CC BY
108
Xhmh^ PACTHTE^tHoro cbipb.3. 2012. №2. C. 165-172.
UDC 577.19
SCREENING OF RUSSIAN MEDICINAL AND EDIBLE PLANT EXTRACTS FOR ANGIOTENSIN I-CONVERTING ENZYME (ACE I) INHIBITORY ACTIVITY
© S.A. Ivanov, S.A. Garbuz, I.L. Malfanov, L.R. Ptitsyn
Ajinomoto-Genetika Research Institute, 1-st Dorozhny pr. 1-1, Moscow, 117545 (Russia), e-mail: [email protected]
The angiotensin I-converting enzyme (ACE I) inhibitory activities of 108 aqueous ethanol extracts obtained from Russian plants were evaluated in vitro. Activity was assessed with a two-stage colorimetric assay using N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FA-PGG) as a substrate and 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a coloring reagent for the enzymatic cleavage product glycyl-glycine (GG). Extracts from eleven plants, seven of which belong to the family Rosaceae, were found to inhibit ACE I with an IC50 <0,3 mg/ml. Among these, the Geranium pratense extract was the most active and had an IC50 value of 81 ng/ml.
Keywords: ACE I inhibition; plant extracts; colorimetric assay.
Introduction
The renin-angiotensin system is an important target in the regulation of blood pressure and fluid and electrolyte balance [1, 2]. Although there are two types of angiotensin-converting enzymes, ACE I and ACE II, the majority of studies have focused on ACE I. ACE I is associated with the functions mediated by the AT1 and AT2 receptors, such as vasoconstriction, inflammation, and cell growth and proliferation [3, 4]. ACE I (dipeptidyl car-boxypeptidase I, kininase II, EC 3.4.15.1) is a multifunctional, zinc-containing enzyme with varying tissue localization. It converts the decapeptide angiotensin I into the potent vasoconstricting octapeptide angiotensin II and degrades bradykinin, a potent vasodilator [5].
Since the original discovery of ACE I inhibitors in snake venom [6], captopril and many other ACE I-inhibiting compounds have been developed and are currently used as clinical antihypertensive drugs [7, 8]. Because all of these drugs are associated with side effects, such as coughing, allergic reactions, taste disturbances, and skin rashes, researchers have attempted to locate natural ACE I inhibitors, which may improve both the safety and cost of these treatments. Numerous ACE I inhibitors have been isolated from foods [9], enzymatic hydrolysates of proteins [10, 11], and marine organisms [12]. Plants are also a major source of natural hypotensive agents, including hydrolysable tannins [13], proanthocyanidins and flavonoids [14], xanthones [15], fatty acids [16], se-coiridoids [17], and alkaloids [18]. Apart from the antihypertensive effects, some ACE I inhibitors also have beneficial effects on glucose and lipid metabolism, such as decreasing insulin requirements in diabetes, increasing exercise tolerance, and preventing coronary and cerebrovascular events [19].
Screens for ACE I inhibitors have identified a number of plant-derived substances as potential antihypertensive agents [20-23]. The present study examines the ACE I inhibitory activities of plants that are used in Russian Federation in official and folk medicine and as food supplements. The results of this preliminary investigation provide a basis for the further isolation and structural elucidation of phytochemicals with ACE I-inhibiting activity.
* Corresponding author.
Ivanov Sergei Aleksandrovich - Senior Research Fellow, Ph.D., e-mail: [email protected]
Garbuz Stanislav Aleksandrovich - engineer, e-mail: [email protected]
Malfanov Il'iaLeonidovich - Junior Researcher, Ph.D., e-mail: [email protected]
Ptitsyn Leonid Romanovich - Head of Laboratory, Ph.D., e-mail: [email protected]
Experimental conditions
Plant materials. Plants were purchased from a local drugstore or obtained from official suppliers as ready-to-use dried herbal preparations. They were collected from various supplier-dependent regions of the Russian Federation. The voucher specimens were deposited in our laboratory at the Ajinomoto-Genetika Research Institute (ZAO AGRI). Herbal materials were stored in dry and dark conditions.
