Antioxidant potential and carbohydrate digestive enzyme inhibitory effects of five Inula species and their major compounds

South African Journal of Botany 111 (2017) 86–92

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Antioxidant potential and carbohydrate digestive enzyme inhibitory effects of five Inula species and their major compounds N. Orhan a, A. Gökbulut b, D. Deliorman Orhan a,⁎ a b

Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 14 November 2016 Received in revised form 23 February 2017 Accepted 9 March 2017 Available online xxxx Edited by J Van Staden Chemical compounds studied in this article: Caffeic acid (PubChem CID: 689043) Chlorogenic acid (PubChem CID: 1794427) Helenin (PubChem CID: 72724) Hispidulin (PubChem CID: 5281628) Luteolin (PubChem CID: 5280445) Quercetin (PubChem CID: 5280343) Rutin (PubChem CID: 5280805)

a b s t r a c t The present study was designed to examine in-vitro antidiabetic activities of different extracts of flowers, leaves and roots of Inula helenium ssp. turcoracemosa, I. montbretiana, I. peacockiana, I thapsoides ssp. thapsoides and I. viscosa extracts. I. viscosa and I. montbretiana flower, I. thapsoides and I. viscosa leaf and I. helenium root methanol extracts exhibited remarkable α-glucosidase inhibitory activity. Additionally, α-amylase inhibitory activities of the extracts were moderate at only 3000 μg/mL. Based on the results of in-vitro antidiabetic activity tests; antioxidant activities, total phenol and flavonoid contents of the most promising extracts were evaluated. To identify compounds responsible for the antidiabetic activity, major compounds of Inula species were analyzed for their in-vitro enzyme inhibitory activity. Quercetin, luteolin and rutin exhibited a significant inhibition on α-glucosidase at 10 mM concentrations. Consequently, Inula species could potentially be used by diabetic patients for their antidiabetic and antioxidant activities. © 2017 Published by Elsevier B.V. on behalf of SAAB.

Keywords: Antidiabetic Antioxidant Asteraceae Inula Elecampane Yellowhead

1. Introduction Diabetes mellitus is one of the most widespread metabolic disorders in all over the world. According to data published by the International Diabetes Federation in 2010, approximately 300 million people suffered from diabetes in the world (Chang et al., 2013). Chronic hyperglycemia induces the production of excessive amounts of reactive oxygen species in tissues and this progress can lead to various health problems in the kidney, heart, eye, liver and central nervous system, and this progress can cause serious tissue and organ damages. Therefore, the discovery of antidiabetic compounds or extracts with antioxidant potential is essential for treatment of diabetes mellitus. For this reason, the studies

⁎ Corresponding author at: Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, Etiler, 06330 Ankara, Turkey. E-mail addresses: [email protected] (N. Orhan), [email protected] (A. Gökbulut), [email protected] (D. Deliorman Orhan).

http://dx.doi.org/10.1016/j.sajb.2017.03.040 0254-6299/© 2017 Published by Elsevier B.V. on behalf of SAAB.

have been focused on antioxidant, in vitro and in vivo antidiabetic potentials of plants based products. The genus Inula comprises more than one hundred species growing in Africa, Asia and Europe, predominantly in the Mediterranean area. The traditional uses of Inula species have been mentioned firstly by the Roman and Greek medical doctors. The members of the genus have widely been used in Traditional Chinese medicine as well as Ayurvedic and Tibetan medicinal systems for the treatment of various diseases such as bronchitis, diabetes, fever, hypertension and inflammation (Seca et al., 2014). Food and Drug Administration permits the use of alcoholic beverages obtained from I. helenium rhizomes and roots as natural flavouring substances and natural adjuvants in foods (Food and Drug Administration, 2014) and The Council of Europe lists I. helenium as a natural food flavouring. In Turkey, flowers, leaves and roots of I. heterolepis, I. viscosa, I. oculus-christi and I. thapsoides subsp. thapsoides are consumed as food raw or cooked (Ertuğ, 2014). On the other hand, a large number of studies are being carried out on Inula species due to their important

