Journal of Ethnopharmacology 146 (2013) 859–865

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Journal of Ethnopharmacology

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In vivo and in vitro antidiabetic effect of Cistus laurifolius L. and detectionof major phenolic compounds by UPLC–TOF-MS analysis

Nilufer Orhan a, Mustafa Aslan a,n, Murat S- ukuroglu b, Didem Deliorman Orhan a

a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara 06330, Turkeyb Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara 06330, Turkey

a r t i c l e i n f o

Article history:

Received 23 July 2012

Received in revised form

24 January 2013

Accepted 6 February 2013Available online 24 February 2013

Keywords:

a-Amylase inhibitory

Antidiabetic

Cistaceae

Cistus laurifolius

Flavonoids

a-Glucosidase inhibitory

41/$ - see front matter & 2013 Elsevier Irelan

x.doi.org/10.1016/j.jep.2013.02.016

esponding author. Tel.: þ90 312 2023184; fa

ail address: marslan@gazi.edu.tr (M. Aslan).

a b s t r a c t

Ethnopharmacological relevance: In Turkish folk medicine, various parts of Cistus laurifolius L. are used to

treat gastric ulcer and various types of pains. Additionally the tea prepared from the leaves is used to

decrease symptoms of diabetes.

Materials and methods: In the present study, the hypoglycemic effects of aqueous and ethanol extracts

of Cistus laurifolius were investigated in normal, glucose loaded hyperglycemic and streptozocin (STZ)-

induced diabetic rats. a-Glucosidase and a-amylase enzyme inhibitory effects were determined to

evaluate the mechanism of action. Total phenolic content of the extracts were determined by using

Folin–Ciocalteu reagent and Ultra Performance Liquid Chromatography–Time of Flight Mass Spectro-

meter (UPLC–TOF-MS) was used to detect the major phenolic compounds in the extract.

Results: Results indicated that blood glucose levels of the STZ-induced diabetic rats were decreased by

ethanol extract at of 250 and 500 mg/kg doses as compared to control group (16%–34%). In glucose

loaded animals, extracts have shown a weak hypoglycemic effect (11%–20%). Additionally, the ethanol

extract of Cistus laurifolius is found to be a potent inhibitor of a-glucosidase and a-amylase, possibly due

to several polyphenolic compounds present within the extract. Twelve major flavonoids (apigenin,

quercetin, kaempferol, naringenin, quercitrin and their derivatives), gallic, ellagic and chlorogenic acid

in chromatographic fingerprint were analyzed by the on-line UPLC–TOF-MS system.

Conclusions: Due to having inhibitory effect on blood glucose level and carbohydrate digesting enzymes

(a-glucosidase and a-amylase), Cistus laurifolius leaves might be beneficial for diabetic patients.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Diabetes mellitus is a common disease caused by the absoluteor relative absence of insulin (Amos et al., 1997). The burden ofdiabetes is increasing globally and 346 million people worldwidehave diabetes according to World Health Organization (WorldHealth Organization, 2011).

Herbal medications have been used for the treatment ofvariety of ailments by a huge number of people in the world.Many medicinal plants and their formulations are used fortreating diabetes in traditional medicine systems and in ethno-medicinal practices. Scientific investigations on traditional herbalremedies for diabetes may provide valuable leads for the devel-opment of alternative drugs and therapeutic strategies. Alterna-tives are clearly needed because of the inability of currenttherapies to control all of the pathological aspects of diabetes,and the high cost and poor availability of current therapies for

d Ltd. All rights reserved.

x: þ90 312 2235018.

many rural populations, particularly in developing countries(Marles and Farnsworth, 1995).

The genus Cistus (Cistaceae) is one of the characteristic generaof the Mediterranean region, colonizing degraded areas (Attaguileet al., 2000). Cistus laurifolius L. is a common plant and usedagainst various ailments in Turkish traditional medicine. Leavesof the plant are used to treat rheumatic and related inflammatorydiseases, externally as a bath remedy or poultice to reduce pain inrheumatism, against fever in common cold or applied externallyas a plaster on the dorsal part of the body in a line of the kidneysfor urinary inflammations (Yes-ilada et al., 1997). Decoctions offlowers and buds are used for the treatment of peptic ulcers.Meanwhile, infusion prepared from the leaves (2% w/v) is usedas hypoglycaemic in Turkish folk medicine (Baytop, 1999). In aprevious study, it is reported that infusion of branches of Cistus

laurifolius was taken as one teacup twice a day for one week totreat diabetes in Balıkesir, Turkey (Polat and Satıl, 2012).

