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Total phenolic and flavonoid contents and antihypertensive effect of the crude extract and fractions of Calamintha vulgaris
Accepted Manuscript
Total Phenolic and Flavonoid Contents and Antihypertensive Effect of the Crude Extract and Fractions of Calamintha vulgaris Shamim Khan , Taous Khan , Abdul Jabbar Shah PII: DOI: Reference:
S0944-7113(18)30146-6 10.1016/j.phymed.2018.04.046 PHYMED 52485
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
28 August 2017 19 February 2018 16 April 2018
Please cite this article as: Shamim Khan , Taous Khan , Abdul Jabbar Shah , Total Phenolic and Flavonoid Contents and Antihypertensive Effect of the Crude Extract and Fractions of Calamintha vulgaris, Phytomedicine (2018), doi: 10.1016/j.phymed.2018.04.046
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Total Phenolic and Flavonoid Contents and Antihypertensive Effect of the Crude Extract and Fractions of Calamintha vulgaris
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Shamim Khana, Taous Khana, Abdul Jabbar Shaha,*
Cardiovascular Research Group; Department of Pharmacy, COMSATS Institute of Information
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Technology, University Road, Abbottabad-22060, KPK, Pakistan
Running Title: Antihypertensive potential of Calamintha vulgaris
*Corresponding author
Associate Professor,
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Abdul Jabbar Shah, PhD
Department of Pharmacy,
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COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan. (+92) 992-383591-5
Fax:
(+92) 992-383441 [email protected] (Shah AJ)
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E-mail address:
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Tel:
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ABSTRACT Background: Calamintha vulgaris L., has been used medicinally in the management hypertension. Purpose: To investigate the antihypertensive mechanisms of extract of C. vulgaris L., in Sprague-Dawley (SD) rat.
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Study design: Total phenol and total flavonoid contents were determined in the crude extract through HPLC. In vivo and in vitro pharmacological approaches were utilized to test the crude extract and fractions of C. vulgaris in Sprague-Dawley (SD) rats. The effect on mean arterial pressure (MAP) was compared in normotensive and high salt-induced hypertensive rats. Methods: Crude extract and nHexane, chloroform, ethylacetate and aqueous fractions of C.
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vulgaris were tested. In vitro experiments were carried out in isolated rat and rabbit aortae, to probe vascular mechanism(s). Extract was also evaluated for acute toxicity study in mice. Results: Crude extract and fractions of C. vulgaris induced a fall in MAP in normotensive and high salt-induced hypertensive rats at different doses. The effect was more significant in the hypertensive rats (Max. fall, 38.67 ± 2.17 vs 44.16 ± 4.67 mmHg). Among the fractions,
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chloroform was more effective (Max. fall, 53.20 ± 1.23 mmHg) and aqueous the least (Max. fall, 38.66 ± 1.12 mmHg). Normotensive rats pretreated with atropine (2 mg/kg) or L-NAME (100
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µg/kg) ablated fall in MAP to the extract and fractions. In isolated rat aorta, extract induced endothelium-dependent vasodilatory effect, which was ablated with atropine (1 µM), L-NAME (10 µM), atropine +
L-NAME,
TEA (10 µM) pretreatment and denudation of aorta.
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Indomethacin (10 µM) pretreatment ablated vasodilatation at lower concentrations and unmasked a vasoconstrictor effect, followed by relaxation at higher concentrations. Extract and
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fractions inhibited high K+-precontractions and rightward shifted Ca+2 concentration response curves, similar to verapamil. Total phenolic and flavonoid contents were found 39.41 ± 0.18 (mg
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of GAE/g) and 12.03 ± 0.23 (mg of QUE/g), respectively. HPLC analysis showed the presence of quercetin and rutin Conclusion: Results obtained indicate that the antihypertensive effect of C. vulgaris is the outcome of vasodilation, which is mediated through combination of muscarinic receptor-linked NO, activation of TEA-sensitive K+ channels, prostacyclin and Ca+2 antagonism.
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Keywords: Calamintha vulgaris, antihypertensive, endothelium-dependent and –independent, muscarinic receptors-linked NO, prostacyclin (PGI2), Ca+2 antagonist, HPLC analysis Abbreviations: AP: Aerial parts, Ca+2: calcium ion, C. vulgaris: Calamintha vulgaris, CCBs: Calcium channel
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blockers, CRC: Concentration response curves, GAE: Gallic acid equivalent, HPLC: High performance liquid chromatography, K+: Potassium ion, NO: nitric oxide, PGI2: Prostacyclins, QUE: Quercetin equivalent, SD: Sprague-Dawley, TEA: Tetraethyl ammonium bromide,
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VDCCs: Voltage-dependent Ca+ channels
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Introduction Hypertension is a killer occupying an important position in the conventional medicine today and is a prevalent risk factor for cardiovascular disease that affecting >1 billion peoples worldwide (Patel et al., 2011). Literature indicates that complementary and alternative medicine including herbal drugs, has a potential for the management of hypertension due to concomitant
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presence of antihypertensive and side effects minimizing constituents (Ribeiro et al., 2014). Herbal drugs remained a rich source of medicinal agents for years and contributed huge number drugs to clinical practice (Ribeiro et al., 2014).
Calamintha vulgaris L. Druce (Clinopodium vulgare), (Lamiaceae), commonly known as “Wild basil”, found in different parts of Pakistan (Baquar, 1989), Europe and Western and
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Central Asia. C. vulgaris, has gained importance as an alternative remedy for different conditions such as cardiac, inflammation and cancer (Kratchanova et al., 2010). A saponin, clinoposaponin, from C. vulgare is reported as a major constituent (Miyase and Matsushima, 1997). In addition, other constituents identified in C. vulgaris are terpenes, triterpenoids, saponins, phenols, flavonoids (rutin, quercetin, ferrulic acid, rosmarinic acid and chlorogenic acid (Knezevic et al.,
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2014).
Limited pharmacological studies are available on the extract or essential oil of C. vulgaris.
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It has been reported as antihepatomic and antiinflammatory (Rios et al., 2000), antihepatitis (Sparg et al., 2004), antitumor and antioxidant (Kratchanova et al., 2010) and antibacterial (Burk et al., 2012). Biochemical studies on the extract from C. vulgaris showed that it possesses
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anticholinesterase type of constituents (Knezevic et al., 2014). Literature survey indicates that C. vulgare exerts antioxidant effect and this effect is correlated to many activities reported for this
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plant. However, mechanistic in vivo and in vitro studies on the effect of C. vulgaris on blood pressure and vascular tone has not been documented although it has medicinal value in cardiac
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problems. We investigated the methanolic extract and fractions of C. vulgaris on blood pressure in normotensive and high salt-induced hypertensive rats in vivo and explored the underlying vascular mechanisms in vitro.
Material and methods Identification and collection of Calamintha vulgaris
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Aerial parts of Calamintha vulgaris were collected from Abbottabad, KPK, Pakistan, in September, 2013, identified and authenticated by Dr. Zafar Iqbal, Department of Botany, Post Graduate College, Abbottabad. A voucher specimen (Cv-AP-09/13) was deposited in the Graduate Research Lab of Pharmacology and Pharmacognosy, Department of Pharmacy, COMSATS Institute of Information Technology (CIIT), Abbottabad. The authentication of the
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plant was alternatively confirmed through http://www.theplantlist.org.
Preparation of crude extract and fractionation
Shad dried aerial parts (8 kg) of C. vulgaris were coarsely powdered and macerated in sufficient methanol for 15 days with occasional shaking (Azwanida, 2015). It was filtered
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through muslin cloth followed by a qualitative Whatman grade 1 filter paper. The combined filtrate was concentrated in rotary evaporator (-760 mm Hg) at 37 °C (Rotavapour Model Hahn Vapor HS-2005S-N, Korea) coupled with chiller (RW-0525G, Jiec Tech, Korea) and electric aspirator (HS-3000, Korea). The yield of crude extract of C. vulgaris (Cv.Cr) was 11% (w/w). As described previously (Shah and Gilani, 2011), a known quantity of the extract (300 g)
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was dissolved in distilled water (100 ml), equal volume of nHexane was added. The mixture was shaken vigorously and allowed to separate into layers. The upper layer of nHexane was collected
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and concentrated on rotary evaporator to obtain the nHexane fraction, yielding 13%. Similarly, the recovered layer was treated with chloroform, ethyl acetate and respective fractions were
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obtained. The remaining layer was regarded as aqueous layer.
Drugs and standards
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The following drugs and standards were procured from the source specified: acetylcholine chloride, atropine phosphate, norepinephrine bitartrate, Nω-nitro-L-arginine methyl ester (L-
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NAME) hydrochloride, verapamil hydrochloride, tetraethyl ammonium (TEA) bromide and indomethacin (Sigma Chemical Company, St. Louis, MO). Pentothal sodium was purchased from Abbott Laboratories, Pakistan.
Experimental animals All the experiments performed complied with the rulings of Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (NRC, 1996) and approved 5
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by the Ethical Committee of Pharmacy Department, CIIT, Abbottabad. Sprague-Dawley (SD) rats (180–250 g), Balb/c mice (25-30 g) and local rabbits (1-1.5 kg), were used and housed in the Animal House of the Pharmacy Department, CIIT, Abbottabad, under controlled conditions (2325 °C). The animals were allowed food and water ad libitum.