N. Orhan et al. / South African Journal of Botany 111 (2017) 86–92

ethnomedicinal uses. Antidiabetic activity is one of the most important activities of Inula species (Zhang et al., 2012; Seca et al., 2014). I. hupehensis and I. viscosa are reported to be used in folk medicine to treat diabetes (Zeggwagh et al., 2006; Qin et al., 2011). Leaves and flowers of I. viscosa together with I. helenium and I. conyza are included in the list of medicinal plants used traditionally to treat diabetes mellitus in Morocco (Eddouks et al., 2007). Additionally, I. viscosa is mentioned as a plant used for the treatment of diabetes in Israel (Yaniv et al., 1987). Also, standardized extract prepared from leaves of cultivated I. viscosa by proprietary methods of Argo-technology has been used in pharmaceutical and cosmetic industry in Israel (Inulav, 2016). This extract having broad spectrum activity against foliar diseases of crop plants has been utilized as natural pesticide (Wang et al., 2004). Moreover, Inula plants such as I. helenium and I. japonica, take place in commercial herbal preparations (Zhao et al., 2006; Han et al., 2010; Seca et al., 2014). With respect to food and ethnobotanical usage of Inula species on diabetes, we decided to build this work concerning the in-vitro antidiabetic and antioxidant potentials of Turkish Inula plants. Small intestinal α-glucosidase and pancreatic α-amylase are important enzymes supposed to regulate dietary carbohydrate digestion in humans. Inhibition of these enzymes may block the carbohydrate digestion and glucose absorption to suppress hyperglycemia. We used these enzymes for determining the in-vitro antidiabetic activity of the selected Inula species and authentic compounds isolated and quantified from these sources. To our best knowledge, this is the first study about inhibitory activity on carbohydrate digestive enzymes of selected Inula species. The main purpose of this work is to investigate the α-glucosidase and α-amylase inhibitory activities of five Inula taxa together with to evaluate the antioxidant potential, total phenol and flavonoid contents of the most active enzyme inhibitor extracts. Flowers, leaves and roots of I. helenium ssp. turcoracemosa, I. montbretiana, I. peacockiana, I. thapsoides ssp. thapsoides and I. viscosa were extracted separately with water, methanol and ethyl acetate to obtain the crude extracts used in this work. After evaluation of α-glucosidase and α-amylase inhibitory activities; antioxidant activities (metal chelating, ferric reducing, total antioxidant capacity), total phenol and flavonoid contents of the most active extracts were investigated. Moreover, some phenolic acids and flavonoids which were previously determined in Inula taxa were investigated for antidiabetic activity.

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2.3. Assay for α-amylase inhibitory activity The α-amylase inhibitory activity of the selected Inula species was determined by the method of Ali et al. (2006). Porcine pancreatic α-amylase type VI (EC 3.2.1.1, Sigma) was dissolved in distilled water. As substrate solution, potato starch (0.5%, w/v) in phosphate buffer (pH 6.9) was used. Experiments were carried out with three replicates. Plant extract and pure compounds dissolved in DMSO and distilled water were mixed in a tube. The reaction was initiated by the addition of the enzyme solution. Then the tubes were incubated at 37 °C for 3 min. After the addition of substrate, the tubes were incubated at 37 °C for 5 min. Then, DNS (3,5-dinitrosalicylic acid) colour reagent solution was added to the mixture and put into a 85 °C heater. After 15 min, distilled water was added to the tubes and tubes were cooled. Absorbances of the mixtures were read at 540 nm. Acarbose was used as the positive control. The absorbance (A) due to maltose generated was calculated according to following formula: AControl or Sample = ATest − ABlank. The amount of maltose generated was calculated by using the maltose standard calibration curve (0–0.1% w/v) and the obtained net absorbance. Percent of inhibition was calculated as: Inhibition % = [(Maltose Control − Maltose Sample) / Maltose Control)] × 100.

2.4. Assay for α-glucosidase inhibitory activity α-Glucosidase type IV enzyme (Sigma Co., St. Louis, USA) was dissolved in phosphate buffer (0.5 M, pH 6.5). The enzyme solution, extracts and pure compounds were preincubated in a 96-well microtiter plate for 15 min at 37 °C. After that, the substrate solution [pnitrophenyl-α-D-glucopyranoside (NPG), Sigma] was added. The mixture was incubated for 35 min at 37 °C. The increase in the absorption at 405 nm due to the hydrolysis of NPG by α-glucosidase was measured by an ELISA microtiter plate reader (Lam et al., 2008). Acarbose (Bayer Group, Turkey), a potent alpha-glucosidase inhibitor, was used as positive control. The inhibition percentage (%) was calculated by the equation: Inhibition % = [(AControl − ASample) / AControl] × 100. Some of the prepared EtOAc extracts could not be solved in the solvent system of the experiment. Thus, the activity of these extracts could not be tested and these extracts were mentioned as NT (not tested) in the concerning tables (Tables 2–4).

2. Materials and methods 2.5. Metal chelating activity 2.1. Plant materials Plants were collected in their flowering stages from different cities of Turkey. Inula helenium (L.) ssp. turcoracemosa Grierson and I. montbretiana DC. were collected near Ankara, I. peacockiana (Aitch. & Hemsl.) Krovin from Van, I. thapsoides (Bieb. ex Willd.) Sprengel ssp. thapsoides from Erzurum and I. viscosa (L.) Aiton from Isparta. Voucher specimens have been deposited in the Herbarium of Ankara University Faculty of Pharmacy under the herbarium codes of AEF 25193, AEF 25191, AEF 25124, AEF 25123 and AEF 26700, respectively.

The chelating activity of Inula extracts on Fe+2 was determined by the method of Dinis et al. (1994). Extracts were incubated with FeCl2 (2 mM). The reaction was initiated by the addition of 0.2 mL of ferrozine (5 mM) and the total volume was adjusted to 4 mL with ethanol. After 10 min, the absorbance was measured at 562 nm. EDTA was used as a reference compound. The control contained FeCl2 and ferrozine. The percentage of inhibition of the ferrozine-Fe+2 complex formation was calculated using this formula: Metal chelating activity (%) = [(AControl − ASample) / AControl] × 100. Analyses were carried out in triplicate and the results were averaged.