Preparation of plant extracts. Plant materials (10.0 g) were dried, crushed in a mill, and stirred at 200 rpm in 70% aqueous ethanol (1 : 40, w/v) overnight at room temperature. The suspensions were filtered through a paper filter (Whatman no. 4), and the ethanol was removed using a rotary evaporator. After freeze-drying, crude extracts were obtained with an average yield of ca. 31%. The yields ranged from 3,7% for Salsola collina to 94,1% for Beta vulgaris. The extracts were stored in the dark at -20 °C until use. A stock solution of 10 mg/ml was prepared by dissolving the extracts in 10% (v/v) aqueous DMSO with vigorous stirring and sonication. Insoluble matter was removed after sedimentation at 14000 g for 10 min.
Chemicals and equipment. The ethanol used for extraction was of Ph. Eur. quality (Fluka, USA). N-(2-Hydroxy-ethyl)piperazine-N’ -(3 -propanesulfonic acid) (HEPPS), N-[3 -(2-furyl)acryloyl] -L-phenylalanyl-glycyl-glycine (FA-PGG), a 5% (w/v) solution of 2,4,6-trinitrobenzenesulfonic acid in H2O (TNBS), dimethyl sulfoxide (DMSO), N-[(S)-3-mercapto-2-methylpropionyl]-L-proline (captopril), rhodanine, gallic and tannic acids, and ACE I from rabbit as lung acetone powder were purchased from Sigma-Aldrich (USA). UV-visible spectra were recorded on an Ultrospec 3300 Pro spectrophotometer (Amersham Biosciences). For the ACE I and tannin assays, the samples were incubated and reactions were performed in 96-well flat bottom clear polystyrene microplates (Corning, USA) on an Elmi Thermostatic shaker ST-3 (Latvia). Optical densities were measured with a Multiskan Ascent microtiter plate reader (Thermo Electron).
ACE I inhibition assay. To prepare the ACE I stock solution, 1 g of rabbit lung acetone powder was suspended in 10 ml of cold 50 mM HEPPS-NaOH buffer (pH 8,2) supplemented with 5% (v/v) glycerol; the suspension was agitated at 4 °C and 200 rpm on an orbital shaker for overnight. The liquid was removed, and the debris was washed twice with 0,5 ml of the same chilled buffer. The washings were combined with the crude enzyme solution. Insoluble matter was removed by sedimentation at 4 °C and 20800 g for 2 hrs. This concentrated ACE I stock solution, which was a transparent, wine-colored liquid, was stored at -20 °C. Prior to the measurements, the concentrated ACE I solution was diluted ten times in cold assay buffer (see below) to produce an ACE I working solution, which was stored on ice. The ACE I-specific activity was determined from kinetic curves obtained upon FA-PGG cleavage with different enzyme concentrations, and the decrease in absorption of FA-PGG at 340 nm was recorded [24]. The ACE I stock solution, which had an enzymatic activity of 400-450 |amol/(min x mg), was stored for no longer than 6 months at -20 °C. The assay used in this study is based on the cleavage of the synthetic substrate FA-PGG with ACE I followed by reaction of the Gly-Gly (GG) dipeptide product with TNBS. This produces orange-colored 2,4,6-trinitrophenyl-glycyl-glycine (TNP-GG), the absorption of which can be measured at 420 nm with a microtiter plate reader [24, 25]. The ACE I inhibitory activities of the plant extracts were evaluated at extract concentrations of 0,01; 0,1, and 1 mg/ml in 50-|al reaction volumes. The appropriate amounts of the extract stock solutions were mixed with HEPPS-NaOH buffer (50 mM final concentration, pH 8,2), NaCl (300 mM), ZnSO4 (1 |aM), FA-PGG (0,8 mM), and water. The mixture was incubated at 37 °C and stirred at 500 rpm for 5 min. To initiate the cleavage reaction, a sufficient amount of the ACE I working solution, which had been incubated at 37 °C for 5 min, was added to the mixture to achieve a final activity of 6,5 |amol/(min x mg). The mixture was allowed to react at 37 °C and 500 rpm for 30 min. The reaction was terminated by the addition of a 71,5 |al mixture of 40 mM EDTA and 10 mM NaHCO3 (pH 9,8) supplemented with 3,6 mM TNBS as a coloring reagent. After color development at 37 °C and 500 rpm for 20 min, the absorption was measured at 420 nm. The measured absorption was compared to that of a blank solution prepared similarly to the sample, except that the mixture of EDTA and NaHCO3 was poured into the wells prior to addition of the ACE I solution. Mean values were obtained from two duplicate readings. Captopril was used as a positive control. The ACE I inhibitory efficacy (%) was determined from the equation: [1 - (Asample - Ablank sample) / (Acontrol - Ablank control)] x 100, where Acontrol and Ablank control
represent the absorbances of the corresponding mixtures without extract. The inhibitory activities of the plant extracts are expressed in terms of the observed half maximal inhibitory concentrations (IC50), which are the sample concentrations required to inhibit ACE I activity. The IC50 values were determined from dose-response curves.