High salt -induced hypertensive SD rat model (in vivo)
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Pharmacological investigation
The protocol of Van and Montani et al. (2008) was followed with some modifications. Male SD rats were randomly divided into 2 groups (n = 2x30). Group 1 served as control and received standard pellet diet. Group 2 received 8% NaCl in diet for 7 days to induce
measurement.
Measurement of blood pressure in SD rats
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hypertension. At the end of treatment, rats of both groups were used for invasive blood pressure
The rats were anaesthetized with an intra-peritoneal injection of sodium thiopental
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(Pentothal, 40-90 mg/kg). After anesthesia induced, trachea was exposed and cannulated with PE-20 tubing to maintain appropriate respiration. Similarly, carotid artery was cannulated with
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PE-50 tubing, connected to a pressure transducer (MLT 0201) coupled with bridge amplifier (FE 221) and PowerLab (ML 846) Data Acquisition System. This connection was used for blood pressure recording. The left jugular vein was cannulated with similar tubing for intravenous
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injection. After 20 minute period of stabilization, control responses of standards such as acetylcholine (1 g/kg) and norepinephrine (1 g/kg) were obtained. Extract, fractions and
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verapamil (positive control drug) were injected at different doses followed by a flush of normal saline (0.1 ml). Arterial blood pressure was allowed to resume baseline after each injection.
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Changes in blood pressure were recognized as the difference between the steady state values before and the lowest readings after injection. Mean arterial pressure (MAP) calculated as the diastolic blood pressure plus one-third pulse width (Shah and Gilani, 2011).
Tension studies in isolated rat aortic tissues (In vitro) Endothelium-dependent and independent effects
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The procedure of Furchgott and Zawadski (1980) was followed with some modifications; thoracic aortae was isolated from SD rats and care was taken to avoid damage to the endothelium. Approximately 2-3 mm wide rings were mounted in a 10 ml tissue bath containing normal Kreb’s solution, maintained at 37 °C and aerated with carbogen (5% CO2 in O2). A preload of 2 g was applied to each tissue and allowed to equilibrate for 30-45 min. Tissues were
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stabilized with repeated application of PE (1 µM) at interval of 15-20 min. The integrity of endothelium was confirmed by the ability of the tissues produced relaxation to ACh (0.1 µM). In some aortic rings, endothelium was deliberately damaged with gentle rubbing the intimal surface with force. The success of procedure was confirmed when tissues failed to produce relaxation to ACh (0.1 µM). The extract, fractions and standard drugs were added cumulatively on
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phenylephrine (PE) precontractions and effect was calculated as percent of PE control. The underlying mechanism(s) were explored in intact aortic rings pretreated with atropine (1 µM), LNAME (10 µM), combination of atropine + L-NAME, TEA (10 µM) and indomethacin (10 µM). Effect on vascular tone was tested parallel in the denuded aortic rings also.
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Calcium channel blocking activity in isolated rabbit aortic tissues
As described previously (Shah and Gilani, 2011), rabbit aortic rings were stabilized with
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normal Kreb’s solution. This solution was replaced later with Ca+2-free/EGTA solution and control concentration-response curves (CRCs) of CaCl2 (as Ca+2) were obtained in duplicate. After wash out period, aortic rings were preincubated (30-45 min) with the extract, fractions and
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verapamil to see the possible calcium channel blocking effect. A parallel vehicle control was also
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run under similar experimental conditions.
Total phenol content
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Total phenol content
Total phenolic contents in the extract were measured by using spectrophotometric method
(Kaur and Kapoor, 2002). The reaction mixture was prepared by mixing 0.5 ml (1 mg/ml) of methanol extract, 2.5 ml of 10% Folin-Ciocalteu reagent dissolved in water and 2 ml of 7.5% Na2CO3. The blank was prepared by mixing 0.5 ml of methanol, 2.5 ml of 10% Folin-Ciocalteu reagent and 2 ml of 7.5% of Na2CO3. The samples were incubated in the thermostat at 45 °C for 45 min. The absorbance was measured at 765 nm (λmax) in triplicate. The same procedure was 7
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applied for the gallic acid (standard solution). On the basis of measured absorbance, concentration (mg/ml) of phenolics was read from the calibration curve and concentration was expressed as of gallic acid equivalent (mg of GAE/g of extract).
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Total flavonoid content The total flavonoid contents in the extract were determined using spectrophotometric method (Meda et al., 2005). The sample contained 1 ml of methanol extract solution (1 mg/ml) and 1 ml of 2% Aluminium chloride solution dissolved in methanol. The samples were incubated at room temperature for 1 h. The absorbance was measured at 415 nm (λmax) in triplicate. The
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same procedure was used for the standard solution of quercetin and calibration curve was made. Based on the measured absorbance, the flavonoids concentration (mg/ml) was read on the calibration curve and was expressed as of quercetin equivalent (QUE) (mg of QUE/g of extract).
High performance liquid chromatography (HPLC) analysis
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Chromatographic separations of crude extract and standards were carried out using HPLC (Analytical, SHIMADZU, Japan) coupled with UV/ Vis detector. The analytical column used
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was Shim-pack GIST C18 (150 mm × 4.6 mm, 5 µm). Mobile phase used was a mixture of solvent A (Acetonitrile) and solvent B (0.1% formic acid) in 1:1 ratio and eluted using isocratic system at a flow rate of 1 ml/min. The solvent was filtered using 0.45 µm filter and degassed.
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The columnn temperature was maintained at 25 °C at a wavelength of 370 nm. The sample injection volume was 20 µL. Stock solutions of crude methanolic extract of C. vulgaris and its
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fractions were (chloroform, ethylacetate and aqueous) prepared (5 mg/ 20 ml) in HPLC grade methanol. The solutions were pretreated by passing through silica gel (Scharlau) and then
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through 0.45 μm membrane filter. The sample was then injected to HPLC and ran with same solvent system (Kan et al., 2007).
Acute toxicity study Acute toxicity study of crude extract of C. vulgaris was carried by following the methods already described (Mehmood et al., 2011) with some modifications. Animals (Balb/c mice, 25-30 g of either sex) were devided into four groups (5 mice in each) and toxicity study was performed 8
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with increasing doses of Cv.Cr. Group 1 received normal saline as negative control (10 ml/kg, p.o) while groups 2-4 received doses of C. vulgaris (3, 5 and 10 g/kg, p.o). The mice were allowed food and water ad libitum and observed regularly for symptoms of toxicity up to 24 h.
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Statistical analysis The results were expressed as mean ± standard error means (SEM), and median effective concentrations (EC50) values with 95% confidence interval (CI). The data was analyzed by two way ANOVA followed by Bonferroni test. The values of p < 0.05 were considered statistically significant. The software used was Graph Pad Prism version 5 (Graph Pad, San Diego, Ca, USA)
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and SPSS to construct the concentration response curve analyzed by non-linear regression
Results
Effect on blood pressure in normotensive and salt-induced hypertensive anesthetized rats Before the injection of crude extract and fractions of C. vulgaris, standard drugs
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acetylcholine and norepinephrine were tested, they caused a fall and rise in MAP, respectively (Fig. 1A). We found that MAP was 117.64 + 1.74 mmHg (n = 20) and 154.34 + 3.49 mmHg (n =
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20) in normotensive and high salt-induced hypertensive rats, respectively (Fig. 1B), validating the protocol.
In normotensive and hypertensive rats under anesthesia, intravenous injection of Cv.Cr
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caused a fall in MAP (Fig. 1C), at doses of 1, 3, 10 and 30 mg/kg. Percent fall in the MAP observed in the normotensive rats was 2.33 ± 0.61, 4.91 ± 1.22, 21.33 ± 2.11 and 38.67 ± 2.78
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mmHg. However, the fall in the MAP observed in the high-salt hypertensive rat was 6.33 ± 0.75, 15.17 ± 1.88, 33.83 ± 3.95, 44.17 ± 4.67 mmHg. Among the fractions tested, all induced a fall in
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the MAP (Fig. 1), aqueous fraction being least potent at 30 mg/kg dose (26.00 ± 2.38 vs 38.66 ± 2.16 mmHg) (Fig 1H) while other fractions were comparable with the crude extract (Fig. 1). The percent fall in each case was statistically different (p < 0.05) compared to pretreated values. Verapamil (positive control drug) was tested for its antihypertensive effect on normotensive anesthetized rats and showed fall in MAP in a dose-dependent manner (Fig. 1D). The antihypertensive effect of the extract and fractions was statistically significant at doses of 10 and 30 mg/kg, in comparison to the normotensive rats. 9
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To find the possible blood pressure lowering mechanism(s) whether it involves muscarinic linked nitric oxide (NO) pathway, rats were pretreated with atropine (2 mg/kg). In the atropinized rats, the blood pressure lowering effect of the extract and fractions was masked at lower doses while partially ablated at higher doses. Further, anesthetized rats were pretreated with L-NAME (100 µg/kg), this pretreatment inhibited the effect at lower doses while partially
Tension studies in isolated rat aortic tissues (in vitro) Endothelium-dependent and independent effects
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abolished effect at higher doses.
