Effects of PetalsExtracts ofSaffron on Rat Blood Pressure and on Responses Induced by Electrical Field Stimulation in the Rat Isolated Vas Deferens and Guinea–Pigileum
M. Fatehi, T. Rashidabady, Z.F. Hassanabad
Edman Saffron Co.
Iran
Keywords:blood pressure,contraction,guinea-pig ileum,rat vas deferens
Abstract
The effects of petals extracts of Crocus sativus on blood pressure in anaesthetized rats and also on responses of the isolated rat vas deferens and guinea-pig ileum induced by electrical field stimulation (EFS) were studied. Aqueous and ethanol extracts of C.sativus petals reduced the blood pressure in a dose–dependent manner.For example administration of 50mg/100 g of aqueousextract changed the blood pressure from 133.5±3.9 to 117±2.1 (mmHg). EFS of the isolated rat vas deferens and guinea-pig ileum evoked contractions were decreased by aqueous and ethanol extracts of C. Sativus petals. The aqueous extract (560mg/ml) significantly reduced the contractile responses of vas deferens to epinephrine (1μ M)without any change in contraction induced by KCL (300Mm). The present results may suggest that the relaxatory action of C. sativus petals, extract on contraction induced by EFS in the rat isolated vas deferens is a postsynaptic effect.
Introduction
The dried stigma of Crocuse sativus is commercially available in superstores.It is popular because of its delicate aroma and attractive color and can be used as a food additive. C.sativus is cultivated for its red stigmatic lobes that constitute saffron. This plant (C.sativuse) belongs to the Iridaceae family.Saffron blooms only once a year and should be collected within a very short time.It is picked during 3-4 weeks in October–November.
The methods for the cultivation of saffron contribute greatly to its high price. Saffron is cultivated in countries such as Spain, Italy and Iran. The C.sativus requires a strict agroclimatic condition for growth which has an influence on its quality. Recently interest has been focused on the biological activity of various plant extracts. It was previously reported that the extract of C.sativus has antitumor, antimutagenic and cytotoxic activies and inhibits nucleic acid synthesis in human malignant cell (Nair et al. 1991, Abdullaev, 1994). The active compound(s) of this extract and the mechanism (s) of its effects are not exactly known.C. sativus extract consists of many compounds such as α-crocine, a water soluble carotenoid,crocetins,picrocrocin and safranal (Morimoto et al.,1994). Petals of C.sativus are rich in flavonoids and anthocyanines. The protective avtivity of carotenoid against cancer has obtained support from a large number of epidemiological and experimental studies (Gainer et al., 1976; Bendich and Olson, 1989; Moon, 1989; Ziegler, 1989). However, attention given to the pharmacological effects of its petal contents has been little. As a screening project for finding new plant materials with biological activity, it was of interest to study any possible action of various concentration of ethanol and aqueous extracts form petals of C. sativus on rat blood pressure, and also on contraction induced by electrical field stimulation (EFS) in the rat isolated vas deferens and guinea- pig ileum.
Materials and methods
The experiments were performed under the Animals (scientific procedures)Act of 1986 and conform to the National Institutes of Health guidelines for the use of experimental animals. This study was carried out on male Sprague–D awley rats (250 –300 g) and guinea – pigs (300-350g) from Razi Institutes .Mashhad, Iran. Mean arterial blood pressure (MABP) was measured as previously described (Fatehi–Hassanabad) et al., 1995). Briefly, rats were anaesthetized with sodium thiopental (25 mg/g by i.p.injection), right common carotid artery and left jugular vein were catheterized for the measurement of blood pressure and administration of C.sativus petals, extract, respectively. The trachea was annulated and the animals were artificially ventilated using a Palmer respirator (rats 48 strokes/min, stroke volume 1.5 ml/100g body weight).Body temperature was recorded using a rectal thermostat probe and was maintained at 37±0.5C using an incandescent lamp placed over the abdomen ,Animals were allowed to stabilize for 20 min prior to the administration of the extracts .
Rats were killed by a blow to the head and vas deferens was removed and strips, approximately 15mm long, were prepared. The strips were suspended vertically in 10 ml organ baths for isometric recording of mechanical activity .An initial load of 1g was applied to the strips which were then allowed to equilibrate for at least 30min, with the krebs solution being changed every 15 min the Krebs solution had the following composition (mM); Nacl 118/4, KCl, 4/7, MgSO4 7 H2O ¼, KH2PO4 1/2,CaCl2 2/5,NaHCO3 25 and glucose 11/1.
Guinea-pigs were killed by a blow to the head and exsanguination.The ileum was exteriorized, the ileocaecal junction located and approximately 30cm removed. A 10cm segment of the terminal portion was discarded before the contents in the remaining Ileum were flushed out with Krebs solution of the same compassion as mentioned above. Segments of the ileum (2cm) were set up under 1g of tension in 10ml organ baths, ready for isometric recording using an isometric transducers connected to a Washington recorder. Preparations (either rat isolated vas deference or guinea-pigIleum) were incubated in Krebs solution gassed, (95% O2 / 5% CO2)at 37 C for 30 minutes.