Acid butanol assay for proanthocyanidins (PAs). The extract (80 |ag, 1 mg/ml solution in 10% (v/v) DMSO) was mixed with the acid butanol reagent (450 |ag FeCl3 in 56 |al of 6 M HCl and 1,064 ml n-butanol). The mixture was incubated at 95 °C for 50 min and cooled to room temperature. The organic (upper) layer was removed, and its absorption was measured at 550 nm. An unheated mixture of the same composition was employed as a blank. The PA content is expressed in absorption units per 1 mg of extract (A550/mg) [26].
Rhodanine assay for gallotannins (GTs). The extract (100 |ag, 1 mg/ml solution in 10% (v/v) DMSO) was mixed with 2 M sulfuric acid (100 |al). Cleavage was allowed to proceed at 97 °C for 18 hrs. The reaction tube was then cooled on ice, and the acid was neutralized to pH ~7 with 8 M NaOH. The sample was supplemented with the rhodanine reagent (300 |al of 0.67% w/v rhodanine in 80% ethanol) [27]. The mixture was then incubated at 25 °C for 5 min. The reaction was initiated by the addition of 5 M NaOH (200 |al). After color development for 10 min, the sample was appropriately diluted with water, and its absorbance at 520 nm was measured. Gallic acid (2100 |ag) was used to obtain a calibration curve. Gallic and tannic acids (20 |ag) were used as reference compounds throughout the experiment. The amount of gallic acid released (in |ag) was determined from the calibration curve.
Results and discussion
In total, 108 aqueous ethanol plant extracts were obtained and evaluated in vitro for their ability to inhibit ACE I (Table 1). The screen was performed on a 96-well microplate format using a colorimetric approach to estimate inhibitory activity. The artificial tripeptide FA-PGG was used as the cleavage substrate [24]. Hydrolysis can be directly monitored at 340 nm, which has been used for the kinetic [28] and single-reagent microcentrifugal [29] analysis of ACE I activity in serum. However, we found that this approach was unsatisfactory for the study of plant extracts because they have significant absorption at 340 nm (Fig. 1). To obviate interference from extract absorption, we coupled the cleavage reaction product GG with TNBS, a coloring reagent that specifically reacts with primary amino groups of amines, amino acids, and proteins [30]. The TNB-GG product absorbs at 420 nm, a wavelength with lower absorption from the plant extracts (Fig. 1). Serra et al. [25] took a similar approach in their use of hippuryl-glycyl-glycine (HGG) as a substrate. Their procedure required the enzymatic reaction to be quenched with sodium tungstate and sulfuric acid followed by pH adjustment with phosphate buffer (pH 8,5) to allow the coloring reaction to proceed. In the current work, the procedure was simplified by stopping the enzymatic reaction with an EDTA/NaHCO3, pH 9.8, mixture. This approach allowed us to rapidly screen the vast pool of plant extracts in the ACE I assay.