In isolated rat aorta rings, with intact endothelium pre-contracted with PE (1 µM),
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cumulative addition of different concentrations of Cv.Cr caused an endothelium-dependent vasorelaxation with EC50 value of 0.27 mg/ml (0.27-0.42). Pretreatment of intact aortic rings with atropine (1µM), L-NAME (10 µM), combination of atropine+ L-NAME and TEA (10 µM), respectively, inhibited vasorelaxation to Cv.Cr at lower concentrations without modifying effect at higher concentrations. In the presence of indomethacin (10 µM), extract induced a
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vasoconstrictor effect followed by relaxation at higher concentrations (Fig. 3A). Moreover, in the denuded aortic rings Cv.Cr induced relaxation at higher concentrations with EC50 value of 4.22
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mg/ml (3.20-5.42) and shifted the CRCs to the right (Fig. 3A). All the fractions were tested parallel and showed endothelium-dependent vasorelaxation sensitive to different blockers with no dominant potency (Fig. 3). Indomethacin unmasked the vasoconstrictor effect in the fractions
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also. Denudation of the endothelium equally inhibited relaxation to all fractions (Fig. 3).
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Calcium channel blocking activity In isolated rabbit aortic rings, cumulative addition of extract inhibited K+ (80 mM)-induced
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sustained contractions (Fig.4 A), similar to verapamil (Fig. 4 C). All the fractions were tested parallel, aqueous fraction being more potent while crude extract and nHexane were least potent (data not shown). Preincubation of the rabbit aortic rings with crude extract of C. vulgaris caused a rightward shift in the CaCl2 CRCs constructed in Ca+2 -free/EGTA medium, with suppression of maximum response, similar to verapamil (Fig. 4B and 4D). All the fractions tested showed rightward shift in the CaCl2 CRCs (data not shown).
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Total phenolic and flavonoid contents The phenolic and flavonoid contents of C. vulgaris were determined and values are shown in Table 1. The crude methanolic extract of C. vulgaris was found containing significant amount of phenols and flavonoids as demonstrated by its total phenolic and flavonoid contents, with respective values of 39.41 ± 0.18 mg/g gallic acid equivalents (GAE/g) and 12.03 ± 0.23 mg/g
High performance liquid chromatography (HPLC)
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quercetin equivalents (QUE/g).
HPLC fingerprinting further confirmed the presence of quercetin and rutin in the methanol extract of C. vulgaris. The chromatogram of C. vulgaris methanol extract and fractions
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(Chloroform, ethylacetate and aqueous), and standards; quercetin and rutin are shown in Fig. 5CF. The retention time (min) of the peaks of quercetin (3.57) and rutin (2.66) were comparable with the peak retention time of crude extract and fractions.
Acute toxicity study
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Crude extract was found safe up to the dose of 5 g/kg in mice.
Discussion
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This was exciting study that explored the vascular mechanisms underlying the antihypertensive effect of extract and fractions of C. vulgaris. We found that the extract and
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fractions were more effective antihypertensive in the high salt-induced hypertensive rats in vivo, compared to the normotensive rats, particularly at higher doses. We tested four fractions derived
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from the crude extract to see, if there is shift in the antihypertensive constituents to any particular fraction. Our results demonstrated that the chloroform fraction was more and aqueous fraction less effective antihypertensive. Literature on the biological activities of species of Lamiaceae family indicated presence of acetylcholinesterase inhibitory constituents (Knezevic et al., 2014). These constituents indirectly act as cholinomimetics and decrease vascular resistance and hence blood pressure (Takei et al., 2004). Activation of muscarinic receptors located on the vascular endothelial cells leads to vascular relaxation and fall in blood pressure (Gordan et al., 2015). 11
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To have insight into the involvement of vascular muscarinic receptors in the antihypertensive effect of crude extract, normotensive rats were pretreated with atropine, a muscarinic receptor antagonist (Boulanger et al., 1994). This pretreatment ablated (37.80 + 2.88 vs 14.80 + 1.94 mmHg) antihypertensive response to the crude extract, indicating involvement of vascular muscarinic receptors activation. Stimulation of vascular muscarinic receptors is coupled
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with NO pathway through activation of NO synthase (Vanhoutte et al., 2009). We treated rats with L-NAME, NO synthase inhibitor (Pfeiffer et al., 1996), to see if it affects changes in the MAP to the extract. In the L-NAME treated rats, the changes in MAP to the extract were similar to those observed with atropine pretreatment alone. This indicates that extract activates vascular muscarinic receptors without having effect on NO synthase or release of NO. Among the
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fractions tested, ethylacetate fraction was comparable to the crude extract while other three fractions; nHexane, chloroform and aqueous were less effective at higher doses. Blood pressure is the result of vascular resistance and cardiac output (Mayet and Hughes, 2003), we investigated the vascular aspect using in vitro tension studies protocols.
The vascular endothelium plays an important role in homeostasis by modulating vascular
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smooth muscle tone and blood pressure. Endothelium synthesis and releases vasoactive substances such as NO, prostaglandins and endothelium-derived hyperpolarizing factor
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(Campbell et al., 1996). We tested vascular reactivity of the extract and fractions using isolated rat aorta. In isolated thoracic rat aortic rings, extract induced endothelium-dependent vasorelaxation against PE precontractions. This relaxation to the crude extract was ablated with
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denudation of the endothelium, without modifying effect at higher concentrations (>3 mg/ml) indicating that vascular endothelium plays role in its vasorelaxant effect. We investigated the
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involvement of mediators of endothelial origin. Medicinal plants belong to family “Lamiaceae” are known having anticholinesterase constituents (Knezevic et al., 2014). We wondered if these
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constituents might have effect on vascular muscarinic receptors. We tested this hypothesis in rat aortic rings. Aortic rings with intact endothelium were pretreated with atropine, a muscarinic receptor antagonist (Shah et al., 2016). This pretreatment attenuated (by 50%) relaxation to the crude extract at concentration of 3 mg/ml, suggesting that extract induces vasorelaxation through activation of endothelial muscarinic receptors. Stimulation of vascular endothelial muscarinic receptors is known to activate nitric oxide synthase (NOS) and thus induce vasodilatation (Vanhoutte et al., 2009). 12
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To probe the effect of extract and fractions on NOS, we treated intact aortic rings with LNAME. This pretreatment ablated, about 75%, relaxation to the crude extract at similar concentration without modifying effect at higher concentrations. This indicates that extract induces relaxation through activation of muscarinic receptors and NOS. This was further confirmed in aortic rings pretreated with combined atropine and L-NAME, the magnitude of
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relaxation was similar to that observed alone with atropine, indicating muscarinic receptor activation is the predominate pathway underlying the vasodilatory effect of the extract. Consistently, effect of the extract at higher concentrations remained unaffected by L-NAME pretreatment, suggesting involvement of additional mechanisms. We wondered if prostaglandins have played a role. Vascular endothelium is known to produce prostacyclin (PGI2), a known
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vasodilator (Willems and van Aken, 1979). Aortic rings with intact endothelium were treated with indomethacin, a prostaglandins inhibitor (Altura and Altura, 1976). Interestingly, this pretreatment abolished vasorelaxant effect of the extract and unmasked a vasoconstrictor component and tissue got relaxed at higher concentrations. This finding suggests that some of the constituents in the extract activate vasoconstrictor PGs that interfered the relaxation.
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We compared the vascular reactivity of the extract in the aorta from normotensive rats with high salt-induced hypertensive rats. We found that the vasorelaxant effect of the extract was
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ablated to similar magnitude observed in the normotensive rat’s aorta pretreated with different inhibitors. This indicates that high salt induces endothelium damage, making vasoactive substances unavailable and subsequently lead to hypertension (Van Vliet and Montani, 2008).
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Additionally, the extract was also tested against high K+ precontractions, to see effect on vascular smooth muscles. Cumulative addition of the extract induced inhibition of high K+
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precontractions with least potency. K+ at higher (>14 mM) concentration causes depolarization and contract the smooth muscle because potassium at high concentration inactivates all K +
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channels (Ross and Yallampalli, 1996) and allows to open voltage-gated calcium channels (Bolton 1979). This led us to hypothesize that the extract might have constituents act through activation of K+ channels, in addition to its antagonistic effect on Ca+2 moments. Pretreated aortic rings with TEA, a K+ channel inhibitor (Shah and Gilani, 2011) ablated relaxation to the extract, suggesting the involvement of K+ channel activation also. To have further insight into the effect of extract on Ca+2 moment through VDCs, rabbit aortic rings were pretreated with extract in Ca+2 free/EGTA medium. This pretreatment induced a rightward shift in the CaCl2 13
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concentration response curves, similar to verapamil, a standard calcium channel blocker (Opie, 1996). This finding indicates that constituent(s) in the extract also inhibit Ca+2 moments through VDCs. Studies on the fractions indicated that the indomethacin-sensitive vasoconstrictor constituent(s) were equally distributed. Data shows that the chloroform fraction contained
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indomethacin sensitive vasoconstrictor constituent(s) without having indomethacin sensitive vasodilatory constituent(s). The nHexane fraction was a potent vasodilator which is supposed to have potential vasodilatory constituents sensitive to combined inhibition with atropine and LNAME; the difference in the EC50 value was > 90 times. The chloroform and ethylacetate fractions were similar vasodilator like the parent crude extract. The aqueous fraction is least
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potent vasodilator, although different blockers attenuated response to varying degree. We assume that the presence of vasoconstrictor and comparatively less potent vasodilator constituents in the aqueous fraction could possibly explain its least potent antihypertensive nature. It might be due to the release of endothelial-derived contractile factor(s) at lower concentration and relaxing factors at higher concentrations. The relaxing factors are sensitive to the inhibitory effect of
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atropine and indomethacin, as the vasorelaxation was abolished to >50% and >80%, respectively with atropine and indomethacin pretreatment. This indicates that the crude extract contained
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combinations of vasoactive constituents; some are like endothelium-derived relaxing factors, which were equally distributed among the crude extract, ethylacetate and chloroform fractions. However, the vasoconstrictor constituent(s) that were not prominent in the crude extract, shifted
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to the aqueous fraction.