EFSwas applied using a ring electrode every 10s with trains of ten pulses of 0/5 ms duration and a voltage greater than which produced a maximal twitch. EFS were provided by a Grass S88 stimulator. In the rat isolated vas deferens and guinea – pig.
Ileum responses were calculated as a percentage of the contraction evoked by KCL (300mM) which was added to all preparations at the beginning and at theend of each experiment .
C. sativus petals were identified by Botanists in the herbarium of FerdowsiUniversity, Mashhad and the specimen number of the plant is 143-0319-1. The plant was collected from farms in Torbatt-hydaria,Iran, during October 1999.The petals, extract were prepared as follow: 10g of the chopped , dried petals was extracted with 100 ml distilled water, ethanol extract was prepared as the same as aqueous extract except the solvent which was ethanol instead of distilled water. Doses and concentrations of the extracts are expressed as mg of the dried plant material/100g body weight and mg of the dried plant material/ml of organ bath liquid. The volume of the petals, extract injected to the rats(intravenously)or to the organ bath was0.3ml.Injection of 0.3ml of distilled water and ethanol did not modify either the MABP or the responses to EFS in the rat isolated vas deferens and guinea-pig ileum.
Values in the text refer to mean ±S.E. Changes in MABP were compared using Student’s Ttest. Differences between groups have been compared using one way ANOVA followed by a Turkey-Kramer multiple comparison test.A P-value less than 0.05 were considered statistically significant.
Results
Effects of the aqueous and ethanol petals, extract on MABP
In anaesthetized rats, injection of aqueous and ethanol extract caused a dose–dependent reduction in MABP (e.g. injection of the aqueous extract, 50 mg/100g body weight in 0.3 ml. reduced the MABP from 133.5 ±3.9 to 117 ±2.1(.
Rat isolated-vas-deferens
Potassium chloride at concentration of 300mM evoked contraction in the rat isolated vas deferens. EFS induced a contractile response in the rat-isolated vas deferens. Addition of the C.sativus petals, extracts (both aqueous and ethanol extracts) decreased the contractility induced by EFS in a concentration–dependent manner. Preoccupation of the tissue with aqueous extract (560mg/ml) significantly reduced the contractile response to epinephrine (1µm)without any change in response to KCL (300 Mm)
Guinea-pig Isolated Ileum
EFS caused a contraction in the guinea-pig isolated ileum which was significantly reduced by different concentration of the extracts. Incubation of the guinea-pig isolated ileum with atropine (1 µm, for 15 min) completely blocked the contractile responses induced by EFS. However, the contraction caused by 300 mM of KCL was not modified by preoccupation of the tissue with different concentration of the petals, extracts.
Discussion
Saffron is widely grown in Khorasan province,Iran. Due to the low profitability of this product, it is no longer of major industrial interest, but is a family labour activity. Saffron, s stigmas are expensive, therefore, its use is limited. In order to expand the use of this medicinal herb, the effect of its petals, this can be collected easily and cheaply, was investigated. To our knowledge for the first time, the present results show that aqueous and ethanol extracts of saffron (C. sativus)petals reduced the MABP in anaesthetized rats. This reduction could be either due to the effect of the C. sativus. Petals, extracts on the heart itself/total peripheral resistance, orboth. However, administration of the extracts did not significantly modify the heart rate (data not show), therefore, the effect of extracts on peripheral resistance seems to be more important. To investigate the effects of petals, extracts on the tonicity of smooth muscle in different species, two preparation were used (rat isolated vas deferens and guinea-pig isolated ileum).In the rat isolated vas defers; contractile responses to EFS were decreased by the petals, extracts. Contractions of the vas deferens to EFS are mediated by a combination of nor adrenaline and ATP released as co transmitters from sympathetic nerves (Hoyle and Burnstock, 1991).
In this tissue responses to epinephrine but not those to KCL were significantly reduced by the petals, extract. The antagonistic effect of the aqueous extract on adrenergic receptors of rat isolated vas deferens is well confirmed by our experiments. The responses evoked by EFS in the guinea-pig isolated ileum were induced by muscarinic receptors as it was completely blocked by atropine. Contractile responses induced by EFS were significantly reduced by the petals, extract in this preparation. Considering this result, it could be suggested that the inhibitory action of C.sativus petals, extract oncontractile responses of both isolated rat vas deferens and guinea-pig ileum induced by EFS is a postsynaptic effect. The ethanol extract induced greater changes in both arterial blood pressure and contraction evoked by EFS in the rat isolated vas deferens and guinea-pig ileum than the aqueous extract. Further, fractionation of the extracts and bioassay experiments are necessary to find out which compounds (chemical structures) in the petals, extract are responsible for these effects. Hematological and biochemical studies are also needed to assess the toxicity of petals, extract on different organs such as liver and kidney. In summary, petals, extracts reduced the smooth muscle tonicity in different preparations such as rat isolated vas deferens and guinea pig ileum.
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