SUPPLEMENTARY MATERIAL
Endothelium dependent vasorelaxant effect of Alchemilla vulgaris methanol extract: a
comparison with the aqueous extract in rat aorta
S. Takır a*, B. Sezgi b, S. Süzgeç-Selçuk c, E. Eroglu Ozkan d, K.J. Beukelmanb, A. Matd,
B.S. Uydeş-Doğana
a
Department of Pharmacology, Faculty of Pharmacy, Istanbul University, Istanbul,
Turkey
b
Department of Pharmaceutical Sciences, Medicinal Chemistry & Chemical Biology
Section, ß-Faculty, Utrecht University, Utrecht, Netherland
c
Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, Istanbul,
Turkey
d
Department of Pharmacognosy, Faculty of Pharmacy, Istanbul University, Istanbul,
Turkey
* Corresponding author () :
Selçuk Takır
Istanbul University, Faculty of Pharmacy,
Department of Pharmacology
Beyazıt, 34116, Istanbul, TURKEY
Tel&Fax: +90 212 5271825
e-mail: stakir@istanbul.edu.tr or selcuk_takir@yahoo.com
Endothelium dependent vasorelaxant effect of Alchemilla vulgaris methanol extract: a
comparison with the aqueous extract in rat aorta
Abstract
We aimed to investigate the vascular effects of methanol and aqueous extracts of
Alchemilla vulgaris (Rosaceaea). Increasing concentrations of the methanol extract (0.01-10
mg/ml) produced relaxations in noradrenaline (NA: 10-6M) and K+ (40mM) precontracted
aortas while contractions were obtained with the aqueous extract (0.01-10 mg/ml). Responses
to the methanol extract were inhibited in the presence of putative inhibitors of endothelial
vasodilators or after removal of the endothelium. Pretreatment of aortic rings with the
methanol extract (10mg/ml, 20 min.) reduced the maximal contractions to NA and K+
whereas, an enhanced contractility was observed with the aqueous extract (10mg/ml, 20
min.). Total flavonoid content was higher in methanol extract compared to the aqueous
extract. Quercetin was determined particularly high in the methanol extract while gallic acid
in the aqueous extract. Our results indicated that methanol extract of Alchemilla vulgaris
display favourable vascular effects via endothelium-dependent mechanisms.
Key words: Alchemilla vulgaris extract, rat aorta, relaxation, endothelium, flavonoid,
quercetin
1. Experimental
The dried aerial part of A. vulgaris herb (product no:22140) is purchased from Jacob
Hooy & Co. BV in the Netherlands.
1.1.
Preparation of extracts
1.1.1 Methanol extract: Plant material was exhausted in Soxhlet apparatus for 18
hours. Methanol containing extract was lyophilized after condensation in rotavopor.
1.1.2. Aqueous extract: Plant material was let to maceration at room temperature for
24 hours. Then, under reverse refrigerant water bath exhausted for 6 hours at 100oC. The
acquired water containing extract was lyophilized after blowing of the water.
1.2.
Total phenolic compounds
Total soluble phenolic compounds in the methanol and aqueous extracts of A. vulgaris
were determined by Folin-Ciocalteu reagent (Slinkard & Singleton 1977) and expressed as
“mg of gallic acid equivalents”. Absorbance of the mixture was recorded after 2 h at 760 nm.
1.3.
Total flavonoid content
Total flavonoid content in the methanol and aqueous extracts of A. vulgaris was
determined as previously described (Sakanaka et al 2005) and expressed as “mg of quercetin
equivalents”.
1.4.
HPLC-DAD analyses
The methanol and aqueous extracts of A.vulgaris were analyzed by HPLC-DAD
according to the method described in European Pharmacopoeia (2008). The HPLC system
was consisted of a pump (LC-10AD), a diode-array detector (DAD) (SPD-M10A) and an auto
sampler (SIL-10AD). Separation was accomplished with an ACE C18 column, 250 x 4.6mm
i.d., 5 µm (Advanced Chromatography Technologies, Alberdeen, Scotland). The control of
the system and the data analysis procedure were performed with Shimadzu LC Solutions
software.
1.4.1. Chromatographic conditions
The elution conditions were as follows: flow rate: 1 ml/min; column temperature: 40
°C; injection volume: 10 µl; detection: 270nm for gallic acid and 360nm for quercetin. The
solvent system used was a gradient of solvent A (0.3% formic acid in water (v/v)) and solvent
B (0.3% formic acid in acetonitril (v/v)). The following gradient was applied: 0–8 min, 82%
A; 8–18 min, 82–47% A; 18–18.1 min, 47–3% A; 18.1–29 min, 3% A; 29–30 min, 3–82% A
The dried extracts were dissolved in mixture of methanol/water (8:2 v/v). All samples were
filtered through a 0.45 µm filter into a vial for HPLC analysis. Each sample was prepared and
injected for three times.
1.5.
Pharmacological studies
1.5.1. Characteristics of the experimental animals
Male Wistar albino rats (200-250 g) were housed under a standard temperature of
20±2 oC on a 12:12 hour light/dark cycle with free access to standard rat chow and water. All
experimental procedures utilized were performed in accordance to the guidelines of Care and
Use of Laboratory Animals (NIH Publication, 1996). Study was approved by Local Animal
Experimentation Ethics Committee of Istanbul University (25/03/2010, decision no: 56).
