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Evaluation antithrombotic activity and action mechanism of myricitrin
Industrial Crops & Products 129 (2019) 536–541
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Evaluation antithrombotic activity and action mechanism of myricitrin Nan He
a,b,1
, Pengyu Wang
a,b,1
a
d
, Yingying Niu , Jinqiang Chen , Changqin Li
a,b,⁎
, Wen-yi Kang
a,b,c,⁎
T
a
Joint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Kaifeng 475004, China National Center for Research and Development of Edible Fungus Processing Technology, Henan University, Kaifeng 475004, China Kaifeng Key Laboratory of Functional Components in Health Food, Kaifeng 475004, China d Exonano RNA LLC, Rev1 Ventures, 1275 Kinnear Road, Columbus, Ohio, 43212, USA b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Myricitrin Antithrombosis In vivo
Myricitrin was isolated from Cercis chinensis leaves and its antithrombotic activity was evaluated by activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB) in vitro and rat acute blood stasis model in vivo. APTT, PT and TT were significantly prolonged and plasma FIB was significantly shortened in myricitrin group compared with model group in vitro. In rat acute blood stasis model, myricitrin (5 mg/mL and 2.5 mg/mL) could extend APTT, PT and TT, increase the level of 6-keto prostaglandin F1α (6-keto-PGF1α) and nitric oxide synthase (eNOS), decrease the level of plasma FIB, thromboxane B2 (TXB2), endothelin-1 (ET-1), blood sedimentation (ESR), hematocrit (PCV), whole blood viscosity (WBV) and plasma viscosity (PV). All of above revealed that myricitrin had good antithrombotic effects.
1. Introduction Thrombotic disease, which causes high risk of morbidity and mortality, is a serious threat to human health and is gradually increasing in recent years worldwide (Chen et al., 2017; Song et al., 2017). Thrombosis disease is caused by both thrombosis and thromboembolism. There are three types of vascular thrombosis: arterial thromboembolism, venous thromboembolism and microvascular thromboembolism (Pang and Wang, 2011). Many clinical events are caused by thrombotic disease, such as acute myocardial infarction, ischemic stroke, pulmonary embolism and disseminated intravascular coagulation (Li, 2006). The treatments of thrombotic diseases include antithrombotic treatment, thrombolysis, interventional therapy, surgery, antiplatelet and anticoagulant drugs, used alone or in combination, are a cornerstone of clinical treatment for human diseases associated with increased thrombotic risks (Metha et al., 2009; Metharom et al., 2015). However, the lower cure rate and side effects such as higher bleeding risk and gastrointestinal dysfunctions have puzzled researchers (Barrett et al., 2008). Therefore, it is still a difficult and important task to find new antithrombotic drugs, and much more researches focus on natural active ingredient due to its significant effect and lower side effects (Chen
et al., 2015), and had found some natural products with effective antithrombotic (Wang et al., 2017; Gao et al., 2017; Park et al., 2016; Pomin, 2012; Xie et al., 2017). In order to find active components that have antithrombotic effect from natural products, our group isolated and identified active components from leaves of Cercis chinensis. C. chinensis, belonging to family Leguminosae, is distributed to southeast of China and its compounds are mainly flavonoids (Flora of China, 1988; Zhang et al., 2014). Bark and wood of C. chinensis have been used for promoting blood circulation, clearing heat and detoxicating, relieving swelling and pain. The flower of C. chinensis can be used to treat rheumatism. The fruit of C. chinensis can be used to treat cough (Jiangsu New Medical College, 1997). Compounds were isolated from leaves of C. chinensis by our group. Myricitrin was one of the highly abundant compounds isolated from leaves of C. chinensis. There have been many reports about the pharmacology of myricitrin, such as anti-inflammatory, anti-tumor, antihyperlipidemic, antibacterial activity (Wu et al., 2016; Chen et al., 2016; Feng, 2016; Xu and Zhang, 2013; Nostro et al., 2016; He et al., 2016; Innocenti et al., 2010). However, no researches on antithrombotic activity of myricitrin have been reported. This study aimed to investigate the antithrombotic activity and acting mechanism of myricitrin.