Furthermore, either blocker or combination failed to ablate vasodilator response to the
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crude extract and fractions at higher concentrations, which suggests existence of other vasodilatory constituents probably working directly on vascular smooth muscles. We studied
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fractions against high K+-precontractions, to see if affect Ca+2 moments through VDCs. Rabbit aortic rings were precontracted with high K+, cumulative addition of the fractions induced a vasorelaxant effect, similar to verapamil, suggesting presence of calcium channel blocking constituents. This finding was further confirmed when pretreatment of the rabbit aortic rings with fractions induced a rightward non-parallel shift in the CaCl2 CRCs, in Ca+2 free/EGTA medium. Many earlier studies with plant extracts who used similar experimental models, proved that phenolic and flavonoids induce vasorelaxation through multiple mechanisms, including blockade 14
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of voltage-dependent calcium channels (Ko et al., 1991), endothelial protective effect through superoxide scavenging activity (Goto et al., 1996) and involvement of endothelium through NOguanylyl cyclase pathway (Lee et al., 2002) etc. In addition to the well-known antioxidant effect of these natural bioactive substances, they also enhance the production of vasodilating factors including NO (via expression of NOS) and endothelium-derived factor (EDHF).
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Our finding on the chemistry of the extract of C. vulgaris indicating presence of large amount of phenolic and flavonoid compounds, compared to rutin and quercetin. HPLC fingerprints also showed the presence of quercetin in crude extract and fractions with various intensity of absorption. These direct chemical evidences support the multiple vasodilatory mechanisms of the extract and fractions and provide a pharmacological rationale to the
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antihypertensive effect of the extract of C. vulgaris. However, we could not identify and structurally elucidate particular phenol or flavonoid at this stage.
Conclusion
In conclusion, our results indicate that the antihypertensive effect of C. vulgaris is the outcome
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of vasodilation, which is mediated through combination of muscarinic receptor-linked NO release, activation of TEA-sensitive K+ channels, prostacyclin and Ca+2 antagonism. Co-
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existence of the vasoconstrictor constituents might be of therapeutic importance as herbal drugs are considered having side effect neutralizing potential. However further research on the active
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constituents will be interesting to explore this important herbal drug for clinical purpose.
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Funding source: This research work was carried out through a grant to the principle investigator
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from the Higher Education Commission (HEC) of Pakistan (grant No. 20-1554/R&D/10).
Conflicts of interest The authors declared no conflict of interests concerning primary interest such as validity of research, may be influenced b a secondary interest, such as financial gain that might bias this work.
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Furchgott, R.F., Zawadaski, J.V., 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 299, 373-376. Goto, H., Shimada, Y., Tanaka, N., Tanigawa, K., Itoh, T., Terasawa, K., 1999. Effect of extract
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Kan, Y., Gökbulut, A., Kartal, M., Konuklugil, B., Yılmaz, G., 2007. Development and validation of a LC method for the analysis of phenolic acids in Turkish Salvia species. Chromatographia 66, 147. Kratchanova, M., Denev, P., Ciz, M., Lojek, A., Mihailov, A., 2010. Evaluation of antioxidant activity of medicinal plants containing polyphenol compounds. Comparison of two
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extraction systems. Acta Biochim. Pol. 57, 229-234. Miyase, T., Matsushima, Y., 1997. Saikosaponin homologues from Clinopodium spp. The structures of clinoposaponins XII-XX. Chem. Pharm. Bull. 45, 1493-1497.
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Meda, A., Lamien, C.E., Romito, M., Millogo, J., Nacoulma, O.G., 2005. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 91, 571-577.
Mehmood, M.H., Siddiqi, H.S., Gilani, A.H., 2011. The antidiarrheal and spasmolytic activities of Phyllanthus emblica are mediated through dual blockade of muscarinic receptors and
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Opie, L.H., 1996. Calcium channel antagonists should be among the first-line drugs in the
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to the free acid, NG‐nitro‐L‐arginine. Br. J. Pharmacol. 118, 1433-1440. Patel, S.S., Verma, N.K., Ravi, V., Gauthaman, K., Soni, N., 2011. Antihypertensive effect of
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an aqueous extract of Passiflora nepalensis Wall. Int. J. Appl. Res. Nat. Prod. 3, 2-7. Parasuraman, S., Raveendran, R., 2012. Measurement of invasive blood pressure in rats. J.
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Pharmacol. Pharmacother. 3, 172. Rios, J.L., Recio, M.C., Maáñez, S., Giner, R.M., 2000. Natural triterpenoids as antiinflammatory agents. Stud. Nat. Prod. Chem. 22, 93-143.
Ross, G.R., Yallampalli, C., 2006. Endothelium-independent relaxation by adrenomedullin in pregnant rat mesenteric artery: role of cAMP-dependent protein kinase A and calciumactivated potassium channels. JPET 317, 1269–1275.
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Ribeiro, R.M., PinheiroNeto, V.F., Ribeiro, K.S., Vieira, D.A., Abreu, I.C., Silva, S.D., Cartágenes, M.D., Freire, S.M., Borges, A.C., Borges, M.O., 2014. Antihypertensive effect
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other endothelium-derived factors. Medicina (Kaunas) 39, 333-41. Sparg, S., Light, M.E., Van Staden, J., 2004. Biological activities and distribution of plant saponins. J. Ethnopharmacol. 94, 219-243.
Shah, A.J., Gilani, A.H., 2011. Blood pressure lowering effect of the extract of aerial parts of Capparis aphylla is mediated through endothelium-dependent and independent
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mechanisms. Clin. Exp. Hypertens. 33, 470-477.
Shah, A.J., Bukhari, I.A., Gilani, A.H., 2016. Mentha longifolia lowers blood pressure in anesthetized rats through multiple pathways. Bangladesh J. Pharmacol. 11, 784-792. Takei, H., Nakai, Y., Hattori, N., Yamamoto, M., Kurauchi, K., Sasaki, H., Aburada, M., 2004. The herbal medicine Toki-shakuyaku-san improves the hypertension and intrauterine
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growth retardation in preeclampsia rats induced by Nω-nitro-L-arginine methyl ester. Phytomedicine 11, 43-50.
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Van Vliet, B.N., Montani, J.P., 2008. The time course of salt-induced hypertension, and why it matters. Int. J. Obes. 32, S35-S47. Vladimir-Knežević, S., Blažeković, B., Kindl, M., Vladić, J., Lower-Nedza, A.D., Brantner,
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A.H., 2014. Acetylcholinesterase inhibitory, antioxidant and phytochemical properties of selected medicinal plants of the Lamiaceae family. Molecules 19, 767-782.
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Willems, C., van Aken, W.G., 1979. Production of prostacyclin by vascular endothelial
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cells. Pathophysiol. Haemos. Thromb. 8, 266-273.
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Table Legend
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Table 1. Total phenol and flavonoid contents in the methanolic crude extract of Calamintha vulgaris. Total phenol contents 39.41 ± 0.18 (mg of GAE/g)
Total flavonoid contents 12.03 ± 0.23 (mg of QUE/g)
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Plant C. vulgaris extract
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Figure legends
Fig. 1. Shows effect of norepinephrine (NE) and aetylcholine (ACh) on mean arterial pressure (MAP) in normotensive anesthetized rats. Fig. IB shows MAP values in the normotensive rats in comparison with the high salt-induced hypertensive rats. Fig. 1C-H show effect of the 20
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Verapamil, crude extract (Cv.Cr), nHexane (Cv.nHexane), chloroform (Cv.Chl), ethylacetate (Cv.EtAc), and aqueous (Cv.Aq) fractions of Calamintha vulgaris on MAP in the sodium thiopental induced anesthetized rats. Values shown are mean ± SEM (6-7) and p*** < 0.001, represents the significant difference between the% fall in MAP in normotensive and hypertensive
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rats.
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Fig. 2. A-E show effect of the crude extract (Cv.Cr) and nHexane (Cv.nHexane), chloroform (Cv.Chl), ethylacetate (Cv.EtAc) and aqueous (Cv.Aq) fractions of Calamintha vulgaris on MAP in the absence (normotensive) and presence of atropine (2 mg/kg) and L-NAME (100 µg/kg) on mean arterial pressure (MAP) in the sodium thiopental induced anesthetized rats. Values shown are mean ± SEM (6-7) and p*** < 0.001, represents the significant difference between the% fall in MAP in normotensive rats.
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Fig. 3. Aortic rings from normotensive rats were tested for the vascular reactivity to the crude extract and fractions of Calamintha vulgaris. Fig. A-E show effect of the crude extract and nHexane (Cv.nHexane), chloroform (Cv.Chl), ethylacetate (Cv.EtAc) and aqueous (Cv.Aq) fractions of Calamintha vulgaris on vascular tone precontracted with phenylephrine (PE) in the absence (intact) and presence of atropine (1 µM), L-NAME (10 µM), atropine (1 µM) + LNAME (10 µM), tetraethyl ammonium bromide (TEA; 10µM) and indomethacin (10 µM). A vasoconstrictor component was unmasked in the presence of indomethacin. Denudation of the 22
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aortic rings also ablated the vasodilatory effect at lower concentration. Values shown are mean ±
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SEM (n = 6-7), p* < 0.05, p** < 0.01, and p*** < 0.001.
