50 values were obtained using the concentration required for 50% inhibition of enzymatic activity for each compound to determine potency [18]. Enzyme inhibition curves, percent α-amylase, α-glucosidase and lipase inhibition of the samples were plotted as a function of concentration as shown in the Figs. 1–3. Percent enzyme inhibition of the samples was positively correlated with the concentration of samples. Percent α-amylase, α-glucosidase and lipase inhibition (%) increased as concentration of samples increased (r2 = 0.9). Sage and rosemary showed the inhibitory activity against αglucosidase (IC50 88.49 ± 2.35 and 76.80 ± 1.68 μg/mL, respectively) and α-amylase (IC50 107.65 ± 12.64 and 95.65 ± 2.73 μg/mL, respectively). Moreover, the sage and rosemary exhibited inhibitory activity against lipase with IC50 6.20 ± 0.63, 4.31 ± 0.62 μg/mL, respectively. Samples showed greater inhibition against lipase compared to αglucosidase and α-amylase enzymes. The reason of this could be having high carnosic acid content. Loizzo et al. stated that Salvia acetabulosa methanol extracts exerted the highest activity against α-amylase (IC50 91.2 μg/mL, respectively) and α-glucosidase (IC50 76.9 μg/mL) [19]. The methanolic extract from the leaves of Salvia officinalis L. (sage) showed significant inhibitory effect against pancreatic lipase, which is participated in digestion of lipids. The sage extract had an IC50 value as 36 μg/mL for pancreatic lipase [20]. Bustanji et al. [21] conducted a study on the inhibition effect of the rosemary extract for pancreatic lipase. They observed that the rosemary extract had an IC50 value as 13.8 μg/mL for pancreatic lipase. The results showed that the inhibitory activity of the extract on both enzymes is dose dependent. At concentrations of 6.3–200 μg/mL, the percentage of enzyme inhibition ranged from 36.8 to 95.1% [21].

In addition to evaluation of enzyme inhibition of sage and rosemary before consumption, the enzyme inhibition assays were done after in vitro stomach digestion of samples at 1 mg/mL concentration. The percentages of enzyme inhibition for each sample after in vitro stomach digestion are shown in Table 4. The percentages of enzyme inhibition after in vitro stomach digestion were found lower than before in vitro digestion for each enzyme and each sample. The reason of this could be the effects of digestion process, temperature and pH change (acidic or basic environment). Sage with 1 mg/ml concentration showed the lowest enzyme inhibition of 58.34 ± 0.67% for α-amylase enzyme after in vitro stomach digestion. The highest percentage of enzyme inhibition was found as 84.71 ± 0.86% for α-glucosidase inhibition of rosemary after in vitro stomach digestion. Enzyme inhibition curves, percent α-amylase, α-glucosidase and lipase inhibition of the carnosic acid were plotted as a function of concentration as shown in the Fig. 4. Enzyme inhibition (%) increased as concentration of carnosic acid increased (r2 = 0.9). The standard carnosic acid exhibited highest inhibitory activity against lipase enzyme compared to its inhibitory activity against α-amylase and α-glucosidase enzymes (p b 0.05). Sage and rosemary at 1 mg/ml concentration, had 3.76 and 18.72 μg/mL carnosic acid, respectively. Therefore, the enzyme inhibitions of standard carnosic acid were evaluated at 3.76 and 18.72 μg/mL concentrations (Table 5). The percentages of α-amylase enzyme inhibition of standard carnosic acid at the concentrations of 3.76 and 18.72 μg/mL were 4.41 ± 0.41 and 32.55 ± 0.22%, respectively. While the standard carnosic acid at 3.76 and 18.72 μg/mL concentrations provided 8.51 ± 0.41 and 41.46 ± 0.19% α-glucosidase enzyme inhibition respectively, the lipase enzyme inhibition by 3.76 and 18.72 μg/mL of standard carnosic acid was 12.07 ± 0.11 and 60.12 ± 0.44%, respectively. However, sage and rosemary at 1 mg/mL concentration, which included 3.76 and 18.72 μg/mL carnosic acid, showed higher enzyme inhibitory activities against α-amylase, α-glucosidase, lipase than the standard carnosic acid at the same concentration (Table 5). The higher inhibitory activity can be due to other biologically active substances and anti-nutritional factors except carnosic acid. Carnosic acid equivalent inhibition capacity (CAEIC50) was determined for each enzyme and sample (Table 6). While low IC50 value means higher inhibition effect, high equivalent inhibition capacity (EIC) value means higher inhibition effect. Higher CAEIC50 values for both sage and rosemary were observed for lipase compared to α-glucosidase and α-amylase. The lowest CAEIC50 values for both samples were found for α-amylase. The highest CAEIC50 values for α-glucosidase, α-amylase, lipase were found for the rosemary sample. According to the CAEIC50 and

Table 5 Enzyme inhibitions of carnosic acid standard. Concentration of carnosic acid 3.76 μg/mL α-Glucosidase inhibition (%) α-Amylase inhibition (%) Lipase inhibition (%)

Fig. 4. Enzyme inhibition - concentration graph of carnosic acid standard.