2.2. Preparation of extracts and standards Dried and milled flowers, leaves and roots of samples were extracted with water, methanol and ethyl acetate (5% w/v) by magnetic stirrer for 1 h (50 °C, 250 rpm). Extracts were then filtered from filter paper. Methanol (MeOH) and ethyl acetate (EtOAc) extracts were condensed by a rotary evaporator (Buchi-R200) and the aqueous extracts were freeze-dried. Yields of the extracts were calculated and given in Table 1. Phenolic compounds were purchased from Sigma (Germany): chlorogenic acid (C3878), caffeic acid (C0625), quercetin (Q0125), luteolin (L9283), rutin (R5143), and hispidulin (SML0582). Helenin was supplied from Roth (Roth 7677).

2.6. Ferric-reducing antioxidant power Different logarithmic concentrations of the extracts (3, 1, and 0.57 mg/mL) and ascorbic acid as reference were mixed with phosphate buffer (0.2 mol/L, pH 6.6) and K3Fe(CN)6. Tubes were incubated at 50 °C for 20 min, then trichloroacetic acid was added and the mixture was vortexed. Following centrifugation, the supernatant was mixed with same amount of distilled water and FeCl3 and the absorbance at 700 nm was measured (Oyaizu, 1986). Analyses were run in three replicates and the results were averaged.

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Table 1 Yield percentages (w/w) of all extracts from different parts of Inula species and total flavonoid (mg QE/g extract) and total phenol contents (mg GAE/g extract) of active Inula extracts (S.D.: Standard Deviation). Plant name

Plant part

I. helenium

Root Flower Leaf Root Flower Leaf Root Flower Leaf Root Flower Leaf Root Flower Leaf

I. montbretiana

I. peacockiana

I. thapsoides

I. viscosa

Yield %'s extracts Aqueous

MeOH

EtOAc

22.0 8.1 9.1 4.4 3.3 7.1 2.9 9.0 9.8 8.6 4.1 6.2 8.1 8.9 8.9

20.5 16.5 17.5 17.5 10.2 12.1 17.0 10.5 10.6 14.5 18.2 19.6 21.0 13.0 15.1

7.9 4.3 4.9 3.8 2.1 5.1 2.5 3.2 4.1 5.3 3.0 4.6 2.9 6.5 12.9

2.7. Total antioxidant activity by phosphomolybdenum assay This assay is based on the reduction of Mo (VI) to Mo (V) by the sample and the subsequent formation of a green phosphate/Mo (V) complex at acidic pH. Active Inula extracts were added to test tubes containing 3 mL of distilled water and 1 mL molybdate reagent solution (Molybdate reagent: 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). Vortexed tubes were incubated at 90 °C for 90 min. Then, tubes were cooled to room temperature and the absorbances of the samples were measured at 695 nm. Results were expressed as mg ascorbic acid equivalent/g extract (Prieto et al., 1999).

2.8. Determination of total phenol content The extracts (1 mg/mL) were mixed with Folin-Ciocalteu reagent (10%) and samples were incubated for 5 min at room temperature. Then, sodium carbonate solution (7.5%) was added and samples were vortexed immediately. The absorbance of mixture was measured at 735 nm after 30 min at room temperature in a dark place (Zongo et al., 2010). The mean of three readings was used and the total phenol

Total flavonoid content of MeOH extracts (mean ± S.D.)

Total phenol content of MeOH extracts (mean ± S.D.)

29.87 50.11 43.78 NT 42.54 52.59 NT 63.19 46.25 NT 60.30 88.65 NT 81.36 77.50

10.74 ± 1.21 119.80 ± 3.87 164.40 ± 7.55 NT 201.59 ± 14.79 90.52 ± 2.29 NT 131.10 ± 4.50 109.56 ± 1.73 NT 169.05 ± 8.86 181.36 ± 10.94 NT 172.88 ± 7.70 178.16 ± 15.17

± 1.04 ± 3.13 ± 2.58 ± 2.19 ± 1.95 ± 6.11 ± 1.89 ± 2.51 ± 7.02 ± 0.72 ± 6.48

content was expressed in mg of gallic acid equivalents (GAE)/g extracts. Calibration curve equation was; y(Abs.) = 5.306x(Conc.) + 0.0587 and the coefficient of determination was r2 = 0.9986. 2.9. Determination of total flavonoid content The method of Kosalec et al. (2004) was used to determine total flavonoid contents of the active Inula extracts. Dry extracts were dissolved in 80% ethanol (1 mg/mL). 95% ethanol, 1 M sodium acetate and aluminium chloride solution (10%) were added to the samples and the mixture was diluted to 5 mL by distilled water. After 30 min incubation at room temperature, the absorbance of yellow mixtures was measured at 415 nm. Methanol was used as blank. Results were expressed in mg of quercetin equivalents (QE)/g extracts. Calibration curve equation was; y = 2.4214x − 0.051 and the coefficient of determination was r2 = 0.9998. 2.10. Statistical analysis All analyses were carried out in triplicates and the results were averaged. All values are expressed as the mean ± standard deviation (S.D.)