Fig. 4. Concentrations response curves show effect of (A) the crude extract of Calamintha vulgaris (Cv.Cr) and (B) verapamil on high K+ precontractions in isolated rabbit aortic rings. Fig. C-D show effect of Cv.Cr and verapmail on Ca+2 concentrations response curves in Ca+2 free/EGTA. Values shown are mean ± SEM (n = 6-7), p* < 0.05, p** < 0.01, and p*** < 0.001.
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Fig. 5. Representative HPLC chromatogram of Calamintha vulgaris A) rutin B) quercetin C)
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crude extract (Cv.Cr), D), Chloroform (Cv.Chl), E) ethylacetate (Cv.EtAc) and F) Aqueous
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Graphical abstract
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Total Phenolic and Flavonoid Contents and Antihypertensive Effect of the Crude Extract and Fractions of Calamintha vulgaris Shamim Khan , Taous Khan , Abdul Jabbar Shah PII: DOI: Reference:
S0944-7113(18)30146-6 10.1016/j.phymed.2018.04.046 PHYMED 52485
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
28 August 2017 19 February 2018 16 April 2018
Please cite this article as: Shamim Khan , Taous Khan , Abdul Jabbar Shah , Total Phenolic and Flavonoid Contents and Antihypertensive Effect of the Crude Extract and Fractions of Calamintha vulgaris, Phytomedicine (2018), doi: 10.1016/j.phymed.2018.04.046
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Total Phenolic and Flavonoid Contents and Antihypertensive Effect of the Crude Extract and Fractions of Calamintha vulgaris
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Shamim Khana, Taous Khana, Abdul Jabbar Shaha,*
Cardiovascular Research Group; Department of Pharmacy, COMSATS Institute of Information
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Technology, University Road, Abbottabad-22060, KPK, Pakistan
Running Title: Antihypertensive potential of Calamintha vulgaris
*Corresponding author
Associate Professor,
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Abdul Jabbar Shah, PhD
Department of Pharmacy,
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COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan. (+92) 992-383591-5
Fax:
(+92) 992-383441 [email protected] (Shah AJ)
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E-mail address:
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Tel:
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ABSTRACT Background: Calamintha vulgaris L., has been used medicinally in the management hypertension. Purpose: To investigate the antihypertensive mechanisms of extract of C. vulgaris L., in Sprague-Dawley (SD) rat.
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Study design: Total phenol and total flavonoid contents were determined in the crude extract through HPLC. In vivo and in vitro pharmacological approaches were utilized to test the crude extract and fractions of C. vulgaris in Sprague-Dawley (SD) rats. The effect on mean arterial pressure (MAP) was compared in normotensive and high salt-induced hypertensive rats. Methods: Crude extract and nHexane, chloroform, ethylacetate and aqueous fractions of C.
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vulgaris were tested. In vitro experiments were carried out in isolated rat and rabbit aortae, to probe vascular mechanism(s). Extract was also evaluated for acute toxicity study in mice. Results: Crude extract and fractions of C. vulgaris induced a fall in MAP in normotensive and high salt-induced hypertensive rats at different doses. The effect was more significant in the hypertensive rats (Max. fall, 38.67 ± 2.17 vs 44.16 ± 4.67 mmHg). Among the fractions,
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chloroform was more effective (Max. fall, 53.20 ± 1.23 mmHg) and aqueous the least (Max. fall, 38.66 ± 1.12 mmHg). Normotensive rats pretreated with atropine (2 mg/kg) or L-NAME (100
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µg/kg) ablated fall in MAP to the extract and fractions. In isolated rat aorta, extract induced endothelium-dependent vasodilatory effect, which was ablated with atropine (1 µM), L-NAME (10 µM), atropine +
L-NAME,
TEA (10 µM) pretreatment and denudation of aorta.
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Indomethacin (10 µM) pretreatment ablated vasodilatation at lower concentrations and unmasked a vasoconstrictor effect, followed by relaxation at higher concentrations. Extract and
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fractions inhibited high K+-precontractions and rightward shifted Ca+2 concentration response curves, similar to verapamil. Total phenolic and flavonoid contents were found 39.41 ± 0.18 (mg
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of GAE/g) and 12.03 ± 0.23 (mg of QUE/g), respectively. HPLC analysis showed the presence of quercetin and rutin Conclusion: Results obtained indicate that the antihypertensive effect of C. vulgaris is the outcome of vasodilation, which is mediated through combination of muscarinic receptor-linked NO, activation of TEA-sensitive K+ channels, prostacyclin and Ca+2 antagonism.
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Keywords: Calamintha vulgaris, antihypertensive, endothelium-dependent and –independent, muscarinic receptors-linked NO, prostacyclin (PGI2), Ca+2 antagonist, HPLC analysis Abbreviations: AP: Aerial parts, Ca+2: calcium ion, C. vulgaris: Calamintha vulgaris, CCBs: Calcium channel
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blockers, CRC: Concentration response curves, GAE: Gallic acid equivalent, HPLC: High performance liquid chromatography, K+: Potassium ion, NO: nitric oxide, PGI2: Prostacyclins, QUE: Quercetin equivalent, SD: Sprague-Dawley, TEA: Tetraethyl ammonium bromide,
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VDCCs: Voltage-dependent Ca+ channels
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Introduction Hypertension is a killer occupying an important position in the conventional medicine today and is a prevalent risk factor for cardiovascular disease that affecting >1 billion peoples worldwide (Patel et al., 2011). Literature indicates that complementary and alternative medicine including herbal drugs, has a potential for the management of hypertension due to concomitant
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presence of antihypertensive and side effects minimizing constituents (Ribeiro et al., 2014). Herbal drugs remained a rich source of medicinal agents for years and contributed huge number drugs to clinical practice (Ribeiro et al., 2014).
Calamintha vulgaris L. Druce (Clinopodium vulgare), (Lamiaceae), commonly known as “Wild basil”, found in different parts of Pakistan (Baquar, 1989), Europe and Western and
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Central Asia. C. vulgaris, has gained importance as an alternative remedy for different conditions such as cardiac, inflammation and cancer (Kratchanova et al., 2010). A saponin, clinoposaponin, from C. vulgare is reported as a major constituent (Miyase and Matsushima, 1997). In addition, other constituents identified in C. vulgaris are terpenes, triterpenoids, saponins, phenols, flavonoids (rutin, quercetin, ferrulic acid, rosmarinic acid and chlorogenic acid (Knezevic et al.,
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2014).
Limited pharmacological studies are available on the extract or essential oil of C. vulgaris.
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It has been reported as antihepatomic and antiinflammatory (Rios et al., 2000), antihepatitis (Sparg et al., 2004), antitumor and antioxidant (Kratchanova et al., 2010) and antibacterial (Burk et al., 2012). Biochemical studies on the extract from C. vulgaris showed that it possesses
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anticholinesterase type of constituents (Knezevic et al., 2014). Literature survey indicates that C. vulgare exerts antioxidant effect and this effect is correlated to many activities reported for this
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plant. However, mechanistic in vivo and in vitro studies on the effect of C. vulgaris on blood pressure and vascular tone has not been documented although it has medicinal value in cardiac
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problems. We investigated the methanolic extract and fractions of C. vulgaris on blood pressure in normotensive and high salt-induced hypertensive rats in vivo and explored the underlying vascular mechanisms in vitro.
Material and methods Identification and collection of Calamintha vulgaris
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Aerial parts of Calamintha vulgaris were collected from Abbottabad, KPK, Pakistan, in September, 2013, identified and authenticated by Dr. Zafar Iqbal, Department of Botany, Post Graduate College, Abbottabad. A voucher specimen (Cv-AP-09/13) was deposited in the Graduate Research Lab of Pharmacology and Pharmacognosy, Department of Pharmacy, COMSATS Institute of Information Technology (CIIT), Abbottabad. The authentication of the
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plant was alternatively confirmed through http://www.theplantlist.org.
Preparation of crude extract and fractionation
Shad dried aerial parts (8 kg) of C. vulgaris were coarsely powdered and macerated in sufficient methanol for 15 days with occasional shaking (Azwanida, 2015). It was filtered
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through muslin cloth followed by a qualitative Whatman grade 1 filter paper. The combined filtrate was concentrated in rotary evaporator (-760 mm Hg) at 37 °C (Rotavapour Model Hahn Vapor HS-2005S-N, Korea) coupled with chiller (RW-0525G, Jiec Tech, Korea) and electric aspirator (HS-3000, Korea). The yield of crude extract of C. vulgaris (Cv.Cr) was 11% (w/w). As described previously (Shah and Gilani, 2011), a known quantity of the extract (300 g)
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was dissolved in distilled water (100 ml), equal volume of nHexane was added. The mixture was shaken vigorously and allowed to separate into layers. The upper layer of nHexane was collected
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and concentrated on rotary evaporator to obtain the nHexane fraction, yielding 13%. Similarly, the recovered layer was treated with chloroform, ethyl acetate and respective fractions were
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obtained. The remaining layer was regarded as aqueous layer.
Drugs and standards
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The following drugs and standards were procured from the source specified: acetylcholine chloride, atropine phosphate, norepinephrine bitartrate, Nω-nitro-L-arginine methyl ester (L-
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NAME) hydrochloride, verapamil hydrochloride, tetraethyl ammonium (TEA) bromide and indomethacin (Sigma Chemical Company, St. Louis, MO). Pentothal sodium was purchased from Abbott Laboratories, Pakistan.