Whereas, antiulcerogenic (Yesilada et al., 1997; Ustun et al.,2006), analgesic (Ark et al., 2004), antioxidant and hepatoprotective(Kupeli et al., 2006) effects of Cistus laurifolius have been studied, noresearch on its antidiabetic effect has been found so far. Because ofthe widespread utilization of the plant as a remedy, determination

N. Orhan et al. / Journal of Ethnopharmacology 146 (2013) 859–865860

of potential effects on blood glucose level was aimed for theevaluation of its antidiabetic effect. Hence in the present study thehypoglycemic and antidiabetic effects of Cistus laurifolius aqueousand ethanol extracts were examined in the normal, glucose loadedand STZ-induced diabetic rats.

The inhibition of the carbohydrate-hydrolyzing enzymes(a-glucosidase, a-amylase), prolong overall carbohydrate diges-tion time. It causes a reduction in the rate of glucose absorptionand decreases the post-prandial hyperglycemia (Rhabasa-Lhoretand Chiasson, 2004). Our second aim is to investigate a-glucosi-dase and a-amylase enzyme inhibitory effects of Cistus laurifolius

extracts. Moreover, after in vivo and in vitro biological activitystudies, for chemical characterization of extracts total phenolcontents were evaluated. Leaf ethanol extract was employed todetect the major compounds by Waters Acquity UPLC coupledto a Waters Micromass LCT Premier XE Time-of-Flight Massspectrometer.

2. Material and methods

2.1. Plant material

Cistus laurifolius L. (Cistaceae) leaves were collected inKurtbogazı, Ankara, Turkey in June 2009. Voucher specimenswere identified by a comparison with authentic specimens thathad already been identified by Prof. Dr. Mustafa Aslan. Authenti-cated voucher specimen was stored in the herbarium of Faculty ofPharmacy at Gazi University (GUE2489), Ankara, Turkey.

2.1.1. Preparation of the extracts

Leaves were seperated and extracted with distilled hot waterand ethanol 80% on the shaker for 24 h. Extracts are filteredand evaporated to dryness under vacuum (yields: aqueous extract13.5%, ethanol extract 16.5%).

2.2. In vivo studies

2.2.1. Preparation of the test samples

Dried extracts were suspended in 0.5% aqueous carboxy-methylcellulose (CMC) suspension in distilled water prior to oraladministration to animals (10 ml/kg, b.w.) [b.w.: body weight].Tolbutamide was used as the reference drug. Animals in thecontrol group received only the vehicle (10 ml/kg, b.w.).

2.2.2. Animals

Male Wistar-albino rats (150–200 g) were used in the experi-ments. Prior to the experiments, rats were fed with standard foodfor one week to adapt to the laboratory conditions. 12 h before

Table 1Effects of Cistus laurifolius extracts on blood glucose levels in normal and glucose load

Group Dose

(mg/kg)

Mean blood glucose concentration7Standard error of the m

Initial 30th min 60th min (gl. load) 90th

Control – 108.772.6 101.571.6 100.272.4 133.

Tolb. 100 117.577.2 64.673.8nnn (37%) 63.671.5nnn (37%) 77.

Aqueous

extract

250 106.073.8 100.373.8 109.072.2 114.

500 109.572.5 105.073.7 109.274.3 119.

Ethanol

extract

250 99.871.8 100.672.0 119.673.1 120.

500 100.371.3 106.872.3 102.373.3 111.

n po0.05 significant from the control animals.nn po0.01 significant from the control animals.nnn po0.001 significant from the control animals.

the experiments, they were fasted overnight, but allowed freeaccess to water. Six animals were used for each group of study.The animal experiments were processed following the interna-tionally accepted ethical guidelines for the care of laboratoryanimals. The study was approved by the Institutional AnimalEthical Committee of Gazi University.

2.2.3. Determination of the blood glucose levels

Blood glucose concentration (mg/dl) was determined using aGlucometer-elite commercial test (Bayer), based on the Glucoseoxidase method. Blood samples were collected from the tip of tailat the defined time patterns.