18.72 μg/mL b,B

8.51 ± 0.41 4.41 ± 0.41b,C 12.07 ± 0.11b,A

41.46 ± 0.19a,B 32.55 ± 0.22a,C 60.12 ± 0.44a,A

Different capital letters within the same column shows statistically significant difference. Different lower case letters within the same rows shows statistically significant difference (p b 0.05). (n = 6).

938

P. Ercan, S.N. El / International Journal of Biological Macromolecules 115 (2018) 933–939

Fig. 5. Sample chromatograms a) carnosic acid standard b) sage c) rosemary. HPLC conditions: Agilent Technologies 1200 series HPLC equipped with a Diode Array Detector and a Agilent PLRP-S column (300 A, 8 μm, 150 × 4.6 mm), mobile phase acetonitrile–0.1% phosphoric acid solution (55:45, v/v), flow rate 1.0 mL/min, 25 °C, wavelength 210 nm.

IC50 values, rosemary had higher inhibition capacity against digestive enzymes compared to sage. 4. Conclusion

Conflict of interest

In conclusion, carnosic acid content in rosemary (18.72 ± 0.33 mg/g) was found higher than sage (3.76 ± 0.13 mg/g) (p b 0.05). The carnosic acid bioaccessibilities were found as 45.10 ± 1.88% and 38.32 ± 0.21% in sage and rosemary, respectively. Present study demonstrated that the tested sage and rosemary showed the inhibitory activity against α-glucosidase (IC50 88.49 ± 2.35, 76.80 ± 1.68 μg/mL, respectively), α-amylase (IC50 107.65 ± 12.64, 95.65 ± 2.73 μg/mL, respectively) and lipase (IC50 6.20 ± 0.63, 4.31 ± 0.62 μg/mL, respectively). Overall, rosemary samples showed the highest inhibitory activity against lipase enzyme according to IC50 and CAEIC50 values. In summary, this study reported that sage and rosemary can inhibit digestive enzymes in vitro. The consumption of these samples would be used in conjunction with a low calorie diet for body weight management. These foods may provide safe, natural, and cost-effective alternatives to synthetic drugs for obesity and diabetes. As a future study, further isolation and Table 6 CAEIC50 values of samples for α-glucosidase, α-amylase, lipase. Samples

Sage Rosemary

identification of active inhibitory compounds in these samples are needed for developing anti-obesity functional foods.

CAEIC50 values (mM carnosic acid/g sample) α-Glucosidase

α-Amylase

Lipase

4.55 4.78

0.67 0.83

103.75 149.09

The authors declare no competing financial interest. Acknowledgments The authors are deeply grateful for the financial support of Ege University Council of Scientific Research Projects-BAP (Project number: 13MÜH016). Corresponding Author, S.N.El was a participant in the COST Action FA1005 and is a participant in the COST Action FA1403. References [1] C.I. Gamboa-Gómez, N.E. Rocha-Guzmán, J.A. Gallegos-Infante, M.R. MorenoJiménez, B.D. Vázquez-Cabral, R.F. González-Laredo, Review article: plants with potential use on obesity and its complications, EXCLI J. 14 (2015) 809–831 (ISSN 16112156). [2] J.W. Yun, Possible anti-obesity therapeutics from nature – a review, Phytochemistry 71 (2010) 14–15 (1625–1641). [3] R.B. Birari, K.K. Bhutani, Pancreatic lipase inhibitors from natural sources: unexplored potential, Drug Discov. Today 12 (19:20) (2007) 879–889. [4] G.A. Mohamed, S.R.M. Ibrahim, E.S. Elkhayat, R.S.E. Dine, Natural anti-obesity agents, Bull. Fac. Pharm. Cairo Univ. 52 (2014) 269–284. [5] S. Birtić, P. Dussort, F. Pierre, A.C. Bily, M. Roller, Carnosic acid, molecules of interest, Phytochemistry 115 (2015) 9–19. [6] H. Yan, L. Wang, X. Li, C. Yu, K. Zhang, Y. Jiang, L. Wu, W. Lu, P. Tu, High-performance liquid chromatography method for determination of carnosic acid in rat plasma and its application to pharmacokinetic study, Biomed. Chromatogr. 23 (2009) 776–781.