Table 1. Angiotensin I-converting enzyme (ACE I) inhibitory activities of aqueous ethanol plant extracts
Common name, plant part Scientific name ACE I inhibition, %a Sourceb Voucher number
1 2 3 4 5
Absinthe wormwood, aerial parts Artemisia absinthium L. -2,8±2,1 16 114-A
Agrimony, aerial parts Agrimonia eupatoria L. 33,8±1,3 17 91-A
Alder, cones Alnus glutinosa (L.) Gaertn. 51,6±2,2 2 20-A
Alpine bistort, aerial parts Polygonum viviparum L. 24,0±2,3 18 94-P
Anise, fruits Pimpinella anisum L. -15,8±2,7 7 59-P
Anise hyssop, aerial parts Agastache foeniculum (Pursh) Kuntze 3,9±8,7 18 95-A
Aralia Manchurian, roots Aralia mandshurica Rupr. et Maxim. -5,3±2,1 3 6-A
Baikal skull-cap, aerial parts Scutellaria baicalensis Georgi 10,8±4,5 19 96-S
Barberry, roots Berberis vulgaris L. 3,9±0,7 5 8-B
Beggars-ticks, aerial parts Bidens tripartita L. 7,8±0,2 2 67-B
Bergenia, rhizomes Bergenia crassifolia (L.) Fritsch 43,6±3,5 7 28-B
Bilberry, sprouts Vaccinium myrtillus L. 3,0±5,2 4 7-V
Birch, buds Betula pendula Rot. 26,9±5,7 1 1-B
Bird cherry, fruits Prunus padus L. -6,1±0,4 2 22-P
Bistort, aerial parts Polygonum bistorta L. 17,5±2,5 16 83-P
Blackberry, seeds Rubus caesius L. 41,6±5,6 22 97-R
Blackcurrant, fruits Ribes nigrum L. 13,0±2,5 11 33-R
Burdock, roots Arctium lappa L. 2,3±0,5 7 64-A
Camomile, flowers Chamomilla officinalis L. 15,0±1,2 1 69-M
Caragana, aerial parts Caragana jubata (Pall.) Poir. -0,9±0,8 16 99-C
Continue of the table 1
1 2 3 4 5
Caraway, fruits Carum carvi L. 0,4±2,2 7 63-C
Celandine, aerial parts Chelidonium majus L. 5,1±3,3 12 54-C
Chicory, aerial parts Cichorium intybus L. -4,3±1,1 16 85-C
Cinnamon rose, fruits Rosa majalis Herrm. 13,6±2,3 2 5-R
Common aspen, bark Populus tremula L. 10,5±2,9 18 103-P
Common bean, fruit walls Phaseolus vulgaris L. -1,5±0,3 16 104-P
Common centaury, aerial parts Centaurium erythraea Raf. -6,0±6,3 2 3-C
Common elder, flowers Sambucus nigra L. 8,6±1,0 2 70-S
Common wormwood, aerial parts Artemisia vulgaris L. 7,3±8,3 16 105-A
Coriander, fruits Coriandrum sativum L. -1,5±0,6 7 65-C
Creeping thyme, aerial parts Thymus serpyllum L. 9,6±3,5 7 62-T
Crowberry, aerial parts Empetrum nigrum L. 10,8±2,6 10 30-E
Dandelion, roots Taraxacum officinale Wigg 0,6±2,1 2 4-T
Dill, seeds Anethum graveolens L. 22,1±1,8 2 40-A
Dwarf everlasting, aerial parts Helichrysum arenarium (L.) Moench -7,2±5,3 1 26-H
Edelweiss, aerial parts Leontopodium leontopodioides Willd. Beauverd 14,3±0,2 19 106-L
Fennel, fruits Foeniculum vulgare Mill. -3,7±4,1 7 60-F
Field pansy, aerial parts Viola arvensis Murr. 11,2±1,5 2 50-V
Fireweed, aerial parts Epilobium angustifolium L. 51,8±2,9 12 48-E
Fragrant buckler fern, aerial parts Dryopteris fragrans (L.) Schott 3,4±0,1 16 108-D
French lilac, aerial parts Galega officinalis L. 4,6±2,0 6 9-G
Garden angelica, roots Angelica archangelica L. -3,1±2,4 16 109-A
