Journal of Ethnopharmacology 119 (2008) 109116

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

In vitro inhibitory activities of plants used in Lebanon traditional medicine against angiotensin converting enzyme (ACE) and digestive enzymes related to diabetes
Monica R. Loizzo a, , Antoine M. Saab b , Rosa Tundis a , Federica Menichini a , Marco Bonesi a , Vitaliano Piccolo a , Giancarlo A. Statti a , Bruno de Cindio c , Peter J. Houghton d , Francesco Menichini a
a

Faculty of Pharmacy, Nutrition and Health Sciences, Department of Pharmaceutical Sciences, University of Calabria, I-87036 Rende (CS), Italy Faculty of Sciences II, Lebanese University, Chemistry Department, P.O. Box 90656, Fanar, Beirut, Lebanon Department of Modeling Engineering, University of Calabria, I-87036 Rende (CS), Italy d Pharmaceutical Sciences Research Division, Kings College London, 150 Stamford Street, London SE1 9NH, UK
b c

a r t i c l e

i n f o

a b s t r a c t
Aim of the study: In recent years the use of medicinal plants has been growing worldwide and this is particularly true in Lebanon. In the present investigation we report the inhibitory activity against digestive enzymes related to diabetes and angiotensin converting enzyme (ACE) of extracts of nine plant species collected in Lebanon, where they have a traditional use against diabetes. Materials and methods: In order to evaluate if the extraction procedure could inuence the activity we decided to perform different extractions with methanol, n-hexane and chloroform of Calamintha origanifolia, Satureja thymbra, Prangos asperula, Sideritis perfoliata, Asperula glomerata, Hyssopus ofcinalis, Erythraea centaurium, Marrubium radiatum and Salvia acetabulosa and test each of them. Results and conclusions: Marrubium radiatum and Salvia acetabulosa methanol extracts exerted the highest activity against -amylase (IC50 61.1 and 91.2 g/ml, respectively) and -glucosidase (IC50 68.8 and 76.9 g/ml, respectively). The same extracts exhibited a strong inhibitory activity against ACE with IC50 of 72.7 and 52.7 g/ml, respectively. The results support the traditional use of some the species examined. 2008 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 14 December 2007 Received in revised form 21 May 2008 Accepted 7 June 2008 Available online 13 June 2008 Keywords: -Amylase -Glucosidase ACE In vitro screening Extracts Lebanon

1. Introduction Herbal remedies and alternative medicines are used throughout the world and herb-derived medications are still used extensively for the treatment of high blood pressure, diabetes and other illness. Lebanon, located on the eastern Mediterranean shore, presents a climatic and ecological diversity that is unique to the region, and the whole country is recognized as a centre of plant diversity (WWF and IUCN, 1994). The Lebanese people can buy herbs in small stores called Dabbous in which the herbalist suggests plants for a specic disease without a prescription. In this study some herbs recommended in Dabbous in Lebanon for two common conditions, diabetes and hypertension, are examined using some in vitro test systems relevant to these diseases. After the recommendations made by WHO on diabetes mellitus (WHO, 1980), the search for safer and more effective hypoglycaemic pharmaceuticals has continued to be an important area of active research. The search for novel hypoglycaemic compounds from medicinal plants has become an important aspect of this

Corresponding author. Tel.: +39 0984493169; fax: +39 0984493298. E-mail address: mr.loizzo@unical.it (M.R. Loizzo). 0378-8741/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.06.003

and, in view of the difculties in testing in vivo, several in vitro tests have been developed in recent years. Two of these are concerned with the inhibition of the digestive enzymes would delay the degradation of starch and oligosaccharides, which would in turn cause a decrease in the absorption of glucose and consequently inhibit the increase in postprandial blood glucose. In particular, -amylase and -glucosidase participate in glucose digestion and are considered as key enzymes that can control postprandial hyperglycemia (Ali et al., 2006; Lee et al., 2007). -Amylase is present in both salivary and pancreatic secretion (Ramasubbu et al., 2004) and is responsible for cleaving large malto-oligosaccharides to maltose, which is then a substrate for intestinal -glucosidase. Tests for the ability of extracts and compounds to inhibit both -amylase and -glucosidase have been described by several workers (Asano et al., 2004; Conforti et al., 2005; Ali et al., 2006; Kotowaroo et al., 2006). In this study, in vitro models for inhibition of these two enzymes were used to screen extracts from nine plant species recommended by Lebanese store-keepers and also reported to be used in Lebanon for controlling diabetes (Nehm, 1981; Nehm, 2000; Tohm and Tohm, 2002). The species used are shown in Table 1. Diabetes is frequently associated with arterial hypertension, which may eventually result in cerebrovascular accidents and cardiovascular disease (Segura