Abbreviations: APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time; FIB, plasma fibrinogen; 6-keto-PGF1α, 6-keto prostaglandin F1α; eNOS, nitric oxide synthase; TXB2, thromboxane B2; ET-1, endothelin-1; ESR, blood sedimentation; PCV, hematocrit; WBV, whole blood viscosity; PV, plasma viscosity; TXA2, thromboxane A2; AA, arachidonic acid; TP, TXA2 receptor; PGI2, prostacyclin ⁎ Corresponding authors at: Joint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Kaifeng 475004, China. E-mail addresses: [email protected] (N. He), [email protected] (P. Wang), [email protected] (Y. Niu), [email protected] (J. Chen), [email protected] (C. Li), [email protected] (W.-y. Kang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.indcrop.2018.12.036 Received 14 October 2018; Received in revised form 24 November 2018; Accepted 10 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
3.2. Coagulation time test in vitro (Wang et al., 2017)
2.1. Materials
Blood sample (3.6 mL) was drawn from auricular vein of the male New Zealand white rabbits and placed in a centrifuge tube containing 40 μL of 0.109 mol/L sodium citrate, mixed lightly, and centrifuged at 3000 rpm for 15 min and obtained plasma. Different concentration samples of myricitrin (0.70, 0.35, 0.18, 0.09 and 0.04 mg/mL) were tested.
The leaves of Cercis chinensis were collected in Aug, 2016 in the KaiFeng (Henan, China) and was identified by Professor Changqin Li of Henan University (Kaifeng, Henan, China). A voucher specimen was deposited in the Institute of Chinese Materia Medica, Henan University, KaiFeng, Henan, China. Activated Partial Thrimboplastin Time Reagent (1121781), Thrombin Time Reagent (121119), Prothrombin time Reagent (105262), Fibrinogen Assay Reagent (132079) were purchased from Shanghai Sunbio limited company. 6-Keto-PGF1α ELISA Kit (201707), TXB2 ELISA Kit (201707), eNOS ELISA Kit (201707) and ET-1 ELISA Kit (201707) were purchased from NanJing Jiancheng Bioengineering Institute.
3.2.1. APTT assay In briefly, sample of myricitrin (25 μL), plasma (100 μL) and APTT reagents (100 μL) were added successively and incubated at 37℃ for 5 min, and followed by adding 25 mM CaCl2 (100 μL). The clotting time was taken as the value of APTT. Solvent without myricitrin (DMSO: Tween 80: physiological saline in the ratio of 5%: 3%: 92%) was used as control group. Myricitrin milt in above-mentioned solvent was used as medicated group. Breviscapine was used as positive group.
2.2. Animals 3.2.2. PT assay Sample of myricitrin (25 μL) was mixed with 100 μL of plasma and incubated at 37℃ for 3 min, followed by adding 200 μL of PT reagent that was pre-incubated at 37℃. The clotting time was recorded.
Male and female Sprague–Dawley (SD) rats (6–8 weeks, 200–250 g) and male New Zealand white rabbits (6 months, 2.0–2.5 kg) were obtained from the Experimental Animal Center of Henan Province (Zhengzhou, Henan, China). The animals were maintained in a 12 h light/12 h dark cycle, at 25 °C and 45–65% humidity, and fed with standard rodent diet and water adlibitum. All the animal procedures were approved by the Ethical Committee in accordance with "Institute ethical committee guidelines" for Animal Experimentation and Care. Animals were housed in standard cages. The experiment was carried out according to the guidelines of the National Institutes of Health for Care and Use of Laboratory Animals and was approved by the Bioethics Committee of Henan University.
3.2.3. TT assay Sample of myricitrin (50 μL) was mixed with 200 μL of plasma and incubated for 3 min at 37℃, then 200 μL of reagent of TT was added and the clotting time was recorded. 3.2.4. FIB assay Plasma (200 μL) was mixed with sample of myricitrin (100 μL), then buffer (700 μL) was added to obtain mixed solution. The mixed solution (200 μL) was taken and incubated at 37℃ for 3 min after blending, then 100 μL of all the enzyme solution was added and the content of FIB was recorded. PT, APTT, TT, and FIB assays were conducted by Semiautomated Coagulation Analyzer.
