Hypertension is a highly prevalent cardiovascular risk factor for cardiovascular disease that numerous individuals around the world suffer from this condition. Hypertension shortens the life expectancy of people due to heart disease, stroke, impairment of vision and kidney disease .
The exact mechanisms of hypertension is unknown but suggested factors such as endothelial dysfunction, extracellular matrix alterations and activation of renin-angiotensin system (RAS) are involved . The RAS plays an important vital role in physiological regulation of cardiovascular, renal and endocrine functions . This system has been also shown to involve in development and maintenance of different forms of hypertension  so drugs that inhibit the RAS can apply useful therapeutic effects on hypertension and heart failure . In addition, an extensive interaction between the RAS and other blood pressure control systems such as sympathetic nervous system  and baroreceptor reflexes  and nitric oxide (NO) has been demonstrated.
Effect of medicinal plants on RAS especially angiotensin II (AngII), main product of this system, has been reported . One of these plants is Crocus sativus (C. sativus). This plant which is also known as saffron spice has several part including stigma, petal, stamen. The valuable part of this plant is stigma that possesses various beneficial components including crocin, Safranal, crocetin, anthocyanin, flavonoids, vitamins such as riboflavin and thiamine, minerals, carbohydrate, raw fibers, proteins, and fats . Besides its use in food as a spice, C. sativus has been documented to have therapeutic properties . C. sativus has been shown to have the beneficial effects against the disturbances of the nervous system, blood pressure, lungs, kidneys, gastrointestinal tract and endocrine gland . In addition, crocetin a component of C. sativus has been claimed to has cardioprotective effects via exerting antioxidant, anti-inflammatory and anti-apoptotic effects . We previously has been shown that crocin, a main product of C. sativus has ameliorative effect on hypertension induced by AngII . Despite all these findings, the effect of C. sativus on RAS especially AngII has not yet been investigated. Therefore, current study was conducted to determine the effect of the hydroalcoholic extract of C. sativus on cardiovascular parameters in AngII hypertensive rats.
2. Materials and Methods
2.1. Preparation of plant extract
C. sativus was collected from saffron farms of South Khorasan Province (Esfeden city). After identifying by a botanist in the herbarium of the Ferdowsi University of Mashhad (Herbarium No: 143-0319-1), the hydroalcoholic extract was prepared. For this purpose, 10 g of stigma of plants and 400 mL of 70% aqueous alcohol solution in a Soxhlet extractor were mixed for 14 h. Then prepared extract was filtered through a 0.2 mm filter to be sterilized. Finally, the extract was concentrated to 100 mL by a rotatory evaporator and was stored at -200C until being used. The prepared extract was dissolved in saline in order to arrive at desired doses and was then administered.
2.2. Experimental animals
In this study, 36 male wistar rats with 250 ± 20 g of weight were purchased from animal's house of Mashhad University of Medical Sciences, the animals were housed in standard condition (light / dark cycle = 12 h and temperature = 21 ± 4℃). Working with animals was done based on approved procedures by the Animal Protection Committee of Mashhad University of Medical Sciences (Code Number 922834).
The drugs used in current study are include: AngII and urethane purchased from Sigma, USA and losartan (Los) prepared from Darupaksh Company, Iran. The solvent of drugs was saline.
2.4. Experimental groups
The rats were assigned to 6 groups (n = 6 rats/group): (1) Control, (2) AngII, (3) Los + AngII, (4) C. sativus 10 + AngII, (5) C. sativus 20 + AngII and (6) C. sativus 40 + AngII. AngII (300 ng/kg) was intravenously (i.v) administered during 2 min. Los as AT1 blocker (10 mg/kg, i.v) and 3 doses of C. sativus extract (10, 20 and 40 mg/kg) was injected (i.v) 10 min before AngII. The doses of all drugs were selected based on a previous study [8, 13].
2.5. Experimental procedure
In this study, urethane (1.5 g/kg, i.p) was used to anesthetize the animal. After anesthesia the femoral artery and vein catheterized by a polyethylene tube 50 which was filled with heparinized saline to recording of blood pressure and drug injection, respectively. Arterial catheter connected to a blood pressure transducer and cardiovascular parameters [mean arterial pressure (MAP), systolic blood pressure (SBP) and heart rate (HR)] were recorded [14, 15] 15] by a power lab instrument(ID instrument, Australia). After stabilization of cardiovascular parameters, In AngII group, AngII (300 mg/kg) was intravenously administered during 2 min to achieve a high blood pressure. In Los group, Los injected after 10 min AngII (300 mg/kg) injected. In three doses of C. sativus groups firstly extract was injected in separate groups after 10 min AngII (300 mg/kg) injected and cardiovascular parameters recorded [8, 13] 13].