110 Table 1 Plant species studied Plant species Asperula glomerata (M. Bieb) Griseb. Calamintha origanifolia Vis. Family Rubiaceae Lamiaceae

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Traditional use Reduce blood pressure, reduce inammation/edema Reduce blood pressure, antimicrobial activity, diaphoretic and expectorant Reduce blood pressurea , reduce spasm in the smooth muscle of the gastrointestinal tract, sedative, fever-reducing, reduce inammation/edema, digestive and stomachic activities Reduce blood sugarb , reduce blood pressure, reduce inammation, reduce spasm in the smooth muscle of the gastrointestinal tractc , antimicrobial activity Reduce blood sugar, reduce blood pressure, reduce spasm in the smooth muscle of the gastrointestinal tract, analgesic and neurosedative effects, reduce inammation/edema Reduce blood pressure, skin disease, digestive disorder, hemorrhoids. Reduce blood sugard , e , reduce blood pressure, antimicrobial effect, eupeptic and antihydrotic effects, menstrual disorder, insomnia, reduce pain in joints, coronary heart disease, reduced anxiety and depression Reduce blood pressure, reduce pain in joints, antimicrobial activityf Reduce blood pressure, antiinammatory activityg , reduce pain in joints, antimicrobial effects, decongestant of the respiratory tract, astringent property

Part used Aerial parts Steam

Way of use Herbal preparation for internal use Infusion

Place of collection Bakish Bakish

Date of collection 07/2005 08/2005

Voucher sample AS 5 AS 1

Centaurium erythraea L.

Gentianaceae

Aerial parts

Infusion

Kfarkaab

08/2005

AS 7

Hyssopus ofcinalis L.

Labiateae

Dried leaves

Infusion

Kfarkaab

07/2005

AS 6

Marrubium radiatum Devile ex Benth.

Lamiaceae

Aerial parts

Decoction

Tarshich

08/2005

AS 8

Prangos asperula Boiss.

Apiaceae

Aerial parts

Infusion

Bakish

07/2005

AS 3

Salvia acetabulosa L.

Lamiaceae

Aerial parts

Decoction

Tarshich

06/2005

AS 9

Satureja thymbra L.

Lamiaceae

Leaves

Infusion

Tarshish

08/2005

AS 2

Sideritis perfoliata L.

Lamiaceae

Leaves

Infusion

Kfarkaab

08/2005

AS 4

a b c d e f g

Haloui et al. (2000). Miyazaki et al. (2003). Lu et al. (2002). Omidbeygi (1997). Zargari (1997). Chorianopoulos et al. (2006). Charami et al. (2008).

and Ruilope, 2006). Excessive activation of the Renin angiotensin system (RAS) system has been considered to be a main cause of hypertension (Schricker et al., 1994) and this system is regulated by angiotensin converting enzyme (ACE). ACE inactivates the vasodepressor compound bradykinin and activates the potent vasoconstrictor and growth-promoting substance angiotensin II, by the removal of the carboxy-terminal dipeptide of angiotensin I (Paul et al., 2006). The importance of ACE inhibitors in the chronic treatment of various cardiovascular diseases is now well established (Carson, 2000; Shlipak et al., 2001; Remuzzi and Ruggenenti, 2006) and several ACE inhibitors, e.g. captopril, are currently used in the treatment of hypertension. Consequently, the nine species under consideration were also tested for ACE inhibition using a technique now carried out in several laboratories (Duncan et al., 1999; Loizzo et al., 2007). The ways in which herbalists suggested the use of these species in medicine are reported in Table 1.