3. Experimental 3.1. Extraction and isolation Dried and powered leaf (4.5 kg) of C. chinensis was extracted by 70% ethanol under 50℃ three times for 9 h totally. Then, solution was concentrated under reduced pressure to yield total extract. After total extract subjected to D101 macroporous resin column with gradient ethanol, 4 fractions (20%, 40%, 60% and 95% ethanol) were obtained. 20% fraction was subjected to silica gel column and eluted with CH2Cl2MeOH (30:1-3:1) gradient to obtain raw myricitrin and separated through Sephadex LH-20 column with methanol to obtain myricitrin. Myricitrin, EI-MS m/z: 464.38[M]+, 1H-NMR (DMSO-d6, 400 MHz) δ: 12.68 (1H, s), 6.88 (2H, s, H-2′, H-6′), 6.37 (1H, d, J = 2.4 Hz, H-8), 6.20 (1H, d, J = 2.4 Hz, H-6), 5.19 (1H, brs, J = 1.2, H-1′'), 0.84 (3H, d, J = 6.2 Hz, H-6′'); 13C-NMR (DMSO-d6,100 MHz) δ: 177.79 (C-4), 164.27 (C-7), 161.33 (C-5), 157.51 (C-2), 155.44 (C-9), 145.80 (C-3′, C5′), 135.49 (C-4′), 134.29 (C-3), 119.61(C-1′), 107.91 (C-2′, C-6′), 104.04 (C-10), 101.95 (C-1′'), 98.71 (C-6), 93.56 (C-8), 71.29 (C-4′'), 70.59 (C-2′'), 70.40 (C-3′'), 70.04 (C-5”), 17.57 (C-6′') (Zhang et al., 2006) (Fig. 1).
3.3. Assays of the antithrombotic effect of myricitrin in vivo Rats were randomly divided into 6 groups as follows: blank control group, the model group (rats in the group were built in acute blood stasis model), the positive control group (XiangDan injection, which was a traditional Chinese medicine injection used for treating thrombotic diseases), high dose myricitrin (10 mg/kg), moderate dose (5 mg/ kg) and low dose group (2.5 mg/kg). Each group included 8 rats. Rats in blank control and model group were given corresponding blank solvent (DMSO: Tween 80: physiological saline in the ratio of 5%: 3%: 92%), rats in the positive control group were given XiangDan injection (3.6 mL/kg) and experimental groups were given blank solvent with corresponding drugs, respectively. Once one day for 7 days totally. Acute blood stasis model (Li et al., 2011) was built 6 days later as follows: Rats were putted to ice-water (0∼2℃) for 5 min. Adrenaline hydrochloride (0. 8 mg/kg) was injected 2 h later to build acute blood stasis rat model. The second injection of Adrenaline hydrochloride was administered 4 h later and the rats began to fast with free access to water with administration was continued. Myricitrin was injected 16 h later after the second injection of adrenaline hydrochloride. Rats were injected with 10% chloral hydrate anesthesia (300 mg/kg) and the blood samples were taken from the abdominal aorta 30 min later. 1 mL of blood was kept in tube at room temperature for 20 min, then serums was collected after being centrifuged at 3000 r/min for 20 min. The eNOS, ET, 6-Keto-PGF1α, TXB2 in supernatant were measured. Blood sample (2 mL) was used for measuring WBV and PV. A part of blood sample (3 mL) was collected in the vacuum tube with sodium citrate, followed by centrifuging at
Fig. 1. The structure of myricitrin. 537
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Fig. 2. Anticoagulation activity of different concentrations of myricetrin. Data represent mean ± SD, n = 4. Compare with control group: ***P < 0.001, **P < 0.01, *P < 0.05; Compare with Breviscapine group: ### P < 0.001, ##P < 0.01.
Fig. 3. Effect of myricitrinon plasma coagulation parameters in vivo test. Data represent the mean ± SD, n = 8. Compared with control group: *** P < 0.001, or, **P < 0.01; Compared with model group: ### P < 0.001; Compared with Xiangdan injection: &&&P < 0.001.
3000 r/min for 15 min to get plasma. APTT, PT, TT and FIB in plasma were measured. Other blood (2 mL) was used to measure ESR and PCV.
were not as good as those in Breviscapine group.