2.2.4. Effect on normoglycaemic plus glucose-hyperglycaemic model

[NG-OGTT]

A combined methodology of Kato and Miura (1993) is pre-ferred for the activity assessment of extracts to avoid using excessnumber of animals with some modifications in time pattern forblood glucose level determination. After overnight fasting (12 h)the blood glucose level of rats was determined and then testsamples were given. The normoglycaemic animals were dividedinto six groups. Control group received only vehicle (0.5% CMC),Tolbutamide was given to reference group at a dose of 100 mg/kg.Aqueous and ethanol extracts of Cistus laurifolius were given at250 and 500 mg/kg doses.

Test samples were given immediately after the collection ofinitial blood samples. The blood glucose levels were determinedat 30th and 60th min to assess the effect of the test samples onnormoglycaemic animals. After the 60th min measurement, therats were orally loaded with 2 g/kg glucose immediately. Bloodglucose levels were monitored to determine the effects onglucose-hyperglycaemic rats in the following time pattern: 1.5h,2, 3 and 6 h (Table 1).

2.2.5. Effects on streptozotocin induced diabetic rats

Diabetes was induced in rats by intraperitoneal injection of STZat a dose of 55 mg/kg b.w. dissolved in distilled water (1 ml/kg).Before STZ injection, rats were fasted for 12 h. Seven days after theinjection; the blood glucose levels were measured. Each animalwith a blood glucose level above 250 mg/dl was considered tobe diabetic. To evaluate acute antidiabetic effect, test samples(aqueous extract, EtOH extract, and tolbutamide) were adminis-tered orally by using a gastric gavage. Blood glucose levels weredetermined at 30, 60, 120, and 240 min after the administration ofthe test samples.

ed hyperglycaemic (NG-OGTT) rats.

ean (mg/dl) (Inhibition %)

min 120th min 180th min 360th min

375.2 132.574.9 109.371.5 107.673.7

674.8nnn (42%) 93.173.6nnn (30%) 65.572.3nnn (41%) 68.874.6nnn (37%)

773.9n (%15) 107.171.6nn (%19) 104.671.7 96.572.9

275.2n (11%) 121.674.5 107.073.8 101.875.5

075.69n (10%) 116.572.4n (13%) 109.573.7 100.873.6

374.7n (17%) 106.374.3nn (20%) 100.874.0 92.874.7n (16%)

N. Orhan et al. / Journal of Ethnopharmacology 146 (2013) 859–865 861

2.3. In vitro studies

2.3.1. Assay for a-amylase inhibitory activity

The a-amylase inhibition assay was performed using thechromogenic method used by Ali et al. (2006). Porcine pancreatica-amylase (EC 3.2.1.1, type VI, Sigma) was dissolved in ice-colddistilled water. Potato starch in phosphate buffer was used as asubstrate solution. According to the method, 40 ml of plantextract, 160 ml of distilled water and 400 ml of starch were mixedin a tube. The reaction was started by the addition of 200 ml of theenzyme solution. The tubes were incubated at 37 1C for 5 min.After adding 400 ml of starch solution, all tubes were incubated at37 1C for 3 min. After that, 200 ml of this mixture was added into atube containing 100 ml DNS color reagent solution and placed intoan 85 1C heater. After 15 min, 900 ml distilled water was addedand tubes were cooled. a-Amylase activity was determinedby measuring the absorbance of the mixture at 540 nm. Controlincubations were conducted in an identical fashion replacingplant extract with distilled water (40 ml). For blank incubations,the enzyme solution was replaced with distilled water and thesame procedure was carried out as above. The absorbance (A) dueto maltose generated was calculated as

A control or plant extract¼A test�A blank

From the net absorbance obtained, the %(w/v) of maltosegenerated was calculated from the equation obtained from themaltose standard calibration curve (0%–0.1%, w/v, maltose).Percent of inhibition was calculated as

% inhibition¼(mean maltose in sample/mean maltose incontrol)�100–100

2.3.2. Assay for a-glucosidase inhibitory activity

a-Glucosidase activity was assayed according to the reportedmethod by Lam et al. (Lam et al., 2008). The enzyme solution wasprepared by dissolving a-Glucosidase type IV (Sigma Co., St. Louis,USA) from Bacillus stearothermophilus in 0.5 M phosphate buffer.The enzyme solution (20 ml) and solutions of test compounds/extracts (10 ml) were mixed in a 96-well microtiter plate. After15 min preincubation at 37 1C, the substrate solution [10 ml,20 mM p-nitrophenyl-a-D-glucopyranoside (NPG), Sigma] in thesame buffer was added and the solution was incubated for anadditional 35 min at 37 1C. The increment of absorbance at405 nm due to the hydrolysis of NPG by a-glucosidase wasmeasured by ELISA microtiter plate reader. Acarbose (BayerGroup, Turkey) was used as positive control. The inhibitionpercentage (%) was calculated by the equation: Inhibition(%)¼[1�(Asample/Acontrol)]�100.

