World Journal of Pharmaceutical Sciences ISSN (Print): 2321-3310; ISSN (Online): 2321-3086 Published by Atom and Cell Publishers © All Rights Reserved Available online at: http://www.wjpsonline.org/ Original Article

The Attenuation Effects of , Vitexin, Hyperoside and on Carrageenan-Induced Mice Tail Thrombosis Model

Elif SUMBUL2, Rana ARSLAN1*, Nurcan BEKTAS1

1Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey 2Bilecik Public Hospital, Bilecik, Turkey.

Received: 10-04-2016 / Revised: 16-05-2016 / Accepted: 27-05-2016 / Published: 30-05-2016

ABSTRACT

Flavonoids exert different pharmacological actions, such as analgesic, anti-inflammatory and cardioprotective activities Apigenin, vitexin, quercetin and hyperoside are flavonoids found within medicinal plants. We aimed to study selected flavonoids’ antithrombotic effects using a carrageenan-induced tail thrombosis model in mice. To evaluate the antithrombotic effects of selected flavonoids, a carrageenan-induced tail thrombus mouse model was used. Flavonoids were administered intraperitoneally in doses of 50, 100 and 200 mg/kg. Forty μl (1%) carrageenan (type I) injected in the mouse’s hind paw 45 min after drug administration. The wine-colored region at the mouse tail was then measured at 24, 48 and 72 h. The findings showed that all flavonoids tested had antithrombotic effects. A dose of 200 mg/kg of quercetin and hyperoside decreased the length of tail thrombosis significantly (P<0.001) at day three. Apigenin and vitexin were more effective at 50 and 100 mg/kg doses than at the 200 mg/kg dose. The results obtained indicate that selected flavonoids exert preventive anticoagulant effects. Apigenin and vitexin showed antithrombotic effects at all tested doses, while quercetin and hyperoside were effective only at the highest doses. We believe that these flavonoids are promising new antithrombotic agents or could be used as complementary therapy with other anticoagulant drugs.

Keywords: Apigenin, vitexin, quercetin, hyperoside, antithrombotic effects, thrombosis

INTRODUCTION such as analgesic, anti-inflammatory, antihepatotoxic, anti-ulcer, anticancer, Hemostasis is a necessary mechanism to stop antimutagenic, antispasmodic, anti-allergic, bleeding, whereas thrombosis can be either a antiviral and cardioprotective activities [4-7]. pathological or a physiological event, and therefore Flavonoids are also part of a therapeutic diet to may require treatment. Thrombus formation is one help prevent mortality due to coronary artery of the main causes of many cardiovascular disease. It is believed that their inhibitory effects on diseases, such as myocardial infarction, ischemia, low-density lipoprotein oxidation and platelet deep vein thrombosis and angina [1,2]. Thrombus aggregation are responsible for these beneficial formation is a highly complicated physiological effects [8-11]. Additionally, it has been reported process involving many factors, such as platelets that flavonoids exhibit their antithrombotic effects and coagulation factors. Intravascular arterial by inhibiting arachidonic acid metabolism and thrombosis causes multiple types of cardiovascular modulating endothelial nitric oxide and damage and arises primarily from platelet prostacyclin levels [12, 13]. activation. Venous thrombosis, which can cause deep vein thrombosis and pulmonary embolism, Traditional medicine, such as hawthorn plant arises mostly from activation of coagulation factors products, is utilized in the management of [1-3]. thromboembolic and multiple cardiovascular diseases [14, 15]. It was previously demonstrated Flavonoids are large polyphenolic compounds by us [16, 17] that the flavonoid-rich plant derived from medicinal plants, and their health hawthorn (genus Crataegus), commonly used to benefits have been reported in various treat angina, hypertension, arrhythmias, and epidemiological studies. These compounds are congestive heart failure [14,15], possesses an in used to obtain different pharmacological effects, vivo antithrombotic effect in a carrageenan-induced