Table 2 α-Glucosidase and α-amylase inhibitory effects of roots extracts of Inula species. Plant name

Plant part

I. helenium

Root

I. montbretiana

Root

I. peacockiana

Root

I. thapsoides

Root

I. viscosa

Root

Extract type

Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc

α-Glucosidase inhibition (inhibition % ± S.D.)

α-Amylase inhibition (inhibition % ± S.D.)

3000 μg/mL

1000 μg/mL

570 μg/mL

300 μg/mL

100 μg/mL

3000 μg/mL

– 88.69 ± 0.41 2.64 ± 0.01 22.38 ± 5.73 64.73 ± 3.24 NT 10.84 ± 1.84 32.36 ± 5.38 26.22 ± 182 18.63 ± 4.57 51.84 ± 2.81 38.69 ± 2.46 5.16 ± 0.01 50.65 ± 3.06 NT

– 82.24 ± 2.10 – – 24.63 ± 3.97 NT – 18.47 ± 1.48 5.88 ± 1.81 2.71 ± 0.01 25.70 ± 0.59 9.44 ± 3.83 – – NT

– 75.54 ± 1.70 – – 12.12 ± 0.75 NT – 11.12 ± 2.70 – – 2.40 ± 0.01 5.08 ± 4.03 – – NT

– 64.06 ± 1.71 – – 6.62 ± 3.79 NT – – – – – – – – NT

– 27.56 ± 0.57# – – 2.21 ± 3.30# NT – – – – – – – – NT

10.67 ± 0.65# – 9.35 ± 0.20# 8.43 ± 1.76# 0.38 ± 0.00# 13.09 ± 3.46# 13.86 ± 5.22# 3.44 ± 4.59# 2.28 ± 3.22 3.18 ± 1.30# 14.93 ± 1.53# 5.60 ± 1.80# 5.34 ± 1.64# 16.56 ± 1.54# –

α-Glucosidase inhibition (inhibition % ± S.D.) Acarbose

α-Amylase inhibition (inhibition % ± S.D.)

Concentration

100 μg/mL

30 μg/mL

10 μg/mL

3 μg/mL

1 μg/mL

3000 μg/mL

1000 μg/mL

300 μg/mL

Inh. % ± S.D.

98.67 ± 0.17

98.02 ± 0.03

96.13 ± 0.62

92.77 ± 1.05

88.45 ± 3.35

80.74 ± 2.53

64.50 ± 1.72

38.06 ± 1.00

NT: Not tested, −: No activity, S.D.: Standard Deviation. # p b 0.0001 (compared with acarbose values at the same concentration).

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Table 3 α-Glucosidase and α-amylase inhibitory effect of major compounds of active Inula extracts. Compound name

Molecular weight

Caffeic acid Chlorogenic acid Quercetin-2-hydrate Luteolin Rutin Helenin Hispidulin

180.16 354.31 338.27 286.24 610.52 232.32 300.26

Reference

Molecular weight

Acarbose

645.60

α-Glucosidase inh. (inh. % ± S.D.)

α-Amylase inh. (inh. % ± S.D.)

10 mM

3 mM

1 mM

10 mM

4.66 ± 1.76 3.30 ± 1.12 78.36 ± 0.51 51.94 ± 0.00 51.84 ± 0.00 5.89 ± 0.00 21.44 ± 3.52

0.95 ± 1.18 1.20 ± 1.18 27.53 ± 14.70 26.83 ± 2.79 23.50 ± 2.07 13.31 ± 6.54 18.45 ± 3.38

– 1.41 ± 0.80# 10.17 ± 0.29# 10.01 ± 0.01# 10.84 ± 5.13# 14.29 ± 0.99# 14.07 ± 1.23#

– 6.51 ± 2.25# 15.90 ± 1.79# 17.00 ± 1.98# 21.08 ± 1.79# NT NT

α-Glucosidase inh. (inh. % ± S.D.)

α-Amylase inh. (inh. % ± S.D.)

1 mM

0.1 mM

0.01 mM

10 mM

99.74 ± 0.18

98.72 ± 0.04

94.99 ± 0.33

91.43 ± 0.89

NT: Not tested, −: No activity, S.D.: Standard Deviation. # p b 0.0001 (compared with acarbose values at the same concentration).

or standard error of the mean (S.E.M.); linear regression analyses and calculations were done by using Microsoft Excel and GraphPad Instat softwares. 3. Results and discussion MeOH extracts of the five Inula taxa exhibited higher α-glucosidase enzyme inhibitory activity than aqueous and EtOAc extracts (Table 2). α-Glucosidase enzyme inhibitory activity of the MeOH extracts ranged from 32.36 to 88.69% at 3000 μg/mL, and in the order I. peacockiana b I. viscosa b I. thapsoides b I. montbretiana b I. helenium ssp. turcoracemosa. Enzyme inhibitory activity of MeOH extract of I. helenium ssp. turcoracemosa roots was found remarkably high (88.69% at 3000 μg/mL) among all investigated root extracts. In this study, acarbose had 98.67% α-glucosidase inhibition at 100 μg/mL. In our previous study, alantolactone/isoalantolactone (helenin) was isolated as the main constituent of I. helenium ssp. turcoracemosa roots. After identification of helenin by modern spectroscopic methods, quantification was performed using HPLC-DAD. Helenin was found in serious amount in the roots of the plant as 1.6338 ± 0.0198% (w/w) (Gökbulut and Şarer, 2013). This finding made us think that helenin might be the