2.4. Phytochemical studies

2.4.1. Evaluation of total phenolic content:

The extracts (100 ml) were mixed with 0.2 ml Folin–Ciocalteureagent, 2 ml of H2O, and 1 ml of 15% Na2CO3 respectively. Themixture was measured at 765 nm after 2 h at room temperature.The mean of three readings was used and the total phenoliccontent was expressed in mg of gallic acid equivalents/g fraction(Gao et al., 2000). The coefficient of determination was r2

¼0.9957

2.4.2. Phytochemical analysis by UPLC–TOF-MS

i.

Preparation of sample and references:

A few crystals of the reference compounds (apigenin, chloro-genic acid, gallic acid, kaempferol, naringenin, quercetin,quercitrin) were resolved in 2 ml methanol and 20 mg driedethanol extract of Cistus laurifolius was resolved in 20 ml

methanol in ultrasonic bath before analysis. The extract wasfiltered through a 0.45 mm filter membrane. 5 ml of the sampleand reference compounds were injected to the chromato-graphic system and monitored under below chromatographicconditions.

ii.

Chromatographic system:Chromatographic separations were performed on a2.1 mm�100 mm Acquity UPLC BEH, 1.7 mm C18 column(Waters Corp, Milford, MA) using an Acquity UltraPerfor-mance Liquid Chromatography system (Waters Corp, Milford,MA). All solvents were filtered through a 0.45 mm filter andwere then degassed by sonication in an ultrasonic bath beforeuse. The mobile phase was composed of aqueous formic acid(A; %0.1, v/v) and acetonitrile-formic acid (B; %0.1, v/v); A:Bwas as follows: 0 min, 98:2; 6 min, 90:10; 10 min, 55:45;14 min, 45:55; 18 min, 10:90; 25 min, 0:100; the flow ratewas 1.0 ml/min and the column temperature was maintainedat 40 1C. A 1 min re-equilibration time was used after eachanalysis. The total runtime was 26 min at a flow rate of0.25 ml/min.

iii.

TOF instrumentation:Mass spectrometry was performed on a Micromass LCT Pre-mier XE (Waters MS Technologies, Manchester, UK) orthogo-nal acceleration Time-of-Flight mass spectrometer operationin both positive and negative ion mode with electrosprayionization (Z-spray). The desolvation gas flow was set to 700 l/h at a temperature of 300 1C. The cone gas flow was set to 11 l/h and the source temperature was set to 100 1C. The capillaryvoltage was set to 1700 V (in ESþ mode) and 2000 V (inES- mode), the cone voltage was set to 15 V, respectively. Theaperture 1 voltage was set to 5 V. The LCT Premier XE wasoperated in W optics mode with 412,500 resolution. Allanalyses were acquired using the lock spray to ensure accu-racy and reproducibility; leucine-enkephaline was used as thelock mass (m/z 556.2771).

iv.

Data processing:The mass spectrometric data were collected in full scan modethe m/z were from 100 to 1000 in both negative and positiveion. The data were collected and analyzed by MassLynxV 4.1 software (Micromass, Manchester, UK) to search forexpected compounds with accurate mass and fragment ionsinformation.

2.5. Statistical analysis

Results of in vivo studies are presented as means7standarderror of the mean (S.E.M.). Statistical differences between thetreatments and the controls were tested by one-way analysis ofvariance (ANOVA) followed by the Student–Newman–Keuls testusing the ‘‘Instat’’ statistic computer program. A difference in themean values of po0.05 was considered to be statisticallysignificant. In in vitro experiments, all analyses were carried outin triplicates and the results were averaged. All values wereexpressed as the mean7standard deviation (SD); linear regres-sion analyses and IC50 calculations were done by using SigmaPlot12.0 software.

3. Results

3.1. In vivo studies

The effects of Cistus laurifolius extracts and tolbutamideon blood glucose levels of normoglycaemic plus glucose-hyperglycaemic (NG-OGTT) rats are shown in Table 1. Extractsshowed a slight activity that appeared just after the glucose

Table 2Effects of Cistus laurifolius extracts on blood glucose levels in STZ-induced diabetic rats.