*Corresponding Author Address: Dr. Rana ARSLAN, Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470, Eskisehir, TURKEY; e-mail: [email protected] Sumbul et al., World J Pharm Sci 2016; 4(6): 308-313 tail thrombosis model in mice. Flavonoids such as 5%CMC+0.7 ml saline). The vehicle was apigenin, vitexin, , quercetin and hyperoside administered at the same volume in the control are found in Crataegus species [7,8,10,11,18]. group. The vitexin, apigenin, hyperoside and However, there have been limited studies of their quercetin were injected intraperitoneally (i.p.) at antithrombotic effects, and these studies were doses of 50, 100 and 200 mg/kg, 45 min before performed using in vitro experiments. In in vitro carrageenan injection. Intraperitoneal heparin (100 studies, Guerrero et al. demonstrated that apigenin IU) was used as a reference drug in the positive and genistein strongly inhibited agonist-induced control group. The length of tail thrombosis was aggregation of platelets and effects related to measured using a ruler and was photographed at arachidonic acid and collagen-induced platelet 24- to 72-h intervals. responses, such as aggregation and secretion, with a less substantial effect on thromboxane A2 (TXA2) Statistical analysis: Statistical analyses were synthesis [19]. Another study demonstrated that carried out using GraphPad Prism version 5.0 apigenin modified platelet aggregation by (GraphPad Software, San Diego, California, USA). inhibiting adenylyl cyclase [20]. Later research Data obtained from the experiments were expressed found that although apigenin alone had a negligible as mean values ± standard error of mean (S.E.M.). effect on platelet aggregation, it potentiated the Statistical differences between the treatment and antiplatelet effects of aspirin [21]. However, it is control groups were evaluated using one-way possible that the results of these studies may differ ANOVA, followed by Dunnett’s multiple from those of in vivo studies, because the comparison tests. The results were expressed as the coagulation cascade is different in in vitro and in differences between treatment and control groups vivo conditions. Thus, we aimed to investigate the and were considered significant when P≤0.05. in vivo antithrombotic effect of apigenin, vitexin, quercetin and hyperoside using a carrageenan- RESULTS induced tail thrombosis model in mice. The subplantar injection of carrageenan resulted in METHODS the thrombosis tail infarction becoming visible in the control group. The lengths of tail thrombosis at Animals: Male and female Swiss mice (weighing 24, 48 and 72 h are shown in Figure 1. The 30–35 g) housed in an acclimatization, controlled inhibition activities of flavonoids tested in temperature (22 ± 2°C) 12-h light/12-h dark cycle carrageenan-induced tail thrombosis in mice are room were used. The animals were supplied by the seen in Table 1. Heparin (100 IU) prevented the tail Anadolu University Center for Animal vein thrombosis from developing significantly Experiments and Research, Eskişehir, Turkey. (P<0.01) compared with the control group’s Animal care and research protocols were based on treatment. Quercetin and hyperoside decreased the the principles and guidelines adopted by the Guide length of tail thrombosis significantly at a dose of for the Care and Use of Laboratory Animals (NIH 200 mg/kg (P<0.001) at day three relative to the publication No. 85-23, revised in 1985) and doses of 50 and 100 mg/kg and compared with the approved by the Local Ethics Committee of control group. At day three, the inhibition of Anadolu University, Eskişehir, Turkey. thrombosis (%) for 200 mg/kg quercetin was 75% on average and for 200 mg/kg hyperoside was 73% Materials: Carrageenan type I, carboxymethyl on average. The length of tail thrombosis cellulose (CMC), apigenin, vitexin, hyperoside and significantly decreased with doses of 50, 100 and quercetin were purchased from Sigma-Aldrich (St. 200 mg/kg apigenin (P<0.001, P<0.001 and Louis, Missouri, USA). Low-molecular-weight P<0.05, respectively) in the treatment groups at day heparin was purchased from Sanofi-Aventis three. However, apigenin appeared most effective (Istanbul, Turkey). at the 100 mg/kg dose. Table 1 shows that, at all days, apigenin decreased thrombosis by on average The Carrageenan-induced tail thrombosis 76% and 89% at doses of 50 and 100 mg/kg. The model: In order to detect the thrombolytic effects administration of vitexin also significantly of selected flavonoids, a carrageenan-induced tail prevented the developing of tail vein thrombosis thrombus model was used. Forty μl (1%) (P<0.001, P<0.001 and P<0.05, respectively) at carrageenan (type I) was dissolved in physiological 24- and 48-h time points. Although the saline and injected into the mouse hind paw 45 min effectiveness of 200 mg/kg vitexin faded out, 50 after drug administration [42,43]. and 100 mg/kg vitexin injected into the treated mice remained effective (P<0.001) at 72 h. At day Drug administration: Swiss albino mice were three, the 50 mg/kg dose of vitexin inhibited randomly divided into 14 groups (n = 7–9). thrombosis by 84% on average (Table 1). Flavonoids were dissolved in a vehicle (0.3 ml