2. Materials and methods 2.1. Chemicals The following chemicals were obtained from SigmaAldrich S.p.a. (Milano, Italy): vanillin, potato starch, sodium phosphate, sodium chloride, -amylase from porcine pancreas (EC 3.2.1.1), glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20), maltose, sodium acetate, sodium potassium tartrate, 3,5-dinitrosalicylic acid, o-dianisidine colour reagent (DIAN), glucose oxidase peroxidase enzyme solution (PGO), FolinCiocalteu reagent, ACE from rabbit lung (EC 3.4.24.15), dansyltriglycine, dansyl-l-glutamine, HEPES buffer, Na2 EDTA, Na2 CO3 and captopril. Methanol, chloroform, n-hexane, acetic acid, perchloric acid, sulphuric acid, DMSO, basic bismuth nitrate, potassium iodide, phosphoric acid, NaH2 PO4 buffer, acetonitrile, natural products reagent (NP), polyethylene glycol reagent (PEG) and thin-layer chromatogra-

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phy plates (TLC) were obtained from VWR International s.r.l. (Milano, Italy). Acarbose was obtained from Serva (Heidelberg, Germany). 2.2. Plant material Calamintha origanifolia Vis. (Lamiaceae), Satureja thymbra L. (Lamiaceae), Prangos asperula Boiss (Apiaceae), Sideritis perfoliata L. (Lamiaceae), Asperula glomerata (M. Bieb) Griseb (Rubiaceae), Hyssopus ofcinalis L. (Labiateae), Erythraea centaurium Rafn. (Gentianaceae), Marrubium radiatum Devile ex Benth (Lamiaceae) and Salvia acetabulosa L. (Lamiaceae) were selected from a list obtained from informal interviews with herbal shopkeepers in Beirut and other parts of Lebanon and collected in 2005 from various localities in Lebanon. The selected species were recommended by at least two herbalists and authenticated by Dr. Katia Saad, Biology Department, Faculty of Sciences II, Lebanese University. A voucher specimen of each species was deposited in the Herbarium, Faculty of Sciences II, Lebanese University (Table 1). The dried plant materials were ground and stored in brown glass bottles until extraction at 25 C. 2.3. Preparation of plant extracts The air-dried and ground sample (1 g) was extracted with 15 ml of methanol (MeOH), chloroform or n-hexane by macTable 2 Constituents of n-hexane extracts determined by GCMS Compounda Monoterpenes -Thujene -Pinene Sabinene -Phellandrene -3-Carene -Terpinene 1,8-Cineole -Terpinene p-Cresol Sesquiterpenes -Copaene -Humulene -Cadinene -Cadinene Lepidozene Fatty acid esters and alkanes Methyl palmitate Palmitic acid Ethyl palmitate Methyl linoleate Methyl linolenate Eicosane Oleic acid Ethyl linoleate Ethyl oleate Docosane Ethyl laurate Tricosane Alloimperatorin Pentacosane Heptacosane Nonacosane Steroids Stigmast-5-en-3-ol tR b 6.97 7.13 7.94 8.49 8.62 8.71 8.98 9.45 9.78 13.71 14.43 15.15 15.21 16.61 18.44 18.73 18.95 19.72 19.78 19.84 20.09 20.19 20.23 20.29 20.38 21.08 21.94 22.36 23.87 25.92 34.54 x x x x x x x x x x x x A B C x x x

eration using vortexing for 30 s and sonication for 1 min. The material was then centrifuged at 3000 rpm for 20 min. The supernatant was transferred into a 25 ml round bottom ask and then taken to dryness with a rotary vacuum evaporator (Choi et al., 2004). The extraction was performed three times. Percent yields (w/w) ranged from 11.1 to 13.9 for methanol extracts, from 5.9 to 7.2 for chloroform extracts and from 12.5 to 14.6 for n-hexane extracts. 2.4. TLC proles of extracts Plant extracts were evaluated using specic spray reagents used for various compound types in silica gel TLC systems. The n-hexane and chloroform extracts were developed with n-hexane/acetone (85:15) and n-hexane/EtOAc (9:1) then visualized with vanillinsulphuric acid reagent (VS) (Wagner and Bladt, 1996). The methanol extracts were developed with EtOAc/HCOOH/AcOH/H2 O (100:10:10:20) and then sprayed with NP-polyethylene glycol reagent (NP/PEG) (Wagner and Bladt, 1996).

2.5. GC/MS analysis The identication of the compounds from n-hexane and chloroform extracts was made using GCMSanalysis on an Hewlett

I x x x

x x x x

x x x

x x

x x

x x

x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

x x x x

x: presence of compound in (A) Calamintha origanifolia; (B) Satureja thymbra; (C) Prangos asperula; (D) Sideritis perfoliata; (E) Asperula glomerata; (F) Hyssopus ofcinalis; (G) Erythraea centaurium; (H) Marrubium radiatum; (I) Salvia acetabulosa. a Compounds eluted from non-polar SE-30 MS column. b Retention time (min).