Group Dose (mg/kg) Mean blood glucose concentration7Standard error of the mean (mg/dl) (Inhibition %)

Initial 30th min 60th min 120th min 240th min.

Control – 428.6714.9 459.0710.3 449.679.9 452.877.3 427.0715.3

Tolbutamide 100 440.0727.4 384.278.0n (16.5%) 380.3716.8n (16.0%) 339.3713.0nn (25.0%) 323.0719.0nn (25.0%)

Aqueous extract 250 427.5715.0 414.078.0 409.0719.0 413.2712.3 399.2723.0

500 448.0715.6 447.479.0 429.078.0 435.479.1 380.277.8

Ethanol extract 250 432.0710.3 413.2713.4 379.3716.1n (16.0%) 355.8713.3nn (22.0%) 283.6716.9nnn (34.0%)

500 423.2724.4 381.6713.1n (17.0%) 323.8717.7nnn (28.0%) 301.0718.0nnn (33.0%) 282.4713.0nnn (34.0%)

n po0.05 significant from the control animals.nn po0.01 significant from the control animals.nnn po0.001 significant from the control animals.

Table 3a-Amylase inhibitory activity of Cistus laurifolius extracts.

Test Material Extract Dose (mg/ml) Inhibition % SD

Acarbose – 3000 �73.7 0.6

1000 �67.2 0.6

300 �51.8 2.9

100 �32.6 0.3

30 �10.6 2.0

Cistus laurifolius Aqueous extract 3000 �60.5 0.7

1000 �39.3 2.2

300 �17.0 2.6

100 �18.9 2.0

30 � –

Ethanol extract 3000 �75.3 0.3

1000 �71.7 0.6

300 �52.2 1.8

100 �10.0 0.3

30 – –

SD: Standard deviation.

N. Orhan et al. / Journal of Ethnopharmacology 146 (2013) 859–865862

loading (10%–19%), while the reference drug tolbutamide, pos-sessed potent activity during the experiment (30%–42%). How-ever, Cistus extracts did not show any remarkable effect on bloodglucose levels of normoglycaemic animals.

In streptozotocin induced diabetic rats, tolbutamide andextracts showed significant antidiabetic activity as mentioned inTable 2. At the 30th min measurement, a decrease was observedon blood glucose levels of Tolbutamide and ethanol extract(500 mg/kg) groups. Blood glucose concentrations of animals inethanol extract groups decreased at all measurements (16%–34%).The effect of ethanol extract was higher than Tolbutamide at500 mg/kg. Maximum antidiabetic effect was observed, 240 minafter the administration (34%) of ethanol extract. However aqu-eous extract did not show any remarkable effect on STZ-induceddiabetic rats.

3.2. In vitro studies

Inhibitory effect of Cistus laurifolius extracts on a-glucosidaseand a-amylase activity was also investigated. Effects were com-pared with the commercially available a-glucosidase inhibitor,Acarbose. a-Amylase inhibitory activity of the extracts wasevaluated at five different logarithmic doses (3000, 1000, 300,100, 30 mg/ml) and results were given in Table 3. Acarbose andextracts showed a remarkable and dose dependent inhibitoryeffect on a-amylase enzyme. Cistus laurifolius ethanol extractexhibited a higher inhibitory effect (75.3%) than acarbose(73.7%) at 3000 mg/ml concentration.

a-Glucosidase inhibitory activity of the extracts and acarbosewas evaluated at seven different logarithmic doses between

10.000 and 0.3 mg/ml. Extracts have shown promising and dosedependent inhibitory effect on a-glucosidase enzyme. IC50 valueswere calculated as IC50 Acarbose: 0.0009 (mg/ml), IC50 Cistus

laurifolius ethanol extract: 0.0063 (mg/ml) and IC50 Cistus laur-

ifolius aqueous extract: 0.1664 (mg/ml). Ethanol extract haspossessed outstanding inhibitory effect.

3.3. Phytochemical studies

Total phenolic contents of extracts were evaluated and thedata expressed in mg equivalent of gallic acid to 1 g of extract.Total phenolic content was found higher in Cistus laurifolius

ethanol extract (323.977.1) than the aqueous extract(289.9712.9).