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Sumbul et al., World J Pharm Sci 2016; 4(6): 308-313 Photographs of tail thrombosis for each groups at were demonstrated in in vitro studies, and it has 48 h are shown in Figure 2-3 (photographs at 24 been suggested that Crataegus species inhibit and 72 h not shown). The findings obtained in the platelet aggregation [32,33]. Crataegus species present study indicate that apigenin and vitexin are contain various types of flavonoids, such as highly potent against carrageenan-induced tail hyperoside, apigenin, apigenin 7-glucoside, vitexin thrombosis in mice. and quercetin [34,31]. Thus, the in vivo effects of hyperoside, apigenin, vitexin and quercetin on DISCUSSION thrombosis were investigated and it was found that these flavonoids are effective at different doses in In this study, the preventive anticoagulant effects mice. of apigenin, vitexin, quercetin and hyperoside were examined using a carrageenan-induced tail Many different mechanisms may play a role in the thrombosis model in mice. Apigenin and vitexin antithrombotic activity of flavonoids, such as showed antithrombotic effects at all tested doses, inhibiting TXA2 release, decreasing the level of while quercetin and hyperoside were effective only Ca+2 in platelets or blocking glycoprotein IIb/IIIa at the highest doses. receptors [35]. It has been demonstrated that apigenin and quercetin inhibit collagen-induced Physiologically, thrombosis is a similar event to aggregation in platelet-rich plasma in vitro [36]. hemostasis, but thrombosis can be a pathological Apigenin, genistein, luteolin and quercetin block process, whereas hemostasis is a physiological the TXA2 receptors (TP), similarly to the TP reaction of metabolism. In thrombosis, a thrombus receptor antagonist SQ29548. Aspirin, which forms in a vessel and obstructs blood flow through suppresses the TXA2 pathway, shows more potent the circulatory system. This blockage causes antiplatelet effects when combined with apigenin different cardiovascular health problems [22,23]. [37,38]. In this study, thrombosis was induced by Thus, the management of thrombosis with carrageenan. It has been reported that the anticoagulant drugs is vital in treating thrombotic activity of carrageenan results from the cardiovascular diseases [24,25]. Many recent activation of Hageman factor, also known as factor studies have focused on the investigation of new XII, which is followed by intravasal coagulation agents that are effective for treating thrombosis, [39]. Other proposed mechanisms for carrageenan- because available antithrombotic drugs have many induced thrombosis include local blood vessel side effects owing to their narrow therapeutic index inflammation causing the release of interleukin-1 [26,27]. (IL-1) and tumor necrosis factor (TNF), which may destroy the functioning of normal endothelial cells The carrageenan-induced tail vein thrombosis that maintain the balance between model used in this study employed carrageenan, a hemagglutination and fibrinolysis, and endothelial polysaccharide polymer extracted from various red cell damage due to promoting thrombus formation seaweeds (Rhodophyceae). Carrageenan is used for by reducing relaxing factors and increasing gelling, thickening, and emulsifying food during constrictor factors [40,41]. It is thought that preparation and in experimental pharmacological hyperoside, apigenin, vitexin and quercetin thereby studies of anti-inflammatory and antithrombotic prevent thrombosis by inhibiting platelet and other effects. Various types of carrageenan aggregation through antagonizing TXA2 or have been used, such as λ, κ, ι, ε and μ. It is known Hageman factor which initiate the thrombosis that the κ type of carrageenan induces tail thrombus formation, or reducing the effectiveness or levels of in mice and enables researchers to observe the TNF and IL-1, which were released via local blood progression of thrombosis in a time-dependent inflammation following carrageenan-induced manner [28,29]. In this study, type I carrageenan thrombosis in this study. Therefore, reducing the was chosen because it contains large amounts of κ effects of thrombus formation induced by type carrageenan. Tail thrombosis was successfully carrageenan could be accomplished using the tested induced in mice and became visible within 24 h. At substances through any of the mechanism proposed a later time, tail necrosis due to thrombosis was here. We contemplate that the antithrombotic observed. It is known that the formation of a activity of these flavonoids could be used as carrageenan-induced thrombus causes the release monotherapy or as a complementary or of inflammatory factors [30]. combination treatment for thrombosis.