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Packard 6890N gas chromatograph equipped with a non-polar capillary column (SE-30, 30 m 0.25 mm i.d. 0.25 m lm thickness) and interfaced with a Hewlett Packard 5973N mass selective detector, operating in electron ionisation (EI) (70 eV). The carrier gas was helium and the solvent delay, the time gap of a given analysis in which the mass spectrometer is turned off, was 3 min. Extracts were diluted to a nal volume of 1 ml with methanol (approximately 1 mg/ml). One microliter of sample was dissolved in the appropriate solvent and injected into the gas chromatograph. The injector and detector temperatures were 250 and 280 C, respectively. The analytical conditions worked with the following program: oven temperature was programmed from 60 to 280 C at a rate of 16 C/min; the nal temperature of 280 C was held for 10 min. Identication of the compounds were based on the comparison of the mass spectral data on computer matching against

Wiley 138 and Wiley 275 Library. GC/MS results are summarized in Tables 2 and 3. 2.6. Determination of content of total phenolics The content of total phenolics in the MeOH extracts was determined by the FolinCiocalteu assay, chlorogenic acid being used as standard. Extracts were added into 50 ml extraction tubes and then homogenized with the extraction solvent for 30 s with a sonicator. One hundred microliters (three replicates) of the extract so formed were mixed with 0.2 ml FolinCiocalteu reagent, 2.0 ml of H2 O and 1 ml of 15% Na2 CO3 , left to incubate at room temperature for 2 h and the absorbance was then measured at 765 nm using a PerkinElmer Lambda 40 UV/Vis spectrophotometer. The total phenolic content was expressed as chlorogenic

Table 3 Constituents of chloroform extracts determined by GCMS Compounda Monoterpenes -Thujene -Pinene Sabinene -Phellandrene -Terpinene m-Cymene 1,8-Cineole -Terpinene 2-Heptanoic acid, methyl ester Terpinen-4-ol Pulegone Sesquiterpenes -Humulene Spathulenol Fatty acids and alkanes Neophytadiene 2-Heptadecanone Methyl palmitate Palmitic acid Ethyl palmitate Stearic acid 8,11-Octadecadienoic acid, methyl ester 8-Octadecenoic acid, methyl ester Methyl stearate Ethyl linoleate Ethyl oleate Linolenic acid Triacontane Tricosane Tetracosane Pentacosane Heptacosane Oleic acid, 2-hydroxyethyl ester Methyl lignocerate Eicosane Hexatriacontane Nonacosane Methyl cerotinate Octacosane Steroids and triterpenoids Stigmast-5-en-3-ol -Amyrin 3-Keto-urs-12-ene -Amyrin Stigmasta-3,5-dien-7-one tR b 6.97 7.13 7.94 8.50 8.66 8.87 9.07 9.45 9.55 11.28 12.07 14.43 15.73 17.72 18.23 18.40 18.73 18.90 19.23 19.68 19.72 19.88 20.15 20.18 20.57 20.66 21.04 21.44 22.31 23.87 23.90 24.09 24.72 24.85 25.85 26.27 28.83 34.32 35.33 35.93 36.78 37.12 A B x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x C D E F G H I

x x x x

x x x x

x x

x x

x x

x x x x x x x x

x x x

x x x x

x x x

x: presence of compound in (A) Calamintha origanifolia; (B) Satureja thymbra; (C) Prangos asperula; (D) Sideritis perfoliata; (E) Asperula glomerata; (F) Hyssopus ofcinalis; (G) Erythraea centaurium; (H) Marrubium radiatum; (I) Salvia acetabulosa. a Compounds eluted from non-polar SE-30 MS column. b Retention time (min).