Ethanol extract that have both the highest antidiabetic effectand the highest phenolic content, was analyzed by LC–TOF-MS todetect its phenolic compounds. After optimization of UPLC con-ditions, satisfactory results were obtained with a mobile phasecomposed of aqueous formic acid (A; 0.1%, v/v) and acetonitrile-formic acid (B; 0.1%, v/v) by using gradient elution for 26 min.Total ion chromatogram of Cistus laurifolius ethanol extract isgiven in Fig. 1. After analyzing the references under sameconditions, peaks in the total ion chromatogram of the extractwere compared to the peaks of references. Chromatograms ofboth positive and negative ionization ESI mode were checked andaccording to retention times and molecular masses, compoundsin the extract were identified. The name of the identified com-pounds, their molecular formula, retention times, experimentaland calculated mass data were given in Table 4. Additionally,extracted TOF-MS chromatograms of some identified compoundswere given in Fig. 2. In conclusion; flavonoids (apigenin,dimethoxyapigenin, two structural analogs of methoxyapigenin,naringenin, quercitrin, quercetin, methoxyquercetin, two struc-tural analogs of dimethoxyquercetin, two structural analogs ofdimethoxykaempferol), chlorogenic acid, gallic and ellagic acidwere found as the main phenolic constituents in Cistus laurifolius

ethanol extract by UPLC–TOF-MS analysis. Literatures were care-fully examined and in view of isolated compounds from Cistus

laurifolius (Sadhu et al., 2006) and salviifolius (Kuhn et al., 2011),estimated names of structural analogs (methoxyapigenin,dimethoxyapigenin, methoxyquercetin, dimethoyxyquercetin,dimetoxykaempferol) are given in parentheses (Table 4).

4. Discussion

The ethnobotanical information reports many plants that maypossess anti-diabetic potential, of which Momordica charantia,Pterocarpus marsupium, and Trigonella foenum greacum have beenreported to be beneficial for treatment of type 2 diabetes. More

Fig. 1. Total ion chromatogram (TIC) of the main components in Cistus laurifolius extract.

Table 4Compounds determined by UPLC–TOF-MS in Cistus laurifolius extract.

No. Compound Molecular

formula

Selected

ion

m/z

Experimental

m/z

Calculated

Tolerance (ppm in

generated

molecular formula)

Retention time

(min)

1 Gallic acid C7H6O5 [M�H]� 169.0133 169.0137 5 3.12

2 Chlorogenic acid C16H18O9 [M�H]� 353.0875 353.0873 5 11.21

3 Ellagic acid C14H6O8 [M�H]� 300.9987 300.9984 5 12.82

4 Quercetin 3-O-a-rhamnoside C21H20O11 [M�H]� 447.0921 447.0927 5 13.22

5 (3)-Methoxyquercetin C16H14O7 [M�H]� 317.0663 317.0661 5 13.69

6 Quercetin C15H10O7 [M�H]� 301.0340 301.0348 5 14.16

7 Apigenin C15H10O5 [M�H]� 269.0445 269.0450 5 14.66

8 Naringenin C15H12O5 [M�H]� 271.0601 271.0606 5 14.73

9 (3,7 or 3,40) Dimethoxyquercetin C17H14O7 [M�H]� 329.0663 329.0661 5 15.17

10 (3,7 or 3,40) Dimethoxyquercetin C17H14O7 [M�H]� 329.0663 329.0663 5 15.95

11 (7 or 40) Methoxyapigenin C16H12O5 [M�H]� 283.0597 283.0606 5 16.29

12 (7 or 40) Methoxyapigenin C16H12O5 [M�H]� 283.0600 283.0606 5 16.70

13 (3,7 or 3,40) Dimethoxykaempferol C17H14O6 [M�H]� 313.0706 313.0712 5 16.70

14 (3,7 or 3,40) Dimethoxykaempferol C17H14O6 [M�H]� 313.0700 313.0712 5 17.01

15 (3,40 or 7,40) Dimethoxyapigenin C16H14O7 [MþH]þ 299.0916 299.0919 5 19.41

N. Orhan et al. / Journal of Ethnopharmacology 146 (2013) 859–865 863

than 1200 species of plants have been screened for activity on thebasis of ethnomedicinal uses (Patel et al., 2012). Cistus laurifolius

is one of the plants used in Turkey for treatment of diabetes foundin the ethnobotanical field studies. Although many differentbiological activities of Cistus laurifolius were investigated this isthe first study on in vivo and in vitro antidiabetic activity of thisplant. Ethanol extract of Cistus laurifolius leaves was found to haveboth promising antidiabetic activity on STZ-induced diabetic ratsand inhibitory activity on digestive enzymes (a-amylase anda-glucosidase).