possible compound responsible for this significant activity. So, αglucosidase inhibitory activity of helenin was investigated with the same experiment procedure and a weak activity (5.89–14.29%) was determined (Table 3). Interestingly, α-glucosidase inhibition decreased when the concentration of helenin was increased. Although helenin is the major compound of the root extract, the activity should not be attributed to this isomeric mixture. On the other hand, total phenol and flavonoid contents of I. helenium ssp. turcoracemosa roots were determined too low. The higher inhibitory activity should be more likely due to the synergistic effect of sesquiterpenes, phenolics and the other secondary metabolites of the root. Actually, the inhibitory activity of all Inula extracts on α-amylase enzyme was weak (0.0–14.93%). In this enzyme model, acarbose showed 80.74% inhibition at 3000 μg/mL. MeOH extracts of I. viscosa roots inhibited α-amylase with 16.56% at 3000 μg/mL as the most potent one. The α-glucosidase and α-amylase inhibitory activity results of the root extracts were given in Table 2. All of the flower MeOH extracts inhibited α-glucosidase more than 77.0% at 3000 μg/mL concentrations (Table 4). I. viscosa MeOH extract showed the highest inhibition with a ratio more than 90%. This remarkable inhibitory activity of the flower extracts should be attributed to the

Table 4 α-Glucosidase and α-amylase inhibitory effects of flower extracts of Inula species. Plant name

Plant part

I. helenium

Flower

I. montbretiana

Flower

I. peacockiana

Flower

I. thapsoides

Flower

I. viscosa

Flower

Extract type

Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc

α-Glucosidase inhibition (inhibition % ± S.D.)

α-Amylase inhibition (inhibition % ± S.D.)

3000 μg/mL

1000 μg/mL

570 μg/mL

300 μg/mL

100 μg/mL

3000 μg/mL

18.94 ± 6.93 77.09 ± 1.29 50.15 ± 2.98 12.05 ± 3.52 87.05 ± 0.92 NT 9.70 ± 4.87 77.46 ± 3.25 21.24 ± 4.44 14.69 ± 1.03 78.28 ± 1.18 26.92 ± 1.58 54.33 ± 1.25 90.90 ± 2.02 NT

7.53 ± 3.42 29.63 ± 4.55 2.64 ± 1.31 – 44.97 ± 3.83 NT – 40.57 ± 1.41 – 2.89 ± 7.04 31.83 ± 2.64 3.02 ± 2.08 15.47 ± 1.69 36.43 ± 3.24 NT

– 11.27 ± 4.60 2.26 ± 1.77 – 26.73 ± 0.63 NT – 24.05 ± 0.44 – – 12.65 ± 3.32 – 6.27 ± 5.31 20.52 ± 11.01 NT

– 5.94 ± 6.28 – – 12.19 ± 6.20 NT – 7.07 ± 0.96 – – 8.26 ± 9.35 – – 1.22 ± 0.67 NT

– – – – – NT – 2.98 ± 3.47# – – 5.57 ± 0.50# – – – NT

9.31 ± 2.86# 3.65 ± 2.58# 3.58 ± 2.56# 24.51 ± 2.38# 4.13 ± 1.68# 10.75 ± 2.23# 15.23 ± 1.69# – 39.94 ± 1.36# 9.82 ± 1.41# – 15.77 ± 3.71# – 4.62 ± 1.38# 4.23 ± 3.80#

α-Glucosidase inhibition (inhibition % ± S.D.) Acarbose

α-Amylase inhibition (inhibition % ± S.D.)

Concentration

100 μg/mL

30 μg/mL

10 μg/mL

3 μg/mL

1 μg/mL

3000 μg/mL

1000 μg/mL

300 μg/mL

Inh. % ± S.D.

98.67 0.17

98.02 ± 0.03

96.13 ± 0.62

92.77 ± 1.05

88.45 ± 3.35

80.74 ± 2.53

64.50 ± 1.72

38.06 ± 1.00

NT: Not tested, −: No activity, S.D.: Standard Deviation. # p b 0.0001 (compared with acarbose values at the same concentration).