3.4. Statistical analysis
4.2. Effects on plasma coagulation parameters in vivo
All the experimental results were expressed as mean ± standard deviation (SD). Statistical analysis was performed with the SPSS19.0. Comparison between any two groups was evaluated using one-way analysis of variance (ANOVA).
In Fig. 3, APTT, PT, and TT of model group were significantly shortened (P < 0.01, P < 0.001, P < 0.001, respectively) and the level of FIB was significantly elevated (P < 0.01) compared with those of control group, which suggested that acute blood stasis model was successfully established. Xiangdan injection group, myricitrin (10 mg/ kg) and myricitrin (5 mg/kg) could significantly prolong APTT, PT, TT (P < 0.001) and elevate the level of FIB (P < 0.001) compared with model group. However, effects of myricitrin (10 mg/kg) and myricitrin (5 mg/kg) on Plasma Coagulation Parameters were not as good as those of the Xiangdan injection group in vivo.
4. Results 4.1. Effects on plasma coagulation parameters in vitro In Fig. 2, all the concentrations of myricitrin significantly (P < 0.001) elevated the level of FIB, suggesting an extremely significant anticoagulant activity compared with the control group. All of treatments but myricitrin (0.7 mg/mL) significantly prolonged APTT, PT and TT (P < 0.001). No significant differences in APTT assay were found between myricitrin (0.09 mg/mL) and control group. However, the effect of all the experimental groups in APTT, PT, TT, FIB assay
4.3. Effect on TXB2 and 6-Keto-PGF1α In Fig. 4, the level of TXB2 was significantly increased (P < 0.001) whereas that of 6-Keto-PGF1α was significantly reduced (P < 0.001) 538
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Fig. 5. Effects of myricitrinon ET-1 and eNOS. Data represent mean ± SD, n = 8. Compared with control group: **P < 0.01; Compared with model group: ###P < 0.001; Compare with positive group: △ P < 0.05.
Fig. 4. Effects of myricitrinon TXB2 and 6-Keto-PGF1α. Data represent mean ± SD, n = 8. Compared with control group: ***P < 0.001; Compared with model group: ###P < 0.001.
compared with control group. The rate of TXB2/6-Keto-PGF1α was significantly elevated, which indicated that acute blood stasis model was successfully established. Compared with model group, myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) significantly reduced the level of TXB2 (P < 0.001) and the rate of TXB2/6-Keto-PGF1α (P < 0.001) whereas increased the level of 6-Keto-PGF1α (P < 0.001). There were no differences in TXB2 and 6-Keto-PGF1α assay between myricertrin (10 mg/kg) and model groups. In addition, the effects of myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) on TXB2 and 6-Keto-PGF1α were similar to those of Xiangdan injection.
Fig. 6. Effects of myricitrinon ESR and PCV. Data represent mean ± SD, n = 8. Compare with control group: *P < 0.05; Compare with model group: ###P < 0.001, or, ##P < 0.01, or, #P < 0.05.
4.4. Effects on ET-1 and eNOS
4.6. Hemorheology parameters
In Fig. 5, ET-1 was significantly increased (P < 0.001) and eNOS (U/mL) was significantly decreased (P < 0.001) compared with that of control group. Except myricitrin (10 mg/kg) that showed no difference in ET-1 with model group, all the other groups of myricitrin significantly decreased the level of ET-1(P < 0.001) and increased the level of eNOS (P < 0.001) compared with the model group. Besides, myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) could significantly decrease the level of ET-1(P < 0.05) compared with positive group.
In Fig. 7, WBV at all shear rates (P < 0.001) as well as PV (P < 0.001) were significantly increased in model group compared with control group, which suggested that acute blood stasis model was established. Myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) significantly decreased WBV at all shear rates (P < 0.001 and P < 0.01, respectively) and PV (P < 0.05) while myricitrin (10 mg/kg) has no significant effect on WVB at all shares (P > 0.05) compared with model group.
4.5. Effects on ESR and PCV
5. Discussion
In Fig. 6, ESR and PCV of model groups were significantly improved (P < 0.01 and P < 0.05, respectively) compare with control group. There was no significant difference between myricitrin (10 mg/kg) and model group (P > 0.05). Compared with model group, myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) significantly decreased ESR (P < 0.05) and PCV (P < 0.01, P < 0.001, respectively). The effects of myricitrin (5 and 2.5 mg/g) were similar to positive group.