1.5.2. Preparation of isolated rat aortic rings
The rats were sacrificed by stunning followed by decapitation. The thoracic aorta was
carefully excised and placed in Krebs Ringer-bicarbonate solution of the following
composition (mM): NaCl 118, KCl 4.7, KH2PO4 1.2, NaHCO3 25, MgSO4.7H2O 1.2, CaCl2
2.5, glucose 10 and disodium EDTA 0.026. Rings of 3–4 mm in length were suspended
between two stainless steel L-shaped hooks in 10 ml jacketed organ baths containing Krebs
Ringer-bicarbonate solution at 37 oC and aerated with a mixture of 95% O2 and 5% CO2. The
aortic rings were equilibrated for 1.5 hours at a tension of 1g. Responses were recorded on
computer controlled Powerlab data acquisition system (ADInstruments) by means of a force
displacement transducer (Grass FT03E). After the equilibration, preparations were contracted
twice with 40 mM K+ for standardization. In some preparations endothelium was removed by
gently rubbing the luminal surface of the rings with a roughed polyethylene tube.
1.5.3. Experimental protocol
Following standardization, concentration-response curves of NA (10-8-10-4 M) and K+
(10-100 mM) were obtained. The functional endothelium and vascular relaxation capacities of
the preparations were checked by Ach (10-8-10-4M) and SNP (10-8-10-4M), respectively, on
NA (10-6 M) precontracted rings.
Effects of the extracts were investigated parallelly in aortic rings precontracted
submaximally with NA (10-6M) or K+ (40mM). Increasing concentrations (0.01-10mg/ml) of
the extracts were administered cumulatively when contractions to spasmogens reached a
plateau. The role of endothelium in the effects of methanol extract was investigated in rings
removed of endothelium or pretreated with putative inhibitors of endothelial vasodilators such
as, nitric oxide (NO) synthase inhibitor, LG-nitro-L-arginine (L-NOARG, 10-4M), guanylate
cyclase inhibitor, ODQ (10-5M) or cyclooxygenase inhibitor, indomethacin (10-5M) for 20
min.(Uydes-Dogan et al 2005) .
The effects of the extracts on the contractile reactivity were also assessed in rat aorta.
In these experiments, concentration-response curves of NA (10-8-10-4M) and K+ (10-100mM)
were obtained in the absence (control) and presence of the extracts and compared,
respectively. For the purpose, rings were pretreated with the extracts or vehicle (Krebs) for
20 minutes.
1.6.
Chemicals
All drugs used were purchased from Sigma-Aldrich (Taufkirchen, Germany) except
for ODQ (Tocris). The stock solutions of ODQ were prepared in dimethylsulfoxide (DMSO)
whereas indomethacin in 5% (w/v) sodium bicarbonate solution. ACh and NA were dissolved
in 0.001N HCl and ascorbic acid (1 mg/ml) was added to NA solution to prevent oxidation.
Stock solutions of all other drugs as well as the methanol and aqueous extracts of A. vulgaris
were dissolved in distilled water and then diluted with fresh Krebs solution on day of
experimentation. In the HPLC analysis; HPLC grade acetonitrile, methanol, sodium nitrite,
aluminium chloride and sodium hydroxide was purchased from Merck (Darmstadt, Germany),
and ortho-phosphoric acid (85%) from Fluka (Steinheim, Switzerland). Milli-Q ultrapure
water was obtained from Millipore (Billerica, MA).
1.7.
Statistical analysis
Data were presented as “mean±S.E.M” while “n” is the number of rat aortic rings used
in the experiments. The responses to spasmogens were expressed as “g” contraction. The
responses to Ach, SNP and A. Vulgaris extracts were indicated as percent (%) decreases
(relaxation) or increases (contraction) of the precontractile tone. Sensitivities of the aorta
(EC50) to spasmogens were calculated as the effective concentration that elicit 50% of the
maximal response by using non-linear regression curve fit and expressed as “pD2”. Statistical
analyses were determined by Student's paired and unpaired t-tests as well as by one way
analysis of variance (ANOVA) followed by Tukey Kramer post hoc test where appropriate. A
“p” value less than “0.05” was considered statistically significant.
References
European Pharmacopoeia. 2008. Herbal Monographs; St. John’s Wort (Hyperici
herba) pp. 3839–42.
NIH Publication 1996. Guide for the Care and Use of Laboratory Animals, No. 85-23.
Sakanaka S, Tachibana Y, Okada Y. 2005. Preparation and antioxidant properties of
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Slinkard K, Singleton VL. 1977. Total phenol analysis: automation and comparison
with manual methods. Am. J. Enol. Vitic. 28: 49-55.
Uydes-Dogan BS, Takir S, Ozdemir O, Kolak U, Topçu G, Ulubelen A. 2005. The
comparison of the relaxant effects of two methoxylated flavones in rat aortic rings. Vasc.
Pharmacol. 43: 220–226.
Figure S1: HPLC chromatograms for gallic acid (A) and quercetin (D) standards; methanol extract (B) and aqueous extract (C) at 270nm;
methanol extract (E) and aqueous extract (F) at 360nm.