Gmelin's wormwood, aerial parts Artemisia gmelinii Web. ex Stechm. -0,1±0,8 19 110-A
Goldenrod, aerial parts Solidago dahurica Kitag. -4,5±5,4 16 112-S
Great burnet, roots Sanguisorba officinalis L. 55,4±2,8 16 115-S
Guelder rose, bark Viburnum opulus L. -10,7±5,1 8 18-V
fruits 5,1±4,5 8 19-V
Hop, cones Humulus lupulus L. 2,4±1,6 4 86-H
Horse-tail, aerial parts Equisetum arvense L. 16,1±1,5 15 66-E
Hyssop, aerial parts Hyssopus officinalis L. -10,7±0,4 18 116-H
Jacob's ladder, aerial parts Polemonium caeruleum L. 0,2±4,4 19 119-P
Japanese pagoda tree, fruits Sophora japonica L. 5,2±1,1 16 120-S
Juniper, fruits Juniperus communis L. -0,5±1,2 1 57-J
Kelp, fronds Saccharina japonica Aresch. 14,4±0,4 1 43-S
Knotgrass, aerial parts Polygonum aviculare L. 12,2±6,3 1 2-P
Lentil, seeds Lens culinaris Medik. -6,1±0,5 21 124-L
Linden, flowers Tilia cordata Mill. 17,9±2,8 1 73-T
Long-leaved speedwell, aerial parts Veronica longifolia L. -8,2±3,2 19 125-V
Madder, rhizomes with roots Rubia tinctorum L. -8,2±3,2 4 53-R
Maize, stigmata Zea mays L. 11,5±0,1 1 44-Z
Marjoram, aerial parts Origanum majorana L. 6,3±1,0 2 55-O
Marsh cinquefoil, aerial parts with roots Comarum palustre L. 16,9±5,6 13 58-C
Marsh cudweed, aerial parts Gnaphalium uliginosum L. 7,8±1,0 2 24-G
Marshmallow, roots Althaea officinalis L. 1,1±3,1 15 68-A
Meadow crane's-bill, aerial parts Geranium pratense L. 57,6±3,7 16 126-G
Meadow-sweet, aerial parts Filipendula ulmaria (L.) Maxim. 33,4±1,2 10 31-F
Milkvetch, aerial parts Astragalus membranaceus (Fisch.) Bunge 3,7±1,6 19 127-A
roots 5,1±1,6 16 128-A
Peppermint, leaves Mentha piperita L. 17,4±2,2 7 56-M
Oak, bark Quercus robur L. 28,4±2,8 1 71-Q
Patrinia, aerial parts Patrinia rupestris (Pall.) Dufr. 1,7±0,4 19 130-P
Pine, buds Pinus sylvestris L. 6,4±0,7 2 49-P
Plantain, leaves Plantago major L. -1,5±0,3 2 47-P
Prince's pine, aerial parts Chimaphila umbellata (L.) Barton 18,5±7,6 16 132-C
Pumpkin, seeds Cucurbita pepo L. -5,0±3,4 2 39-C
Raspberry, fruits Rubus idaeus L. 10,8±0,5 11 34-R
leaves 45,9±0,4 8 36-R
Continue of the table 1
1 2 3 4 5
Red beet, roots Beta vulgaris L. -7,3±2,1 11 32-B
Red clover, aerial parts Trifolium pratense L. 5,5±8,5 16 133-T
Red-root gromwell, roots Lithospermum erythrorhizon Sieb. et Zucc. 12,0±8,6 18 135-L
Redshank, aerial parts Persicaria maculosa L. 10,6±0,6 7 17-P
Rowanberry, fruits Sorbus aucuparia L. 6,5±0,9 2 21-S
Sage, leaves Salvia officinalis L. 8,4±2,4 1 45-S
Saltwort, aerial parts Salsola collina Pall. 1,8±4,2 16 138-S
Saussurea, aerial parts Saussurea controversa D.C. 1,2±2,5 16 139-S
Schisandra, seeds Schisandra chinensis (Turcz.) Baill. 2,6±8,1 16 140-S
Shepherd's purse, aerial parts Capsella bursa-pastoris (L.) Medik. 23,3±9,4 2 72-C
Shrubby cinquefoil, aerial parts Dasiphora fruticosa (L.) Rydb. 24,8±2,7 16 142-D
Siberian fir, bark Abies sibirica Ledeb. 28,0±7,9 16 143-A