M.R. Loizzo et al. / Journal of Ethnopharmacology 119 (2008) 109116 Table 4 Total phenolic content (mg/g) measured using FolinCiocalteu method Samples Calamintha origanifolia Satureja thymbra Prangos asperula Sideritis perfoliata Asperula glomerata Hyssopus ofcinalis Erythraea centaurium Marrubium radiatum Salvia acetabulosa Total phenolic content 75.7 81.3 84.9 80.8 81.5 83.1 66.8 80.5 80.7 0.20 0.06 0.17 0.08 0.13 0.11 0.12 0.05 0.01

113

acid equivalents in mg per g dry material (Gao et al., 2000) (Table 4). 2.7. Assessment of digestive enzymes activities related to diabetes 2.7.1. -Amylase inhibition The bioassay method was adopted and modied from SigmaAldrich (Conforti et al., 2005). A starch solution 0.5% (w/v) was obtained by stirring 0.125 g of potato starch in 25 ml of 20 mM sodium phosphate buffer with 6.7 mM sodium chloride, pH 6.9 at 65 C for 15 min. The -amylase solution was prepared by mixing 0.0253 g of the enzyme in 100 ml of cold distilled water. Plant extracts were dissolved in the same buffer to give nal concentrations from 1 mg/ml to 12.5 g/ml. The colorimetric reagent was prepared by mixing sodium potassium tartrate solution (12.0 g of sodium potassium tartrate tetrahydrate in 8.0 ml of 2 M NaOH) and 96 nM of 3,5-dinitrosalicylic acid solution (0.88 g of 3,5-dinitrosalicylic acid in 46 ml of deionized water) 1:1 (v/v). Both control and plant extracts (40 l) were added to starch solution (400 l) and left to react with -amylase solution (200 l) in alkaline conditions at 25 C. Acarbose was used as positive control. The reaction was measured over 3 min and the generation of maltose was quantied by measuring the absorbance at 540 nm of 3-amino-5-nitrosalicylic acid, the product formed from the reduction of 3,5-dinitrosalicylic acid. In the presence of an -amylase inhibitor, less maltose would be produced and the absorbance value will increase less rapidly. Preliminary experiments were carried out to establish optimal conditions and these were found to be: starch 0.25% (w/v); -amylase 1 unit/ml; inhibitor concentration 1 mg/ml. The -amylase inhibition was expressed as percentage of inhibition and calculated by the following equations: reaction rate (%) = [maltose] test 100 [maltose] control

inhibition (%) = 100 reaction rate (%) S.D. A calibration curve using a range of concentrations of maltose was constructed to ensure that, over the range of concentrations studied, the dose-inhibition ratio was linear. Results are shown in Fig. 1ac. 2.7.2. -Glucosidase inhibition The -glucosidase inhibition was measured through a modied SigmaAldrich bioassay method (Kapustka et al., 1981). Briey, maltose solution 4% (w/v) was prepared by dissolving 12 g of maltose in 300 ml of 50 mM sodium acetate buffer. The enzyme solution was prepared by mixing 1 mg of -glucosidase in 10 ml of ice-cold distilled water. Plant extracts were dissolved in DMSO to give nal concentrations from 1 mg/ml to 5 g/ml. The colorimetric reagent o-dianisidine colour solution (DIAN) was prepared by dissolving one tablet in 25 ml of distilled water, while the PGO enzyme-colour reagent solution was prepared fresh by dissolving one capsule in