Experimental animals All the experiments performed complied with the rulings of Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (NRC, 1996) and approved 5
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by the Ethical Committee of Pharmacy Department, CIIT, Abbottabad. Sprague-Dawley (SD) rats (180–250 g), Balb/c mice (25-30 g) and local rabbits (1-1.5 kg), were used and housed in the Animal House of the Pharmacy Department, CIIT, Abbottabad, under controlled conditions (2325 °C). The animals were allowed food and water ad libitum.
High salt -induced hypertensive SD rat model (in vivo)
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Pharmacological investigation
The protocol of Van and Montani et al. (2008) was followed with some modifications. Male SD rats were randomly divided into 2 groups (n = 2x30). Group 1 served as control and received standard pellet diet. Group 2 received 8% NaCl in diet for 7 days to induce
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Measurement of blood pressure in SD rats
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hypertension. At the end of treatment, rats of both groups were used for invasive blood pressure
The rats were anaesthetized with an intra-peritoneal injection of sodium thiopental
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(Pentothal, 40-90 mg/kg). After anesthesia induced, trachea was exposed and cannulated with PE-20 tubing to maintain appropriate respiration. Similarly, carotid artery was cannulated with
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PE-50 tubing, connected to a pressure transducer (MLT 0201) coupled with bridge amplifier (FE 221) and PowerLab (ML 846) Data Acquisition System. This connection was used for blood pressure recording. The left jugular vein was cannulated with similar tubing for intravenous
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injection. After 20 minute period of stabilization, control responses of standards such as acetylcholine (1 g/kg) and norepinephrine (1 g/kg) were obtained. Extract, fractions and
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verapamil (positive control drug) were injected at different doses followed by a flush of normal saline (0.1 ml). Arterial blood pressure was allowed to resume baseline after each injection.
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Changes in blood pressure were recognized as the difference between the steady state values before and the lowest readings after injection. Mean arterial pressure (MAP) calculated as the diastolic blood pressure plus one-third pulse width (Shah and Gilani, 2011).
Tension studies in isolated rat aortic tissues (In vitro) Endothelium-dependent and independent effects
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The procedure of Furchgott and Zawadski (1980) was followed with some modifications; thoracic aortae was isolated from SD rats and care was taken to avoid damage to the endothelium. Approximately 2-3 mm wide rings were mounted in a 10 ml tissue bath containing normal Kreb’s solution, maintained at 37 °C and aerated with carbogen (5% CO2 in O2). A preload of 2 g was applied to each tissue and allowed to equilibrate for 30-45 min. Tissues were
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stabilized with repeated application of PE (1 µM) at interval of 15-20 min. The integrity of endothelium was confirmed by the ability of the tissues produced relaxation to ACh (0.1 µM). In some aortic rings, endothelium was deliberately damaged with gentle rubbing the intimal surface with force. The success of procedure was confirmed when tissues failed to produce relaxation to ACh (0.1 µM). The extract, fractions and standard drugs were added cumulatively on
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phenylephrine (PE) precontractions and effect was calculated as percent of PE control. The underlying mechanism(s) were explored in intact aortic rings pretreated with atropine (1 µM), LNAME (10 µM), combination of atropine + L-NAME, TEA (10 µM) and indomethacin (10 µM). Effect on vascular tone was tested parallel in the denuded aortic rings also.
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Calcium channel blocking activity in isolated rabbit aortic tissues
As described previously (Shah and Gilani, 2011), rabbit aortic rings were stabilized with
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normal Kreb’s solution. This solution was replaced later with Ca+2-free/EGTA solution and control concentration-response curves (CRCs) of CaCl2 (as Ca+2) were obtained in duplicate. After wash out period, aortic rings were preincubated (30-45 min) with the extract, fractions and
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verapamil to see the possible calcium channel blocking effect. A parallel vehicle control was also
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Total phenol content
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Total phenol content
Total phenolic contents in the extract were measured by using spectrophotometric method
(Kaur and Kapoor, 2002). The reaction mixture was prepared by mixing 0.5 ml (1 mg/ml) of methanol extract, 2.5 ml of 10% Folin-Ciocalteu reagent dissolved in water and 2 ml of 7.5% Na2CO3. The blank was prepared by mixing 0.5 ml of methanol, 2.5 ml of 10% Folin-Ciocalteu reagent and 2 ml of 7.5% of Na2CO3. The samples were incubated in the thermostat at 45 °C for 45 min. The absorbance was measured at 765 nm (λmax) in triplicate. The same procedure was 7
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applied for the gallic acid (standard solution). On the basis of measured absorbance, concentration (mg/ml) of phenolics was read from the calibration curve and concentration was expressed as of gallic acid equivalent (mg of GAE/g of extract).
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Total flavonoid content The total flavonoid contents in the extract were determined using spectrophotometric method (Meda et al., 2005). The sample contained 1 ml of methanol extract solution (1 mg/ml) and 1 ml of 2% Aluminium chloride solution dissolved in methanol. The samples were incubated at room temperature for 1 h. The absorbance was measured at 415 nm (λmax) in triplicate. The
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same procedure was used for the standard solution of quercetin and calibration curve was made. Based on the measured absorbance, the flavonoids concentration (mg/ml) was read on the calibration curve and was expressed as of quercetin equivalent (QUE) (mg of QUE/g of extract).
High performance liquid chromatography (HPLC) analysis
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Chromatographic separations of crude extract and standards were carried out using HPLC (Analytical, SHIMADZU, Japan) coupled with UV/ Vis detector. The analytical column used
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was Shim-pack GIST C18 (150 mm × 4.6 mm, 5 µm). Mobile phase used was a mixture of solvent A (Acetonitrile) and solvent B (0.1% formic acid) in 1:1 ratio and eluted using isocratic system at a flow rate of 1 ml/min. The solvent was filtered using 0.45 µm filter and degassed.
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The columnn temperature was maintained at 25 °C at a wavelength of 370 nm. The sample injection volume was 20 µL. Stock solutions of crude methanolic extract of C. vulgaris and its
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fractions were (chloroform, ethylacetate and aqueous) prepared (5 mg/ 20 ml) in HPLC grade methanol. The solutions were pretreated by passing through silica gel (Scharlau) and then
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through 0.45 μm membrane filter. The sample was then injected to HPLC and ran with same solvent system (Kan et al., 2007).
Acute toxicity study Acute toxicity study of crude extract of C. vulgaris was carried by following the methods already described (Mehmood et al., 2011) with some modifications. Animals (Balb/c mice, 25-30 g of either sex) were devided into four groups (5 mice in each) and toxicity study was performed 8
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with increasing doses of Cv.Cr. Group 1 received normal saline as negative control (10 ml/kg, p.o) while groups 2-4 received doses of C. vulgaris (3, 5 and 10 g/kg, p.o). The mice were allowed food and water ad libitum and observed regularly for symptoms of toxicity up to 24 h.
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Statistical analysis The results were expressed as mean ± standard error means (SEM), and median effective concentrations (EC50) values with 95% confidence interval (CI). The data was analyzed by two way ANOVA followed by Bonferroni test. The values of p < 0.05 were considered statistically significant. The software used was Graph Pad Prism version 5 (Graph Pad, San Diego, Ca, USA)
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and SPSS to construct the concentration response curve analyzed by non-linear regression
Results
Effect on blood pressure in normotensive and salt-induced hypertensive anesthetized rats Before the injection of crude extract and fractions of C. vulgaris, standard drugs
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acetylcholine and norepinephrine were tested, they caused a fall and rise in MAP, respectively (Fig. 1A). We found that MAP was 117.64 + 1.74 mmHg (n = 20) and 154.34 + 3.49 mmHg (n =
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20) in normotensive and high salt-induced hypertensive rats, respectively (Fig. 1B), validating the protocol.
In normotensive and hypertensive rats under anesthesia, intravenous injection of Cv.Cr
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caused a fall in MAP (Fig. 1C), at doses of 1, 3, 10 and 30 mg/kg. Percent fall in the MAP observed in the normotensive rats was 2.33 ± 0.61, 4.91 ± 1.22, 21.33 ± 2.11 and 38.67 ± 2.78
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mmHg. However, the fall in the MAP observed in the high-salt hypertensive rat was 6.33 ± 0.75, 15.17 ± 1.88, 33.83 ± 3.95, 44.17 ± 4.67 mmHg. Among the fractions tested, all induced a fall in
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the MAP (Fig. 1), aqueous fraction being least potent at 30 mg/kg dose (26.00 ± 2.38 vs 38.66 ± 2.16 mmHg) (Fig 1H) while other fractions were comparable with the crude extract (Fig. 1). The percent fall in each case was statistically different (p < 0.05) compared to pretreated values. Verapamil (positive control drug) was tested for its antihypertensive effect on normotensive anesthetized rats and showed fall in MAP in a dose-dependent manner (Fig. 1D). The antihypertensive effect of the extract and fractions was statistically significant at doses of 10 and 30 mg/kg, in comparison to the normotensive rats. 9
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To find the possible blood pressure lowering mechanism(s) whether it involves muscarinic linked nitric oxide (NO) pathway, rats were pretreated with atropine (2 mg/kg). In the atropinized rats, the blood pressure lowering effect of the extract and fractions was masked at lower doses while partially ablated at higher doses. Further, anesthetized rats were pretreated with L-NAME (100 µg/kg), this pretreatment inhibited the effect at lower doses while partially
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abolished effect at higher doses.