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phenolic acid and flavonoid contents of the plants. In our previous study, quantification of some phenolic acids and flavonoids of Inula taxa were performed (Gökbulut et al., 2013). Rutin and quercetin amounts of I. helenium flowers and luteolin amount of I. montbretiana flowers were determined quite high (0.027–0.24% and 0.07–0.054%). HPLC analysis results revealed that I. viscosa flowers contain rutin, quercetin, luteolin and kaempferol ranging from 0.03 to 0.07% (w/w). Hispidulin which was previously isolated from I. viscosa was another flavonoid thought to be responsible for the important inhibitory activity (Grande et al., 1985). So that, α-glucosidase inhibitory activities of quercetin, luteolin, rutin and hispidulin were investigated with the same procedure and charming results were obtained. Quercetin exhibited 78.36% α-glucosidase inhibition, while luteolin, rutin and hispidulin showed 51.94, 51.84 and 21.44% at 10 mM concentrations, respectively. Tadera et al. (2006) investigated the α-glucosidase and α-amylase inhibitory activities of flavonoids, and indicated that the decreasing order of the inhibitory activity of the six flavonoid groups was concluded to be anthocyanidin ≥ isoflavone ≥ flavonol ≥ flavones ≥ flavanone ≥ flavan-3-ol. The structure of the A, B and C rings was closely related to the inhibitory activity. For A and C rings, hydroxylation at 3 and 5 position of flavon enhanced the inhibitory activity. These results support our findings in terms of α-glucosidase inhibitory activities of investigated flavonoids in our experiment model (quercetin N luteolin N hispidulin). Among the extracts obtained from flower, leaf and root of all Inula species, I. montbretiana flower aqueous (24.51%) and I. peacockiana flower EtOAc (39.94%) extracts showed the highest α-amylase enzyme inhibitory activity. For the α-amylase inhibitory activity of flavonoids, luteolin and quercetin were quoted to be the potent inhibitors (Tadera et al., 2006). The α-amylase inhibitory activity of the extracts should be due to luteolin and quercetin which were determined in all the flower extracts of Inula species (Gökbulut et al., 2013). The α-glucosidase and α-amylase inhibitory activity results of the flower extracts were given in Table 4. The α-glucosidase and α-amylase inhibitory activity results of the leaf extracts were given in Table 5. MeOH extracts of the leaves of the plants exhibited strong α-glucosidase inhibitory activity pretty much the same as the results obtained from flower extracts. The enzyme inhibitory activity of I. thapsoides ssp. thapsoides (97.14%, 3000 μg/mL) and I. viscosa MeOH (92.87%, 3000 μg/mL) extracts was found to be close to that of Acarbose (98.67%, 100 μg/mL) used as reference. αGlucosidase enzyme inhibitory activity of other Inula species ranged

from 80.62 to 72.96%, and in the order I. helenium ssp. turcoracemosa N I. peacockiana N I. montbretiana. According to our previous study, I. viscosa leaves contain quercetin, luteolin and rutin ranging from 0.02 to 0.1% (w/w) (Gökbulut et al., 2013). The higher activity of the I. viscosa flower extract should be attributed to these flavonoids, also total flavonoid and total phenol concentrations of I. viscosa and I. thapsoides ssp. thapsoides leaves were found remarkably high and these findings should be taken in consideration while evaluating the enzyme inhibitory activity of the extracts. α-Amylase inhibitory activity of all Inula leaf extracts was found to be in the range of 0.99–15.77%. Among the tested leaf extracts, I. viscosa MeOH (15.77%) and I. peacockiana (14.60%) EtOAc extracts demonstrated the highest inhibitory activity. Gökbulut et al. (2013) also found high amount of chlorogenic and caffeic acids in all the parts of the investigated Inula taxa (0.14–0.82% and 0.003–0.046%). Hence, α-glucosidase and α-amylase inhibitory activities of chlorogenic and caffeic acids were tested. But, a weak activity was observed for these compounds in both methods (Table 3). Several studies reported in vivo antidiabetic activity of some Inula species. In one of these studies, I. britannica has a preventive effect on autoimmune diabetes by regulating cytokine production (Kobayashi et al., 2002). Additionally, Hong et al. (2012) reported that I. britannica flower polysaccharides could significantly reverse the decrease of plasma glucose, glycogen and the decrease of blood lipid dose-dependently in diabetic mice. Chronic treatment with MeOH extracts of the roots of I. racemosa produced significant reduction in blood sugar level in alloxan induced hyperglycemia (Ajani et al., 2009). Aqueous extract of the flowers of I. japonica was reported to possess antidiabetic activity in alloxaninduced diabetic mice (Shan et al., 2006). In-vivo hypoglycaemic effect of decoction of I. viscosa herb was investigated after single and repeated oral administration at 20 mg/kg dose. The extract was found to have a promising hypoglycaemic activity both on normoglycaemic and diabetic rats. According to the results of this study, authors reported that I. viscosa extract had no effect on plasma insulin levels in both normal and diabetic rats. So they concluded that the underlying hypoglycaemic mechanism seems to be extra-pancreatic (Zeggwagh et al., 2006). The results of our study can help to explain this situation that I. viscosa leaf and flower extracts revealed spectacular α-glucosidase inhibitory activity. Total phenol and flavonoid contents of the most potent Inula extracts were determined. The total phenol content of the active MeOH extracts was estimated by using Folin Ciocalteu reagent and

Table 5 α-Glucosidase and α-amylase inhibitory effects of leaf extracts of Inula species. Plant name

Plant part

I. helenium

Leaf

I. montbretiana

Leaf

I. peacockiana

Leaf

I. thapsoides

Leaf

I. viscosa

Leaf

Extract type

Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc Aqueous MeOH EtOAc

α-Glucosidase inhibition (inhibition % ± S.D.)

α-Amylase inhibition (inhibition % ± S.D.)