According to phytochemical studies, tannins (ellagic acid),sterols, phenolic acids, lignan glycosides and phenolic compoundsmainly flavonoids (including derivatives of apigenin, quercetin andkaempferol) were isolated from ethanol extract of leaves of Cistus

laurifolius (Sadhu et al., 2006). Among these, 3-O-methylquercetin,3,7-O-dimethylquercetin and 3,7-O-dimethylkaempferol were iso-lated as the anti-inflammatory ingredients from the ethanol extractof the plant (Kupeli and Yesilada, 2007). Additionally, quercetin3-methyl ether has been reported to be effective at inhibiting thealdose reductase activity (Enomoto et al., 2004).

In a previous study on Cistus salvifolius, under bioassay-guidance using PPAR transactivation assays and glucose uptakein adipocytes; trans-cinnamic acid, 7-methoxyapigenin, 40-meth-oxyapigenin, 7,40-dimethoxyapigenin, 3,40-dimethoxykaempferol,3,7,40-trimethoxykaempferol and 3,7,30-trimethoxyquercetin wereisolated as the active compounds. Due to their PPARg-activatingproperties, they were tested for their antidiabetic propertiesin vitro. Except for 40-methoxyapigenin, all six compounds testedwere able to enhance both basal and insulin-stimulated glucoseuptake. According to the results of the PPARg-transactivation and

the glucose uptake assays, trans-cinnamic acid seems to be a majoractive compound in Cistus salvifolius, which confirms other studiesfocusing on the antihyperglycemic activities of cinnamic acidderivatives in vitro and in vivo (Kuhn et al., 2011). AdditionallyLiang et al. reported that apigenin and kaempferol increased thePPARg activity in a dose dependent manner (Liang et al., 2001).

Phenolic compounds, especially flavonoids, are among theclasses of compounds that have received the most attention withregard to their antidiabetic properties (Coman et al., 2012).Several papers have been published on the antidiabetic effect offlavonoids such as quercetin, isoquercetin, rutin, apigenin andnaringenin (Vessal et al., 2003; Tadera et al., 2006; Li et al., 2009;Coman et al., 2012). Published data suggest that there might bedirect effects of flavonoids on insulin secretion, as well as onprevention of beta-cell apoptosis, and they could even act viamodulation of proliferation (Pinent et al., 2008). Additionally,gallic acid and derivatives of ellagic acid were found to be the a-glucosidase inhibitory constituents from stem bark of Terminalia

superba (Wansi et al., 2007).Control of blood glucose levels is critical in the early treatment

of diabetes mellitus and reduction of its complications. Onetherapeutic approach is the prevention of carbohydrate absorp-tion after food intake, which is facilitated by inhibition of theenteric enzymes including a-glucosidase and a-amylase presentin the brush borders of intestine (Toeller, 1994; Inzucchi, 2002).The inhibition of both these enzymes has been a strong option inthe prevention of diabetes.

According to the results of the present study, the ethanolextract of Cistus laurifolius possesses a potent antidiabetic activityand could be used as a safe remedy for the treatment of diabetes.

Fig. 2. Extracted TOF-MS chromatograms of identified compoundsn of Cistus laurifolius extract. nOnly spectrums of compounds found more than 3.5e3 in Cistus laurifolius

extract is given.

N. Orhan et al. / Journal of Ethnopharmacology 146 (2013) 859–865864

N. Orhan et al. / Journal of Ethnopharmacology 146 (2013) 859–865 865

Moreover, the ethanol extract of Cistus laurifolius is a potentinhibitor of a-glucosidase and a-amylase and improves hypergly-cemia in type 2 diabetic rats, possibly due to several polyphenoliccompounds (chlorogenic, ellagic and gallic acid, quercetin,apigenin, naringenin and their derivatives) present within theextract. It could be suggested that there may be a positivecorrelation between the phenolic content and a-glucosidaseinhibitory activity of the plant. Due to having inhibitory effecton blood glucose level and carbohydrate digesting enzymes(a-glucosidase and a-amylase), Cistus laurifolius leaves may bebeneficial for diabetic patients.

Acknowledgment

This study was financially supported by the Research Fund ofGazi University (02/2011-22). We are thankful to Bayer Group forproviding us with Acarbose.

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