In isolated rat aorta rings, with intact endothelium pre-contracted with PE (1 µM),
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cumulative addition of different concentrations of Cv.Cr caused an endothelium-dependent vasorelaxation with EC50 value of 0.27 mg/ml (0.27-0.42). Pretreatment of intact aortic rings with atropine (1µM), L-NAME (10 µM), combination of atropine+ L-NAME and TEA (10 µM), respectively, inhibited vasorelaxation to Cv.Cr at lower concentrations without modifying effect at higher concentrations. In the presence of indomethacin (10 µM), extract induced a
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vasoconstrictor effect followed by relaxation at higher concentrations (Fig. 3A). Moreover, in the denuded aortic rings Cv.Cr induced relaxation at higher concentrations with EC50 value of 4.22
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mg/ml (3.20-5.42) and shifted the CRCs to the right (Fig. 3A). All the fractions were tested parallel and showed endothelium-dependent vasorelaxation sensitive to different blockers with no dominant potency (Fig. 3). Indomethacin unmasked the vasoconstrictor effect in the fractions
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also. Denudation of the endothelium equally inhibited relaxation to all fractions (Fig. 3).
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Calcium channel blocking activity In isolated rabbit aortic rings, cumulative addition of extract inhibited K+ (80 mM)-induced
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sustained contractions (Fig.4 A), similar to verapamil (Fig. 4 C). All the fractions were tested parallel, aqueous fraction being more potent while crude extract and nHexane were least potent (data not shown). Preincubation of the rabbit aortic rings with crude extract of C. vulgaris caused a rightward shift in the CaCl2 CRCs constructed in Ca+2 -free/EGTA medium, with suppression of maximum response, similar to verapamil (Fig. 4B and 4D). All the fractions tested showed rightward shift in the CaCl2 CRCs (data not shown).
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Total phenolic and flavonoid contents The phenolic and flavonoid contents of C. vulgaris were determined and values are shown in Table 1. The crude methanolic extract of C. vulgaris was found containing significant amount of phenols and flavonoids as demonstrated by its total phenolic and flavonoid contents, with respective values of 39.41 ± 0.18 mg/g gallic acid equivalents (GAE/g) and 12.03 ± 0.23 mg/g
High performance liquid chromatography (HPLC)
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quercetin equivalents (QUE/g).
HPLC fingerprinting further confirmed the presence of quercetin and rutin in the methanol extract of C. vulgaris. The chromatogram of C. vulgaris methanol extract and fractions
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(Chloroform, ethylacetate and aqueous), and standards; quercetin and rutin are shown in Fig. 5CF. The retention time (min) of the peaks of quercetin (3.57) and rutin (2.66) were comparable with the peak retention time of crude extract and fractions.
Acute toxicity study
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Crude extract was found safe up to the dose of 5 g/kg in mice.
Discussion
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This was exciting study that explored the vascular mechanisms underlying the antihypertensive effect of extract and fractions of C. vulgaris. We found that the extract and
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fractions were more effective antihypertensive in the high salt-induced hypertensive rats in vivo, compared to the normotensive rats, particularly at higher doses. We tested four fractions derived
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from the crude extract to see, if there is shift in the antihypertensive constituents to any particular fraction. Our results demonstrated that the chloroform fraction was more and aqueous fraction less effective antihypertensive. Literature on the biological activities of species of Lamiaceae family indicated presence of acetylcholinesterase inhibitory constituents (Knezevic et al., 2014). These constituents indirectly act as cholinomimetics and decrease vascular resistance and hence blood pressure (Takei et al., 2004). Activation of muscarinic receptors located on the vascular endothelial cells leads to vascular relaxation and fall in blood pressure (Gordan et al., 2015). 11
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To have insight into the involvement of vascular muscarinic receptors in the antihypertensive effect of crude extract, normotensive rats were pretreated with atropine, a muscarinic receptor antagonist (Boulanger et al., 1994). This pretreatment ablated (37.80 + 2.88 vs 14.80 + 1.94 mmHg) antihypertensive response to the crude extract, indicating involvement of vascular muscarinic receptors activation. Stimulation of vascular muscarinic receptors is coupled
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with NO pathway through activation of NO synthase (Vanhoutte et al., 2009). We treated rats with L-NAME, NO synthase inhibitor (Pfeiffer et al., 1996), to see if it affects changes in the MAP to the extract. In the L-NAME treated rats, the changes in MAP to the extract were similar to those observed with atropine pretreatment alone. This indicates that extract activates vascular muscarinic receptors without having effect on NO synthase or release of NO. Among the
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fractions tested, ethylacetate fraction was comparable to the crude extract while other three fractions; nHexane, chloroform and aqueous were less effective at higher doses. Blood pressure is the result of vascular resistance and cardiac output (Mayet and Hughes, 2003), we investigated the vascular aspect using in vitro tension studies protocols.
The vascular endothelium plays an important role in homeostasis by modulating vascular
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smooth muscle tone and blood pressure. Endothelium synthesis and releases vasoactive substances such as NO, prostaglandins and endothelium-derived hyperpolarizing factor
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(Campbell et al., 1996). We tested vascular reactivity of the extract and fractions using isolated rat aorta. In isolated thoracic rat aortic rings, extract induced endothelium-dependent vasorelaxation against PE precontractions. This relaxation to the crude extract was ablated with
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denudation of the endothelium, without modifying effect at higher concentrations (>3 mg/ml) indicating that vascular endothelium plays role in its vasorelaxant effect. We investigated the
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involvement of mediators of endothelial origin. Medicinal plants belong to family “Lamiaceae” are known having anticholinesterase constituents (Knezevic et al., 2014). We wondered if these
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constituents might have effect on vascular muscarinic receptors. We tested this hypothesis in rat aortic rings. Aortic rings with intact endothelium were pretreated with atropine, a muscarinic receptor antagonist (Shah et al., 2016). This pretreatment attenuated (by 50%) relaxation to the crude extract at concentration of 3 mg/ml, suggesting that extract induces vasorelaxation through activation of endothelial muscarinic receptors. Stimulation of vascular endothelial muscarinic receptors is known to activate nitric oxide synthase (NOS) and thus induce vasodilatation (Vanhoutte et al., 2009). 12
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To probe the effect of extract and fractions on NOS, we treated intact aortic rings with LNAME. This pretreatment ablated, about 75%, relaxation to the crude extract at similar concentration without modifying effect at higher concentrations. This indicates that extract induces relaxation through activation of muscarinic receptors and NOS. This was further confirmed in aortic rings pretreated with combined atropine and L-NAME, the magnitude of
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relaxation was similar to that observed alone with atropine, indicating muscarinic receptor activation is the predominate pathway underlying the vasodilatory effect of the extract. Consistently, effect of the extract at higher concentrations remained unaffected by L-NAME pretreatment, suggesting involvement of additional mechanisms. We wondered if prostaglandins have played a role. Vascular endothelium is known to produce prostacyclin (PGI2), a known
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vasodilator (Willems and van Aken, 1979). Aortic rings with intact endothelium were treated with indomethacin, a prostaglandins inhibitor (Altura and Altura, 1976). Interestingly, this pretreatment abolished vasorelaxant effect of the extract and unmasked a vasoconstrictor component and tissue got relaxed at higher concentrations. This finding suggests that some of the constituents in the extract activate vasoconstrictor PGs that interfered the relaxation.
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We compared the vascular reactivity of the extract in the aorta from normotensive rats with high salt-induced hypertensive rats. We found that the vasorelaxant effect of the extract was
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ablated to similar magnitude observed in the normotensive rat’s aorta pretreated with different inhibitors. This indicates that high salt induces endothelium damage, making vasoactive substances unavailable and subsequently lead to hypertension (Van Vliet and Montani, 2008).
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Additionally, the extract was also tested against high K+ precontractions, to see effect on vascular smooth muscles. Cumulative addition of the extract induced inhibition of high K+
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precontractions with least potency. K+ at higher (>14 mM) concentration causes depolarization and contract the smooth muscle because potassium at high concentration inactivates all K +
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channels (Ross and Yallampalli, 1996) and allows to open voltage-gated calcium channels (Bolton 1979). This led us to hypothesize that the extract might have constituents act through activation of K+ channels, in addition to its antagonistic effect on Ca+2 moments. Pretreated aortic rings with TEA, a K+ channel inhibitor (Shah and Gilani, 2011) ablated relaxation to the extract, suggesting the involvement of K+ channel activation also. To have further insight into the effect of extract on Ca+2 moment through VDCs, rabbit aortic rings were pretreated with extract in Ca+2 free/EGTA medium. This pretreatment induced a rightward shift in the CaCl2 13
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concentration response curves, similar to verapamil, a standard calcium channel blocker (Opie, 1996). This finding indicates that constituent(s) in the extract also inhibit Ca+2 moments through VDCs. Studies on the fractions indicated that the indomethacin-sensitive vasoconstrictor constituent(s) were equally distributed. Data shows that the chloroform fraction contained
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indomethacin sensitive vasoconstrictor constituent(s) without having indomethacin sensitive vasodilatory constituent(s). The nHexane fraction was a potent vasodilator which is supposed to have potential vasodilatory constituents sensitive to combined inhibition with atropine and LNAME; the difference in the EC50 value was > 90 times. The chloroform and ethylacetate fractions were similar vasodilator like the parent crude extract. The aqueous fraction is least
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potent vasodilator, although different blockers attenuated response to varying degree. We assume that the presence of vasoconstrictor and comparatively less potent vasodilator constituents in the aqueous fraction could possibly explain its least potent antihypertensive nature. It might be due to the release of endothelial-derived contractile factor(s) at lower concentration and relaxing factors at higher concentrations. The relaxing factors are sensitive to the inhibitory effect of
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atropine and indomethacin, as the vasorelaxation was abolished to >50% and >80%, respectively with atropine and indomethacin pretreatment. This indicates that the crude extract contained
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combinations of vasoactive constituents; some are like endothelium-derived relaxing factors, which were equally distributed among the crude extract, ethylacetate and chloroform fractions. However, the vasoconstrictor constituent(s) that were not prominent in the crude extract, shifted
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to the aqueous fraction.