Fig. 1. (a) -Amylase and -glucosidase inhibitory activity of methanol extracts from Lebanese traditional medicinal plants (IC50 g/ml). Calamintha origanifolia (A), Satureja thymbra (B), Prangos asperula (C), Sideritis perfoliata, (D), Asperula glomerata (E), Hyssopus ofcinalis (F), Erythraea centaurium (G), Marrubium radiatum (H), and Salvia acetabulosa (I). IC50 values are mean S.D. (n = 3). -Amylase: CM , EM , IC50 > 1000 g/ml. One-way ANOVA analysis: ***p < 0.0001; Bonferronis multiple comparison test: **p < 0.001 (A-IM vs. acarbose); -glucosidase: CM , GM , IC50 > 1000 g/ml. One-way ANOVA analysis: ***p < 0.0001; Bonferronis multiple comparison test: **p < 0.001 (A-IM vs. acarbose). (b) -Amylase and -glucosidase inhibitory activity of chloroform extracts from Lebanese traditional medicinal plants (IC50 g/ml). Calamintha origanifolia (A), Satureja thymbra (B), Prangos asperula (C), Sideritis perfoliata (D), Asperula glomerata (E), Hyssopus ofcinalis (F), Erythraea centaurium (G), Marrubium radiatum (H), Salvia acetabulosa (I). IC50 values are mean S.D. (n = 3); -amylase: CC and DC IC50 > 1000 g/ml. One-way ANOVA analysis: ***p < 0.0001; Bonferronis multiple comparison test: **p < 0.001 (A-IC vs. acarbose); -glucosidase: CC , DC and EC , IC50 > 1000 g/ml. One-way ANOVA analysis: ***p < 0.0001; Bonferronis multiple comparison test: **p < 0.001 (A-IC vs. acarbose). (c) -Amylase and -glucosidase inhibitory activity of hexane extracts from Lebanese traditional medicinal plants (IC50 g/ml). Calamintha origanifolia (A), Satureja thymbra (B), Prangos asperula (C), Sideritis perfoliata (D), Asperula glomerata (E), Hyssopus ofcinalis (F), Erythraea centaurium (G), Marrubium radiatum (H), Salvia acetabulosa (I). IC50 values are mean S.D. (n = 3); -amylase: B, D, G, IC50 > 1000 g/ml. One-way ANOVA analsyis: ***p < 0.0001; Bonferronis multiple comparison test: **p < 0.001 (A-IH vs. acarbose); -glucosidase: CH , GH , IC50 > 1000 g/ml. One-way ANOVA analysis: ***p < 0.0001; Bonferronis multiple comparison test: **p < 0.001 (A-IH vs. acarbose).

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100 ml of ice-cold distilled water. In the rst step both control and plant extracts were added to the maltose solution and left to equilibrate at 37 C. The reaction was started by adding -glucosidase solution (50 l) and tubes were left to incubate at 37 C for 30 min. At the end perchloric acid solution 4.2% (w/v) 1 ml was added to stop the reaction. In the second step of the procedure the generation of glucose was quantied by the reduction of DIAN. The supernatant of tube of step I was mixed with DIAN and PGO and was left to incubate at 37 C for 30 min. The absorbance of DIAN was measured spectrophotometrically at 500 nm. The -glucosidase inhibition was expressed as percentage of inhibition and calculated by the following equations: reaction (%) = [glucose] test 100 [glucose] control
Fig. 2. ACE inhibitory activity of extracts from Lebanese traditional medicinal plants (IC50 g/ml). Calamintha origanifolia (A), Satureja thymbra (B), Prangos asperula (C), Sideritis perfoliata (D), Asperula glomerata (E), Hyssopus ofcinalis (F), Erythraea centaurium (G), Marrubium radiatum (H), Salvia acetabulosa (I). IC50 values are mean S.D. (n = 3); One-way ANOVA analysis: ***p < 0.0001; Bonferronis multiple comparison test: **p < 0.001 (A-IM , A-IH , A-IC vs. captopril).

inhibition (%) = 100 reaction rate (%) S.D. A calibration curve using a range of concentrations of glucose was constructed to ensure that, over the range of concentrations studied, the dose-inhibition ratio was linear. 2.8. Angiotensin converting enzyme (ACE) inhibition The in vitro ACE inhibitory activity was measured using the method previously described (Loizzo et al., 2007). Briey, the chromophoreuorophore labelled substrate dansyltriglycine was cleaved by ACE into dansylglycine, which is quantitatively measured by HPLC. Each compound was dissolved in HEPES assay buffer (pH 7.0), to obtain nal concentrations ranging from 330 to 50 g/ml. The ACE solution (25 l) was pre-incubated with a test or control solution (25 l) for 5 min at 37 C. The enzyme reaction was started by adding a combined solution (25 l) of the substrate dansyltriglycine (7.86 mM), and the internal standard, dansyl-l-glutamine (0.353 mM) for the time of incubation, previously chosen by plotting a calibration curve. The reaction was stopped by adding a solution of 0.1N Na2 EDTA (50 l). The dansylglycine was quantied by reversed phase HPLC with UV detection at 250 nm. Instrumentation: HPLC PerkinElmer Series 410 LC pump; Injector PerkinElmer 20 l loop; Detector PerkinElmer UV/VIS LC290 spectrophotometric; solvent system: ALTECH SN 1250-99, Part. N 288215 BIN II 43, HYPERSIL ODS 5u Lot. no. 5002. 150 mm 4.6 mm SN:1250-99; mobile phase: isocratic system10 mM NaH2 PO4 buffer (pH 7)/acetonitrile (88:12); ow rate 2 ml/min; run time 30 min. A linear calibration curve was constructed for dansylglycine by plotting the area under the curve for values of 20 l injections of the substrate from 0.2 to 25 g/ml. The decreased concentration of dansylglycine in the test reaction compared with the control reaction was expressed as percentage inhibition and calculated from the equation: inhibition (%) = 100 (dansylglycine)T 100 (dansylglycine)C