Siberian swollen pistil, roots Phlojodicarpus sibiricus Fisch. ex Spreng. 0,9±2,3 19 144-P
Sidebells wintergreen, aerial parts Orthilia secunda (L.) House 17,9±1,6 19 145-O
Sieversian wormwood, aerial parts Artemisia sieversiana Willd. 18,2±2,4 19 146-A
Silver speedwell, aerial parts Veronica incana L. -0,5±8,7 18 147-V
Silverweed cinquefoil, aerial parts Potentilla anserina L. 48,4±6,8 18 148-P
Spiny cocklebur, aerial parts Xanthium spinosum L. 13,7±12,4 16 149-X
St. John's wort, aerial parts Hypericum perforatum L. 1,2±4,1 1 81-H
Stinging nettle, leaves Urtica dioica L. 2,1±0,3 9 27-U
Strawberry, leaves Fragaria vesca L. 35,9±4,3 16 84-F
Tansy, flowers Tanacetum vulgare L. 2,7±4,2 9 29-T
Tormentil, rhizomes Potentilla erecta (L.) Racusch. 48,1±2,4 2 23-P
Water pepper, aerial parts Persicaria hydropiper (L.) Delarbre 28,4±0,6 2 16-P
Welted thistle, roots Carduus crispus L. 14,6±13,9 16 151-C
Wintergreen, aerial parts Pyrola rotundifolia L. 15,4±1,7 13 51-P
Wormwood sage, aerial parts Artemisia frigida Willd. 4,5±1,2 16 152-A
Yarrow, aerial parts Achillea millefolium L. 5,6±0,5 2 25-A
Yellow bedstraw, aerial parts Galium verum L. 17,8±5,6 18 153-G
Yellow sophora, roots Sophora flavescens Ait. 9,8±4,6 19 154-S
Yellow sweet clover, aerial parts Melilotus officinalis (L.) Pall. 11,0±4,3 8 52-M
a Data were obtained with an extract concentration of 0.1 mg/ml and are shown as the mean + SD (n = 4 or 6). b Sources: 1, OAO Krasnogorskleksredstva; 2, ZAO Zdorov’e; 3, OAO Tverskaya farmacevticheskaya fabrika; 4, OOO Apeks;
5, OOO Nasturciya; 6, OOO Tonus; 7, ZAO Ivan-Chaj; 8, OOO Kompaniya Khorst; 9, ZAO Tekhmedservis; 10, OOO CSI; 11, OOO Prestizh; 12, OOO Travy Bashkirii; 13, IP Chugunov A.I.; 14, OOO PFK Irej; 15, ZAO APF Fito-EM; 16, OOO Shalfej;
17, ZAO Evalar; 18, IP Orokto; 19, PT Daurskaya zagotovitel’naya kompaniya; 20, SPK Dary Sibiri; 21, OOO Agroal’yans;
22, OOO Yagodavest.
Eleven extracts, which were obtained from alder, bergenia, blackberry, fireweed, great burnet, meadow crane's-bill, meadow-sweet, raspberry (leaves), silverweed cinquefoil, strawberry, and tormentil, were found to inhibit ACE I with IC50 <0,3 mg/ml, fulfilling the criterion for classification as «active» [31] (Table 2). The IC50 values ranged from 0,081 mg/ml for the meadow crane's-bill extract to 0,28 mg/ml for strawberry extract. The ACE I inhibition of the agrimony extract was observed to be 34% under 0,1 mg/ml (Table 1), with a corresponding IC50 value of 0,55 mg/ml.
Agrimony was therefore classified as «low active». The shape of the dose-response curves obtained for the active plant extracts (Fig. 2) resembled those of pure flavonoids and their glycosylated derivatives [32]. Captopril (a positive control) had an IC50 value of 3,27+0,18 nM, which falls into the range of IC50’s (1,61-8,91 nM) reported for assays based on FA-PGG cleavage ([24] and references herein).