3000 μg/mL

1000 μg/mL

570 μg/mL

300 μg/mL

100 μg/mL

3000 μg/mL

48.89 ± 9.70 80.62 ± 0.81 NT 7.53 ± 4.80 72.96 ± 1.27 NT 17.17 ± 2.60 73.08 ± 3.63 NT 5.76 ± 6.61 97.14 ± 0.40 NT 63.37 ± 0.74 92.87 ± 2.58 NT

24.43 ± 6.46 63.18 ± 2.77 NT – 50.34 ± 5.66 NT 7.92 ± 5.03 54.69 ± 3.94 NT – 68.91 ± 5.10 NT 31.47 ± 2.58 51.70 ± 7.18 NT

7.16 ± 0.96 48.35 ± 4.82 NT – 35.81 ± 7.01 NT – 34.34 ± 1.89 NT – 36.87 ± 5.78 NT 15.58 ± 3.13 20.51 ± 3.54 NT

– 31.20 ± 2.62 NT – 15.60 ± 3.30 NT – 10.61 ± 1.62 NT – 11.02 ± 4.14 NT 6.54 ± 2.83 8.84 ± 0.02 NT

– – NT – 9.40 ± 5.80# NT – 1.81 ± 0.79# NT – – NT – 2.30 ± 0.82# NT

7.19 ± 1.71# 4.34 ± 3.26# 4.73 ± 3.22# 5.87 ± 1.18# 4.25 ± 3.14# – – 4.25 ± 4.23# 14.60 ± 4.36# 0.99 ± 0.60# – 7.68 ± 2.66# – 15.77 ± 2.98# 4.35 ± 4.23#

α-Glucosidase inhibition (inhibition % ± S.D.) Acarbose

α-Amylase inhibition (inhibition % ± S.D.)

Concentration

100 μg/mL

30 μg/mL

10 μg/mL

3 μg/mL

1 μg/mL

3000 μg/mL

1000 μg/mL

300 μg/mL

Inh. % ± S.D.

98.67 0.17

98.02 ± 0.03

96.13 ± 0.62

92.77 ± 1.05

88.45 ± 3.35

80.74 ± 2.53

64.50 ± 1.72

38.06 ± 1.00

NT: Not tested, −: No activity, S.D.: Standard Deviation. # p b 0.0001 (compared with acarbose values at the same concentration).

N. Orhan et al. / South African Journal of Botany 111 (2017) 86–92

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Table 6 Antioxidant activity of active Inula extracts. Plant name

I. helenium

I. montbretiana I. peacockiana I. thapsoides I. viscosa

References

EDTA Ascorbic acid Trolox

Plant part

Root Flower Leaf Flower Leaf Flower Leaf Flower Leaf Flower Leaf

Extract type

Metal-chelating capacity (inhibition % ± S.E.M.)

Ferric-reducing antioxidant power (absorbance at 700 nm ± S.E.M.)

3000 μg/mL

3000 μg/mL

– 17.64 ± 1.38 4.60 ± 0.53 – – 7.05 ± 0.31 5.21 ± 0.92 – 51.64 ± 1.81 18.51 ± 0.74 11.35 ± 1.53

MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH MeOH

1000 μg/mL – – – – – – – – 17.84 ± 1.19 – –

0.231 2.501 2.501 2.133 1.639 1.744 1.831 2.231 2.698 2.212 2.137

± ± ± ± ± ± ± ± ± ± ±

Total antioxidant (AAE ± S.E.M.)

1000 μg/mL #

0.007 0.025# 0.068# 0.071# 0.099# 0.066# 0.084# 0.010# 0.058# 0.040# 0.081#

0.099 0.535 1.054 0.979 0.583 0.812 0.878 1.273 0.873 1.281 0.598

± ± ± ± ± ± ± ± ± ± ±

570 μg/mL #

0.013 0.035# 0.063# 0.070# 0.046# 0.046# 0.006# 0.042# 0.083# 0.034# 0.084#

0.041 0.384 0.594 0.527 0.442 0.621 0.543 0.924 0.626 0.917 0.526

± ± ± ± ± ± ± ± ± ± ±

3000 μg/mL #

0.006 0.019# 0.030# 0.037# 0.013# 0.009# 0.009# 0.031# 0.004# 0.026# 0.050#

10.82 ± 1.68 # 186.17 ± 29.13# 173.06 ± 22.13# 123.89 ± 16.84# 405.77 ± 20.74 182.89 ± 20.47# 205.83 ± 41.07# 304.16 ± 41.85# 294.33 ± 14.29# 225.50 ± 31.26# 543.43 ± 25.60#

Metal-chelating capacity (inhibition % ± S.E.M.)

Ferric-reducing antioxidant power (absorbance at 700 nm ± S.E.M.)

Total antioxidant (AAE ± S.E.M.)