Prism Version 4.0 for Windows (GraphPad Software, San Diego, CA, USA) (www.graphpad.com). The doseresponse curve was obtained by plotting the percentage inhibition versus concentration. 3. Results and discussion This study was designed to establish the inhibitory activity of 27 extracts from nine Lebanon traditional medicinal plants against amylase and -glucosidase, digestive enzymes related to diabetes, and against ACE. In order to characterize the constituent proles of the extracts used in this study, TLC and GC/MS analyses were performed. The n-hexane and chloroform extracts contained several compounds detectable with the vanillinsulphuric acid spray reagent, so it is likely that they are of a terpenoid nature, conformed by GC/MS analyses. Polyphenols were detected in methanol extracts when examined under UV light 365 nm and after spraying with NP reagent. Marrubium radiatum methanol extract exerted the highest inhibitory activity against both -amylase and -glucosidase enzymes, with IC50 values of 61.1 and 68.8 g/ml, respectively. Total soluble phenolic constituents of Marrubium radiatum methanol extract investigated by the FolinCiocalteu method revealed the value of 80.5 mg/g. Interesting, n-hexane and chloroform extracts obtained from the same plant did not exhibit signicant activity on -amylase, while a good activity was found on -glucosidase (IC50 of 114.7 and 128.5 g/ml, respectively) (Fig. 1ac). Both extracts, analysed by GC/MS, were characterized by the presence of sabinene and -terpinene as monoterpenes, fatty acids and their esters and steroids (Tables 2 and 3) l. Novaes et al. (2001) showed a signicant in vivo hypoglycaemic effect by the related species Marrubium vulgare, used in Brazilian and Mexican traditional medicine. A recent clinical trial on patients with type II non-controlled diabetes mellitus demonstrated that Marrubium vulgare leaf extract reduced the plasma glucose level by 0.64% (Herrera-Arellano et al., 2004), providing some support for its reported use in type II diabetes. Interestingly, the Marrubium radiatum chloroform extract studied here showed activity against ACE with IC50 of 72.8 g/ml. Previously, El Bardai et al. (2004) reported the ability of a water extract of Marrubium vulgare to induce vascular relaxant activity in spontaneously hypertensive rats, although, because of the difference in polarities of the solvents used, the possibility of the activity being due to similar compounds is rather remote. Among the species analysed, Salvia acetabulosa methanol extract revealed strong inhibitory properties against -glucosidase

where T = test and C = control. The doseresponse curves are shown in Fig. 2, captopril being used as a positive control ACE inhibitor. 2.9. Statistical analysis All experiments were carried out in triplicate. Data are expressed as means S.D. Differences were evaluated by one-way analysis of variance (ANOVA) test completed by a Bonferronis multicomparison test. Differences were considered signicant at **p < 0.001. The concentration giving 50% inhibition (IC50 ) was calculated by non-linear regression with the use of Prism Graphpad