2.6. Statistical Analysis
The results of current study were express as mean ± SEM. The peak changes of cardiovascular parameters calculated and were statistically analyzed by one-way ANOVA followed by Tukey’s post hoc comparisons test. Value of p < 0.05 was considered as statistically significant.
3.1. Effect of saline administration on cardiovascular parameters
To determine the effect of saline on cardiovascular parameters, cardiovascular parameters were recorded before and after saline injection (i.v). Saline administration did not significantly changed MAP (before: 113.15 ± 3.22 mmHg vs after: 108.90 ± 2.2 mmHg), SBP (before: 127.3 ± 3.42 mmHg vs after: 124.4 ± 3.32 mmHg) and HR (before: 318.67 ± 10.1 beats/min vs after: 322.56 ± 11.3 beats/min) in rats.
3.2 Effect of AngII administration on cardiovascular parameters
In this experiment, AngII (300 ng/kg, i.v) injected and cardiovascular parameters were recorded (Fig. 1). Injection of AngII significantly increased maximal changes of SBP, MAP and HR in AngII group than control group (P < 0.001in all parameters, Fig. 2a, b and c).
Figure 1 A sample of cardiovascular responses recorded record after intravenous injection 300 mg/kg of AngII. Injection time marked by a vertical line
Figure2 Peak changes of SBP (A), MAP (B), and HR (C) after infusion 300 ng/kg of AngII and Losartan (Los; 10 mg/kg) + AngII. Expression of Data: mean ± SEM (n = 6); Statistical analysis: One-way ANOVA with Tukey’s post hoc; *p < 0.05, **P < 0.01 and ***p < 0.001 vs control; ++p < 0.01, +++p < 0.001 vs AngII 300 MAP: mean arterial pressure; SBP: systolic blood pressure; HR: heart rate
3.3. Effect of losartan on the cardiovascular parameters in hypertension induced by AngII
Pretreatment with Los led to a significant reduction in cardiovascular responses evoked by AngII. Enhancement of MAP, SBP in AngII group significantly attenuate with respect to Los (P < 0.001; 2a and b). Injection of Los also significantly decreased tachycardia induced by AngII (P < 0.01; (Fig. 2c).
Figure 3 Comparison peak changes of MAP induced by i.v injection of 3 doses of C. sativus extract (10, 20 & 40 mg/kg) + AngII with AngII alone and Los + AngII groups. Expression of Data: mean ± SEM (n = 6). Statistical analysis: One-way ANOVA with Tukey’s post hoc;* P < 0.05, ** P < 0.01 and *** p < 0.001 vs AngII; ++p < 0.01, +++p < 0.001 vs AngII + Los MAP: mean arterial pressure
Figure 4 Comparison peak changes of SBP induced by i.v injection of 3 doses of C. sativus extract (10, 20 & 40 mg/kg) + AngII with AngII alone and Los + AngII groups. Expression of Data: mean ± SEM (n = 6). Statistical analysis: One-way ANOVA with Tukey’s post hoc; *P < 0.05, **P < 0.01 and ***p < 0.001 vs AngII; +p < 0.05, ++p < 0. 01 vs AngII + Los; SBP: systolic blood pressure
Figure 5 Comparison peak changes of HR induced by i.v injection of 3 doses of C. sativus extract (10, 20 & 40 mg/kg) + AngII with AngII alone and Los + AngII groups. Expression of Data: mean ± SEM (n = 6). Statistical analysis: One-way ANOVA with Tukey’s post hoc; *P < 0.05, **P < 0.01 vs AngII; HR: heart rate
3.4. Effect of three doses of C. sativus extract on the cardiovascular parameters in hypertension induced by AngII
Pretreatment with the 3 doses of extract of C. sativus (10, 20 and 40 mg/kg) resulted in significant reduction cardiovascular parameters in AngII hypertensive rats. As has been shown in Fig 3 and 4, enhancement of MAP and SBP in C. sativus + AngII groups significantly decreased compared to AngII alone (P < 0.05 to P < 0.001). Tachycardia in AngII group also decreased by pretreatment of all doses of C. sativus extract (P < 0.05 and P < 0.01) (Fig. 5). Comparing all doses of C. sativus + AngII with Los+ AngII has been shown that MAP and SBP in doses10 and 20 mg/kg of C. sativus are significant than Los + AngII group but dose 40 mg/kg was not significant (Fig. 3 and Fig. 4). Changes of HR in all doses of C. sativus was not significant compared to Los + AngII (Fig 5).
Our result showed that hydroalcoholic extract of C. sativus ameliorates cardiovascular responses by acute hypertension induced by AngII and this effect is comparable with Los.