1000 μg/mL

570 μg/mL

3000 μg/mL

1000 μg/mL

570 μg/mL

3000 μg/mL

100N NT –

96.18 ± 1.20 NT –

NT 2.753 ± 0.034 –

NT 2.583 ± 0.0982 –

NT 2.452 ± 0.094 –

NT 382.50 ± 17.03

AAE: Ascorbic Acid Equivalent, NT: Not tested, −: No activity, S.E.M.: Standard Error of The Mean. # p b 0.0001 (compared with reference's values at the same concentration).

varied widely ranging from 10.74 to 201.59 mg GAE/g extract. I. montbretiana flower, I. thapsoides ssp. thapsoides leaf, I. viscosa leaf and flower extracts exhibited the highest total phenol content. I. thapsoides ssp. thapsoides leaf extract (88.65 ± 7.02 mg QE/g extract) showed the highest amount of flavonoid content followed by I. viscosa leaf (77.50 ± 6.48 mg QE/g extract) and flower (81.36 ± 0.72 mg QE/g extract) extracts. These results support our findings on antidiabetic activity of tested Inula extracts in terms of higher total phenol and flavonoid contents. Table 1 summarizes the total phenol and total flavonoid contents of the most active extracts. Depending on the α-glucosidase and α-amylase inhibitory activity results, the most potent Inula extracts were subjected to in vitro antioxidant activity tests. In our previous study, antioxidant capacity of five Inula taxa was evaluated using DPPH and ABTS radical scavenging assays and extracts exhibited significant antioxidant activities in different concentrations. Nearly all the EtOAc extracts had low antioxidant activity, with high IC50 values, compared with the water and MeOH extracts (Gökbulut et al., 2013). In this study, we checked the antioxidant activity of the most potent enzyme inhibitor extracts using metal chelating activity, ferric reducing antioxidant power and phosphomolybdenum assays. I. thapsoides ssp. thapsoides leaf extract revealed the highest chelating activity with 51.64% inhibition at 3000 μg/mL concentration. According to ferric reducing activity power assay results, nearly all the extracts exhibited high absorbance values (1.639–2.698) at 3000 μg/mL concentrations, close to ascorbic acid (2.452–2.753) which was used as a powerful ferric reducing agent. Total antioxidant power of I. montbretiana leaf (405.77 AAE) and I. viscosa leaf (543.43 AAE) was determined much more than Trolox (382.50 AAE) at 3000 μg/mL concentrations. Among all tested extracts, I. helenium root extracts exhibited the lowest antioxidant activity in all assays used. Antioxidant assay results were given in Table 6. 4. Conclusion In conclusion, we made a study of antidiabetic activity on selected Inula taxa collected from different regions of Anatolia. The most active extracts were evaluated for their antioxidant capacity, total phenol and flavonoid contents. Except for I. montbretiana, I. viscosa, I. peacockiana and I. thapsoides ssp. thapsoides root MeOH extracts, MeOH extracts of all Inula species exhibited the inhibition with a ratio more than 72% on α-glucosidase enzyme which is critically affecting carbohydrate digestion and glucose absorption. The MeOH extracts usually showed significant

antioxidant activity in two in vitro antioxidant models (ferric-reducing antioxidant power and total antioxidant capacity). On the other hand, quercetin, rutin, and luteolin which were previously quantified compounds of these five Inula taxa, exerted potential inhibitory activity against α-glucosidase. As a conclusion, MeOH extracts having potent antioxidant activity of Inula species may be beneficial for the prevention or treatment of oxidative stress induced complications of diabetes mellitus. To the best of our knowledge this is the only study setting light to the α-glucosidase and α-amylase inhibitory activities of Turkish Inula taxa, therefore results are important to fight with diabetes and will guide researchers dealing with ethnobotanical usage of plants on diabetes. As a result, Inula extracts exhibiting both antidiabetic and antioxidant activities can be utilized in preparation of functional food or herbal medicines in pharmaceutical industry. Conflict of interest We declare that there is no conflict of interest. Acknowledgement We are thankful to Bayer Group Turkey for providing us with Acarbose. References Ajani, H.B., Patel, H.P., Shah, G.B., Acharya, S.R., Shah, S.K., 2009. Evaluation of antidiabetic effect of methanolic extract of Inula racemosa root in rats. Pharmacology 3, 118–129. Ali, H., Houghton, P.J., Soumyanath, A., 2006. α-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. Journal of Ethnopharmacology 107, 449–455. Chang, C.L.T., Lin, Y., Bartolome, A.P., Chen, Y.C., Chiu, S.C., Yang, W.C., 2013. Herbal therapies for type 2 diabetes mellitus: chemistry, biology, and potential application of selected plants and compounds. Evidence-based Complementary and Alternative Medicine 2013, 378657 (33 pages). Dinis, T.C., Madeira, V.M., Almeida, L.M., 1994. Action of phenolic derivatives (acetaminophen, salicylate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and Biophysics 315, 161–169. Eddouks, M., Ouahidi, M.L., Farid, O., Moufid, A., Khalidi, A., Lemhadri, A., 2007. The use of medicinal plants in the treatment of diabetes in Morocco. Phytothérapie 5, 194–203. Ertuğ, F., 2014. Etnobotanik. In: Güner, A. (Ed.), Illustrated Flora of Turkey. 1. Türkiye İş Bankası Kültür Yayınları, Istanbul, p. 354. Food and Drug Administration, 2014. Food additives permitted for direct addition to food for human consumption. Code of Federal Regulations. Title 21, Vol. 3. Available from: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart= 172&showFR=1 (Last accessed on 10.11.2016).

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