Tannin analyses of the active herbs revealed GTs in all species (Fig. 3). However, PAs were not conclusively detected in raspberry leaves and fireweed under the assay conditions. Among the studied plants, tormentil was found to be the most enriched in PAs. Monomeric phytochemicals, such as flavonoids, xanthones, phenylpro-panoids, cyanidins, and others; oligomeric hydrolysable tannins; and proanthocyanidins have previously been found to be responsible for in vitro ACE I inhibition (for review, see [33]). Because hydrolysable (GTs) and condensed (PAs) tannins were found in all active plants except raspberry leaves and fireweed (Fig. 3), we speculate that the ACE I inhibitory effects of these herbs are related to the detected phytochemicals. The possible mechanisms of ACE I inhibition may involve non-specific chelation of the zinc ion cofactors within the ACE I active site [34, 35] or protein precipitation [36].
Table 2. ACE I inhibitory activities of the most active plant extracts (IC50 <0,3 mg/ml)
Common name, plant part Family IC50 (mg/ml) a
Meadow crane's-bill, aerial parts Geraniaceae 0,081±0,022
Great burnet, roots Rosaceae 0,084±0,007
Fireweed, aerial parts Onagraceae 0,087±0,017
Alder, cones Betulaceae 0,091±0,013
Silverweed cinquefoil, aerial parts Rosaceae 0,091±0,014
Tormentil, rhizomes Rosaceae 0,110±0,011
Bergenia, rhizomes Saxifragaceae 0,128±0,019
Raspberry, leaves Rosaceae 0,160±0,010
Blackberry, seeds Rosaceae 0,169±0,055
Meadow-sweet, aerial parts Rosaceae 0,264±0,021
Strawberry, leaves Rosaceae 0,275±0,023
a The values shown are the mean ± SD (n=4)
Figure 1. Absorption spectra of selected aqueous ethanol plant extracts and TNP-GG; the color of each is denoted in parenthesis. Spectra were collected with extract concentrations of 0,1 mg/ml (10% v/v solution in aqueous DMSO) and a TNP-GG concentration of 0,1 mM (solution in 10 mM carbonate buffer, pH 9,8)
Figure 2. ACE I inhibition dose-response curves (A and B) for extracts of the eleven most active species
Contradictory data on fruit extracts of several strawberry cultivars (Rosaceae, genus Fragaria) were recently published. In one study, no significant ACE I inhibitory activity was found in seven cultivars grown in Brazil (Dover, Camp Dover, Camarosa, Sweet Charlie, Toyonoka, Oso Grande, and Piedade) [37]. On the other hand, the North American strawberry cultivars Jewel and Ovation had moderate ACE I inhibition activity [38]. The fruit extracts from varying cultivars of yellow, red, and black raspberries (Rosaceae, genus Rubus) displayed good activity against ACE I [39]. Contrary to this report, our study found that red raspberry fruit extract was poor ACE I inhibitor (Table 1); this may be a feature of the raspberry cultivar used in this study. Interestingly, the extracts of strawberry and raspberry leaves were potent inhibitors of ACE I (Table 2), which may indicate that the antihypertensive properties of Rosaceae are present in the leafy parts of the plants rather than in their fruits. Further investigations are required to isolate the active compounds from the plants and to elucidate their chemical structures. These studies would help to shed light on the mechanisms of plant-derived ACE I inhibitors.
Figure 3. Proanthocyanidins (PAs) and gallotannins (GTs) contents of the most active plant extracts. PAs and GTs were analyzed in 80 and 100 ^g of extract, respectively. Gallic and tannic acids (20 ^g each) were used as the corresponding negative and positive controls
Conclusion
This study identified eleven plant species from the families Rosaceae (P. anserine, P. erecta, R. caesius, R. idaeus, S. officinalis, F. ulmaria, and F. vesca), Betulaceae (A. glutinosa), Saxifragaceae (B. crassifolia), Ona-graceae (E. angustifolium), and Geraniaceae (G. pratense) that possess remarkable in vitro ACE I inhibitory activity.
Acknowledgements - the authors thank Dr. K. Ishii for stimulating discussions and are indebted to Ms. A. Sheremet (Dipl. Med.) for revising the scientific names of the plants used in this study.
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Received June 9, 2011