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and -amylase with IC50 values of 76.9 and 91.2 g/ml respectively. This extract revealed the highest potency against ACE with IC50 of 52.7 g/ml. The total phenolic content in this fraction was comparable with Marrubium radiatum (80 mg/g). The activity of Salvia acetabulosa n-hexane extract on both digestive enzymes was weaker, with an IC50 of 205.5 and 212.0 g/ml for -amylase and -glucosidase, respectively. This extract was rich in terpenes and fatty acids and their derivatives (Table 2). The use of chloroform as solvent drastically reduced the inhibitory effect on -amylase, but had little effect on the ability of Salvia acetabulosa to inhibit -glucosidase (Fig. 1b) and did not inuence the ACE inhibitory activity (IC50 of 75.6 g/ml). Several Salvia species, including Salvia acetabulosa have been reported in Iranian folk medicine as hypoglycaemic agents (Omidbeygi, 1997; Zargari, 1997). Perfumi et al. (1991) reported the ability of Salvia fruticosa to reduce blood glucose levels in alloxan-hyperglycaemic rabbits, but not in normoglycaemic animals, without modifying plasma insulin levels. Zarzuelo et al. (1990) demonstrated the potentiation, by Salvia lavandulaefolia extract, of insulin release induced by glucose, and the extract also increased peripheral uptake of glucose, decreased intestinal absorption of glucose and gave hyperplasia of the pancreatic islet cells. At the same time there are several reports in the literature of the use of Salvia species in treatment of hypertension. In particular, Salvia miltiorrhiza root is traditionally used as a decoction for treatment of hypertensive disease in China, Korea, and Japan (Kang et al., 2003) and forms a major component of several commercial preparations used in China. Lithospermic acid (LSB), present in this species has been found to signicantly inhibit ACE in a dosedependent manner (IC50 of 86 g/ml) and may be, at least in part, due to ACE inhibitory effect of LSB (Kang et al., 2003), but it was not detected in the Salvia acetabulosa investigated in the present study. Among the other species analysed in this work, the n-hexane and chloroform extracts of Calamintha origanifolia exerted the highest -glucosidase inhibition activity (IC50 of 63.5 and 102.1 g/ml, respectively). Both extracts also inhibited -amylase with IC50 values of 94.1 and 91.6 g/ml, respectively. Fatty acids, their methyl esters and alkanes were detected in both extracts (Tables 2 and 3). Previously, Lemhadri et al. (2004) demonstrated that the aqueous extract of Calamintha ofcinalis leaves induced signicant hypoglycaemic effect in normal and streptozotocin-induced diabetic rats, without affecting basal plasma insulin concentrations and it is of interest that the Calamintha origanifolia chloroform extract exhibited an interesting activity against ACE with IC50 of 106.2 g/ml. The Erythraea centaurium chloroform extract exhibited a good inhibitory activity on both digestive enzymes with an IC50 of 64.9 and 74.9 g/ml for -amylase and -glucosidase, respectively and also against ACE but its methanol and n-hexane extracts exhibited no inhibition. The Erythraea centaurium chloroform extract revealed the presence of 29 compounds, mainly terpenes and fatty acids (Table 3). Previously, Haloui et al. (2000) reported the diuretic property of this species. Hyssopus ofcinalis extracts were active only on the -glucosidase enzyme, with IC50 values ranging from 127.3 to 908.4 g/ml and this activity of Hyssopus ofcinalis was recently demonstrated in vivo (Miyazaki et al., 2003), but for a MeOH extract, (7S,8S)syringoylglycerol-9-O-(6 -O-cinnamoyl)--d-glucopyranoside and (7S,8S)-syringoylglycerol-9-O--d-glucopyranoside subsequently being identied as active constituents (Matsuura et al., 2004). In the studies reported here, only the chloroform extract exhibited a good activity (IC50 127.3 g/ml), and this is less likely to contain the reported compounds than the methanol extract, so it is unlikely that they are the actives present in this instance. A very weak inhibition was found for the methanol and n-hexane extracts. At the same time Hyssopus ofcinalis n-hexane extract exhibited a strong inhibitory potency against ACE (IC50 values of 52.0 g/ml).

Asperula glomerata methanol extract inhibited both glucosidase and ACE (IC50 128.5 and 165.6 g/ml, respectively) with weaker effects against -amylase (IC50 of 209.7 g/ml). Satureja thymbra chloroform extract exhibited a weak activity of against both digestive enzyme, -amylase and -glucosidase with IC50 of 351.6 and 289.8 g/ml, respectively, and against ACE. 4. Conclusion The aim of the present study was to analyze activities relevant to hypoglycaemic and anti-hypertensive of nine different plant species used in Lebanon as traditional medicinal herbs. It should be noted that hot aqueous extracts are most commonly used and that many of the most active extracts reported here are made with less polar solvents. This investigation highlights the health benets of the two most promising herbs Salvia acetabulosa and Marrubium radiatum, and lends some scientic support to their traditional use. Further bioassay-guided fractionation approaches will be required on these species to identify and/or purify the major active constituents. It should be noted that in vivo tests should also be carried out to investigate whether the in vitro results reported translate into activities that might support the traditional uses of these plants. Acknowledgement The authors thank Dr. Katia Saad, Biology Department, Faculty of Sciences II, Lebanese University for supplying and identify the herb samples used in this study. References
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