P. Ercan, S.N. El / International Journal of Biological Macromolecules 115 (2018) 933–939 [7] S. El, S. Karakaya, Ş. Simsek, D. Dupont, E. Menfaatli, A.T. Eker, In vitro digestibility of goat milk and kefir with a new standardised static digestion method (INFOGEST cost action) and bioactivities of the resultant peptides, Food Fuct. 6 (2015) 7 (2322). [8] M. Minekus, M. Alminger, P. Alvito, S. Balance, T. Bohn, C. Bourlieu, F. Carrière, R. Boutrou, M. Corredig, D. Dupont, C. Dufour, L. Egger, M. Golding, S. Karakaya, B. Kirkhus, S. Le Feunteun, U. Lesmes, A. Macierzanka, A. Mackie, S. Marze, D.J. McClements, O. Ménard, I. Recio, C.N. Santos, R.P. Singh, G.E. Vegarud, M.S.J. Wickham, W. Weitschies, A.A. Brodkorb, Standardised static in vitro digestion method suitable for food – an international consensus, Food Funct. 5 (2014) 1113–1124. [9] L.W. Koh, L.L. Wog, Y.Y. Loo, S. Kasapis, D. Huang, Evaluation of different teas against starch digestability by mamalian glycosidases, J. Agric. Food Chem. 58 (2010) 148–154. [10] X.W. Yang, M.Z. Huang, Y.S. Jin, L.N. Sun, Y. Song, H.S. Chen, Phenolics from Bidens bipinnata and their amylase inhibitory properties, Fitoterapia 3 (2012) 1169–1175. [11] H. Sugiyama, Y. Akazome, T. Shoji, T. Yamaguchi, M. Yasue, T. Kanda, Y. Ohtake, Oligomeric procyanidins in apple polyphenol are main active components for inhibition of pancreatic lipase and triglyceride absorption, J. Agric. Food Chem. 55 (11) (2007) 4604–4609. [12] J.C. Luis, C.B. Johnson, Seasonal variations of rosmarinic and carnosic acids in rosemary extracts, analysis of their in vitro antiradical activity, Span. J. Agric. Res. 3 (1) (2005) 106–112. [13] S. Moreno, T. Scheyer, C.S. Romano, A.A. Vojnov, Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition, Free Radic. Res. 40 (2) (2006) 223–231 (February). [14] C.H. Peng, J.D. Su, C.C. Chyau, T.Y. Sung, S.S. Ho, C.C. Peng, R.Y. Peng, Supercritical fluid extracts of rosemary leaves exhibit potent anti-inflammation and anti-tumor effects, Biosci. Biotechnol. Biochem. 71 (9) (2007) 2223–2232.

939

[15] F.B.E. Berker, G. Topçu, K. Günaydın, N. Öztekin, U. Kolak, S. Başkan, E. Kepekçi, Terpenoid ve hidroksi-antrokinon yapısındaki bitki aktif maddelerinin ayırımı ve tayini için kapiler elektroforez ile analiz yöntemleri geliştirilmesi ve bu yöntemlerin Türkiye bitki florasındaki örneklerin analize uygulaması, TÜBİTAK TBAG Proje 2312 103T053 2006, pp. 1–35http://uvt.ulakbim.gov.tr. [16] J. Prada, J.M. Aguilera, Food microstructure affects the bioavailability of several nutrients, J. Food Sci. 72 (2007) 21–31. [17] E.H.A. Doolaege, D.K. Raes, F.D. Vos, R. Verhe, S.D. Smet, Absorption, distribution and elimination of carnosic acid, a natural antioxidant from Rosmarinus officinalis in rats, Plant Foods Hum. Nutr. 66 (2011) 196–202. [18] A.M. Griffith, Inhibition of α-Amylase and α-Glucosidase by Bioflavonoids, Oregon State University. University Honors College, Honors Baccalaureate of Science in Bioresource Research, 2012 (33 pp.). [19] M.R. Loizzo, A.M. Saab, R. Tundis, F. Menichini, M. Bonesi, V. Piccolo, G.A. Statti, B. de Cindio, P.J. Houghton, F. Menichini, In vitro inhibitory activities of plants used in Lebanon traditional medicine against angiotensin converting enzyme (ACE) and digestive enzymes related to diabetes, J. Ethnopharmacol. 119 (1) (2008) 109–116. [20] K. Ninomiya, H. Matsuda, H. Shimoda, N. Nishida, N. Kasajima, T. Yoshino, T. Morikawaa, M. Yoshikawaa, Carnosic acid, a new class of lipid absorption inhibitor from sage, Bioorg. Med. Chem. Lett. 14 (2004) 1943–1946. [21] Y. Bustanji, A. Issa, M. Mohammad, M. Hudaib, K. Tawah, H. Alkhatib, I. Almasri, B. Al-Khalidi, Inhibition of hormone sensitive lipase and pancreatic lipase by Rosmarinus officinalis extract and selected phenolic constituents, J. Med. Plant Res. 4 (2010) 2235–2242.