Furthermore, either blocker or combination failed to ablate vasodilator response to the
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crude extract and fractions at higher concentrations, which suggests existence of other vasodilatory constituents probably working directly on vascular smooth muscles. We studied
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fractions against high K+-precontractions, to see if affect Ca+2 moments through VDCs. Rabbit aortic rings were precontracted with high K+, cumulative addition of the fractions induced a vasorelaxant effect, similar to verapamil, suggesting presence of calcium channel blocking constituents. This finding was further confirmed when pretreatment of the rabbit aortic rings with fractions induced a rightward non-parallel shift in the CaCl2 CRCs, in Ca+2 free/EGTA medium. Many earlier studies with plant extracts who used similar experimental models, proved that phenolic and flavonoids induce vasorelaxation through multiple mechanisms, including blockade 14
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of voltage-dependent calcium channels (Ko et al., 1991), endothelial protective effect through superoxide scavenging activity (Goto et al., 1996) and involvement of endothelium through NOguanylyl cyclase pathway (Lee et al., 2002) etc. In addition to the well-known antioxidant effect of these natural bioactive substances, they also enhance the production of vasodilating factors including NO (via expression of NOS) and endothelium-derived factor (EDHF).
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Our finding on the chemistry of the extract of C. vulgaris indicating presence of large amount of phenolic and flavonoid compounds, compared to rutin and quercetin. HPLC fingerprints also showed the presence of quercetin in crude extract and fractions with various intensity of absorption. These direct chemical evidences support the multiple vasodilatory mechanisms of the extract and fractions and provide a pharmacological rationale to the
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antihypertensive effect of the extract of C. vulgaris. However, we could not identify and structurally elucidate particular phenol or flavonoid at this stage.
Conclusion
In conclusion, our results indicate that the antihypertensive effect of C. vulgaris is the outcome
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of vasodilation, which is mediated through combination of muscarinic receptor-linked NO release, activation of TEA-sensitive K+ channels, prostacyclin and Ca+2 antagonism. Co-
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existence of the vasoconstrictor constituents might be of therapeutic importance as herbal drugs are considered having side effect neutralizing potential. However further research on the active
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constituents will be interesting to explore this important herbal drug for clinical purpose.
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Funding source: This research work was carried out through a grant to the principle investigator
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from the Higher Education Commission (HEC) of Pakistan (grant No. 20-1554/R&D/10).
Conflicts of interest The authors declared no conflict of interests concerning primary interest such as validity of research, may be influenced b a secondary interest, such as financial gain that might bias this work.
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Bolton, T.B., 1979. Mechanism of action of transmitters and other substances on smooth muscles. Physiol. Rev. 59, 606-718.
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Kaur, C., Kapoor, H.C., 2002. Anti‐oxidant activity and total phenolic content of some Asian vegetables. Int. J. Food Sci. Tech. 37, 153-161.
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Kan, Y., Gökbulut, A., Kartal, M., Konuklugil, B., Yılmaz, G., 2007. Development and validation of a LC method for the analysis of phenolic acids in Turkish Salvia species. Chromatographia 66, 147. Kratchanova, M., Denev, P., Ciz, M., Lojek, A., Mihailov, A., 2010. Evaluation of antioxidant activity of medicinal plants containing polyphenol compounds. Comparison of two
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extraction systems. Acta Biochim. Pol. 57, 229-234. Miyase, T., Matsushima, Y., 1997. Saikosaponin homologues from Clinopodium spp. The structures of clinoposaponins XII-XX. Chem. Pharm. Bull. 45, 1493-1497.
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Meda, A., Lamien, C.E., Romito, M., Millogo, J., Nacoulma, O.G., 2005. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 91, 571-577.
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an aqueous extract of Passiflora nepalensis Wall. Int. J. Appl. Res. Nat. Prod. 3, 2-7. Parasuraman, S., Raveendran, R., 2012. Measurement of invasive blood pressure in rats. J.
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Ribeiro, R.M., PinheiroNeto, V.F., Ribeiro, K.S., Vieira, D.A., Abreu, I.C., Silva, S.D., Cartágenes, M.D., Freire, S.M., Borges, A.C., Borges, M.O., 2014. Antihypertensive effect
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other endothelium-derived factors. Medicina (Kaunas) 39, 333-41. Sparg, S., Light, M.E., Van Staden, J., 2004. Biological activities and distribution of plant saponins. J. Ethnopharmacol. 94, 219-243.
Shah, A.J., Gilani, A.H., 2011. Blood pressure lowering effect of the extract of aerial parts of Capparis aphylla is mediated through endothelium-dependent and independent
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mechanisms. Clin. Exp. Hypertens. 33, 470-477.
Shah, A.J., Bukhari, I.A., Gilani, A.H., 2016. Mentha longifolia lowers blood pressure in anesthetized rats through multiple pathways. Bangladesh J. Pharmacol. 11, 784-792. Takei, H., Nakai, Y., Hattori, N., Yamamoto, M., Kurauchi, K., Sasaki, H., Aburada, M., 2004. The herbal medicine Toki-shakuyaku-san improves the hypertension and intrauterine
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growth retardation in preeclampsia rats induced by Nω-nitro-L-arginine methyl ester. Phytomedicine 11, 43-50.
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Van Vliet, B.N., Montani, J.P., 2008. The time course of salt-induced hypertension, and why it matters. Int. J. Obes. 32, S35-S47. Vladimir-Knežević, S., Blažeković, B., Kindl, M., Vladić, J., Lower-Nedza, A.D., Brantner,
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Table Legend
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Table 1. Total phenol and flavonoid contents in the methanolic crude extract of Calamintha vulgaris. Total phenol contents 39.41 ± 0.18 (mg of GAE/g)
Total flavonoid contents 12.03 ± 0.23 (mg of QUE/g)
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Figure legends
Fig. 1. Shows effect of norepinephrine (NE) and aetylcholine (ACh) on mean arterial pressure (MAP) in normotensive anesthetized rats. Fig. IB shows MAP values in the normotensive rats in comparison with the high salt-induced hypertensive rats. Fig. 1C-H show effect of the 20
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Verapamil, crude extract (Cv.Cr), nHexane (Cv.nHexane), chloroform (Cv.Chl), ethylacetate (Cv.EtAc), and aqueous (Cv.Aq) fractions of Calamintha vulgaris on MAP in the sodium thiopental induced anesthetized rats. Values shown are mean ± SEM (6-7) and p*** < 0.001, represents the significant difference between the% fall in MAP in normotensive and hypertensive
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rats.
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Fig. 2. A-E show effect of the crude extract (Cv.Cr) and nHexane (Cv.nHexane), chloroform (Cv.Chl), ethylacetate (Cv.EtAc) and aqueous (Cv.Aq) fractions of Calamintha vulgaris on MAP in the absence (normotensive) and presence of atropine (2 mg/kg) and L-NAME (100 µg/kg) on mean arterial pressure (MAP) in the sodium thiopental induced anesthetized rats. Values shown are mean ± SEM (6-7) and p*** < 0.001, represents the significant difference between the% fall in MAP in normotensive rats.
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Fig. 3. Aortic rings from normotensive rats were tested for the vascular reactivity to the crude extract and fractions of Calamintha vulgaris. Fig. A-E show effect of the crude extract and nHexane (Cv.nHexane), chloroform (Cv.Chl), ethylacetate (Cv.EtAc) and aqueous (Cv.Aq) fractions of Calamintha vulgaris on vascular tone precontracted with phenylephrine (PE) in the absence (intact) and presence of atropine (1 µM), L-NAME (10 µM), atropine (1 µM) + LNAME (10 µM), tetraethyl ammonium bromide (TEA; 10µM) and indomethacin (10 µM). A vasoconstrictor component was unmasked in the presence of indomethacin. Denudation of the 22
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aortic rings also ablated the vasodilatory effect at lower concentration. Values shown are mean ±
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SEM (n = 6-7), p* < 0.05, p** < 0.01, and p*** < 0.001.
Fig. 4. Concentrations response curves show effect of (A) the crude extract of Calamintha vulgaris (Cv.Cr) and (B) verapamil on high K+ precontractions in isolated rabbit aortic rings. Fig. C-D show effect of Cv.Cr and verapmail on Ca+2 concentrations response curves in Ca+2 free/EGTA. Values shown are mean ± SEM (n = 6-7), p* < 0.05, p** < 0.01, and p*** < 0.001.
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Fig. 5. Representative HPLC chromatogram of Calamintha vulgaris A) rutin B) quercetin C)
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crude extract (Cv.Cr), D), Chloroform (Cv.Chl), E) ethylacetate (Cv.EtAc) and F) Aqueous
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Graphical abstract
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