One of the most important systems affecting blood pressure is the renin-angiotensin system (RAS). AngII as primary effector of RAS and its AT1 receptor has been shown to have several deleterious effects such as hypertension, cardiac and vascular hypertrophy, endothelial dysfunction and vasoconstriction . It has been also reported that AngII by vasoconstriction, induce the release of catecholamines from the adrenal gland and increase of salt retention causing to development of hypertension. In addition, AngII could induce hypertension via activation of phospholipase C enzymes and the increase of cytoplasmic calcium concentration as well as reduction of nitric oxide (NO) [8, 16-18]. In line with these findings, in our study also administration of AngII increased blood pressure in rats [8, 19]. C. sativus which possesses several components with different properties including antitumor, anti-inflammatory, antioxidant, anti-depressant and hypolipidemic effects [11, 20], also can exert protective effects on cardiovascular system . For example, the aqueous extract of C. sativus has been documented to have the hypotensive effect in normotensive and hypertensive anesthetized rats . In our finding also pretreatment with the hydroalcoholic extract of C. sativus attenuate increased MAP, SBP, and HR in AngII hypertensive rats. In addition, radical scavenging effects and antioxidant properties of C. sativus have been well documented  22]. The C. sativus extract has been exhibited to improve isoproterenol-induced myocardial infarction through reduction of oxidative stress . C. sativus and its crocin have been proposed to inhibit lipid peroxidation and increase enzymatic and non- enzymatic antioxidant . An important component of C. sativus is crocin that is a water-soluble carotenoids  and has effect on cardiovascular system. In recent study we show that crocin attenuate cardiovascular effect induced by AngII .
Because we use hydroalcoholic extract therefore this component is soluble in extract and effect of C. sativus on AngII partly medicated by this component. C. sativus also contain another important component such as Crocetin and Safranal that has beneficial effect on cardiovascular system. It has been reported that crocetin could suppress effect of AngII in vascular smooth-muscle cell  and Safranal attenuated cardiovascular parameters in hypertension induced by desoxycorticosterone acetate (DOCA) – salt  . Therefore, it is possible these components of C. sativus inhibit effect of AngII in our experiment. However, future studies are needed to confirm our opinion.
Interaction between NO and AngII in vascular also has been reported so AngII decrease NO bioavailability . Because C. sativus could increase NO, we suggested that this plant by effect on NO production ameliorate effect of AngII and reduced cardiovascular effect.
In the other hand, a part of the effects of AngII on blood pressure is attributed to stimulate the overproduction of reactive species (ROS) and induction of oxidative stress by this substance . Researcher has been suggested that AngII can induce vasoconstriction through enhancing the ROS concentration in smooth muscle of the vessels . Furthermore, in recent studies, RAS and AngII have been shown to involve in inflammatory processes . It has been suggested that using immunosuppressive drugs can protect hypertensive rats against AngII–caused renal damage . In addition, the role of T cells in the pathogenesis of hypertension and vascular dysfunction induced by AngII in mice has been reported . Regarding the antioxidant and anti-inflammatory effects of C. sativus and its components, it is guessed that the possible improving effect of C. sativus against oxidative stress and inflammation induced by AngII contributes in the results of the current study.
In addition, type 1 (AT1) receptor of AngII is considered as an important component of RAS . The evidence reveals that AT1 receptors antagonists can have beneficial effects on the blood pressure caused by the activity of RAS . Losartan is a selective AT1 receptors antagonist which relaxes muscle cell and dilates blood vessels and diminishes blood pressure . Losartan has also been shown to diminish blood pressure in hypertensive rats . It has been documented that losartan induces a renin-dependent hypotensive effect in the patient with essential hypertension . In the current study, pretreatment with losartan restored Ang II- caused blood pressure and tachycardia in rats. Considering this finding it seems that AT1 receptors are involved in AngII effects on cardiovascular parameters in this research. Based on the results of current study, attenuating effects of C. sativus extract against enhanced blood pressure and tachycardia induced by AngII is comparable to that of losartan. Therefore, we suggested that the improving effect of the hydroalcoholic extract of C. sativus on hypertensive induced by AngII is mediated through AT1 receptors. In this study we used acute hypertension, because effect of AngII and C. sativus may be achieved in long term we proposed that effect of C. sativus evaluated in chronic model of hypertension such as Goldblatt hypertension.
In the brief, the current study data showed that the hydroalcoholic extract of C. sativus ameliorate cardiovascular effect of AngII, the main product of RAS. Therefore, we suggested that C. sativus by inhibition of RAS has a preventive effect in hypertension induce by RAS.