Crataegus plant species, which are known to The present in vivo study of the antithrombotic contain various flavonoids, have been used to treat activity of flavonoids supported the results of cardiovascular disease [31]. The antithrombotic previous in vitro studies. All tested flavonoids effects of Crataegus oxycantha (a common possessed antithrombotic activity. It can be seen hawthorn) and Crataegus aronia syn. Azarolus (L) that these flavonoids are potential prophylactic

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Sumbul et al., World J Pharm Sci 2016; 4(6): 308-313 compounds for the treatment of thrombosis or Anadolu University, Eskişehir, Turkey, is could be used as complementary drugs with other gratefully acknowledged (Project code no. anticoagulant medicines. Our findings suggest that 1306F254). This study is Elif Sümbül’s master's these flavonoids could be used to develop thesis. antiplatelet and/or anticoagulation agents and that they should be further assessed in clinical studies. Declaration of interest

Acknowledgments The authors report no conflicts of interest. The authors alone are responsible for the content and Financial support for this work through the writing of this paper. Scientific Research Projects Foundation of

Table 1: The inhibition activity of COE against carrageenan-induced tail-thrombosis in mice. Inhibition of thrombosis (%) Test compounds Doses (mg/kg) 24h 48h 72h 50 35,08621 37,20366 39,70739 Quercetin 100 41,81034 40,59806 43,17248 200 78,7931 74,78448 75,74435 50 85,51724 83,65148 84,42358 Vitexin 100 79,31034 78,42134 79,44045 200 57,73399 53,72537 49,83866 50 79,60591 75,06158 74,12731 Apigenin 100 89,35961 89,19335 89,70373 200 60,68966 58,43596 60,13494 50 34,31034 32,35453 34,163243 Hyperoside 100 48,7931 46,17457 46,63758 200 74,58128 72,84483 74,12731 Heparin 100 IU 73,99015 73,99015 73,99015

Figure 1. The antithrombotic effects of quercetin, vitexin, apigenin, hyperoside and heparin in carrageenan- induced mouse tail thrombosis at 24, 48 and 72 h. Values are presented as the mean ± S.E.M; *P<0.05, **P<0.01 and ***P<0.001 were significant differences from controls (n= 7–8).

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Figure 2. The effects of hyperoside, quercetin, control (0,5% CMC-SF) and heparin on carrageenan-induced mouse tail thrombus length (48 h after carrageenan injection). a: Control [0,5 % CMC – saline (3:7)]; b: 100 IU Heparin; c: quercetin 50 mg/kg; d: quercetin 100 mg/kg; e: quercetin 200 mg/kg; f: hyperoside 50 mg/kg; g: hyperoside 100 mg/kg; h: hyperoside 200 mg/kg. Data represent two animals for each group.

Figure 3. The effects of vitexin and apigenin on carrageenan-induced mouse tail thrombus length (48 h after carrageenan injection- Control and 100IU Heparin group not given again). a: apigenin 50 mg/kg; b: apigenin 100 mg/kg; c: apigenin 200 mg/kg, d: vitexin 50 mg/kg; e: vitexin 100 mg/kg; f: vitexin 200 mg/kg;. Data represent two animals for each group.

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