The balance of coagulation system and anticoagulation system plays an important role in maintaining the normal physiological function of body. Under normal circumstances, a combination of coagulation system and anticoagulation system can guarantee hemostasis effectively when the hemorrhage occurred, prevent formatting the thrombus and 539
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hemokinesis. Thromboxane A2 (TXA2) is potent vasoconstriction and platelet aggregation (Sekiya et al., 1991) and is synthesized form arachidonic acid (AA) to provide in exogenous or endogenous phospholipids by the activated platelets. TXA2 is released from the cell and bines with TXA2 receptor (TP) to stimulate platelet deformation and aggregation, causing the contraction of vascular smooth muscle. The halflife of TXA2 is short because it is rapidly metabolized into stable inert TXB2 (Tian et al., 2010). Contrary to TXA2, prostacyclin (PGI2) can effectively inhibit platelet aggregation and blood vessel expansion. PGI2 is produced by endothelial cells and metabolized to 6-Keto-PGF1α speedy after being released by endothelial cells (Liang et al., 2014; Oberti et al., 1993). The dynamic balance of TXA2 and PGI2 in blood plays an important role in maintaining Cardiovascular homeostasis (Li et al., 2001; Dawood and Khan-Dawood, 2007). By detecting the contents of TXB2, 6-Keto-PGF1α and TXB2/6-Keto-PGF1α, we can indirectly estimate the contents of TXA2, PGI2 and ratio of TXA2/PGI2 in blood. Nitrite oxide (NO) is a powerful signal molecule synthesized by the endothelial nitric oxide synthase (eNOS) in endothelial cells and has various biological functions, such as inhibition of regulating blood pressure, platelet aggregation, dilate blood vessels, inhibition of smooth muscle proliferation and prevention of thrombosis (Forstermann and Sessa, 2012; Li and Förstermann, 2000; Li et al., 2002). Dysregulation of NO/eNOS system will cause a variety of cardiovascular diseases such as hypertension, atherosis, and heart failure, etc. (Bruno et al., 2011). The content of NO could be detected indirectly by testing its rate-limiting enzymes eNOS because NO is metabolized rapidly. ET is a polypeptide composed of 23 amino acid residues synthesized by endothelial cells. The function of ET is the opposite of NO, it can strongly inhibit vessel contraction (Li et al., 2014). ET/NO system plays an important role in maintaining the homeostasis of the cardiovascular system (Kawanabe and Nauli, 2011). In our research, myricitrin could significantly down-regulate ET-1 and up-regulate eNOS, indicating that myricitrin is able to maintain the balanced cardiovascular homeostasis and to protect vascular endothelium by regulating ratio of ET/NO. In addition, hemorheology was measured to study the mechanism underlying the action of myricetrin. Hemorheologyis is related to blood flow and pressure, flow volume, and resistance of blood vessels, including WBV, PV, ESR and PCV and used for diagnosis cardiovascular diseases in clinic (Xie et al., 2017; Tian et al., 2017). Results of our research showed that the plasma viscosity and whole blood viscosity of myricitrin (5 mg/kg and 2.5 mg/kg) were significantly down-regulated compared with the model group, indicating that myricitrin can protect cardiovascular system by regulating WBV and PV. As the important factor influencing blood viscosity, packed cell volume (PCV) refers to the volume of red blood cells. It has been shown that PCV is the main factor influencing the plasma viscosity and that there is a positive correlation between PCV and plasma viscosity (Tian et al., 2017). The erythrocyte sedimentation rate (ESR) is the rate of sedimentation of red blood cells under certain conditions. Our research indicated that myricitrin (5 mg/kg and 2.5 mg/kg) could down-regulate PCV and ESR compared with model group, which revealed that myricitrin had an anticoagulant effect by regulating PCV and ESR.
Fig. 7. Effects of myricitrin on WBV and PV. Data represent mean ± SD, n=8. Compared with control group: ***P < 0.001; Compared with model group: ###P < 0.001, or, ##P < 0.01, #P < 0.05.
block blood vessels to maintain the physiological hemokinesis (Lv et al., 2015). However, the damage of vessel walls, the change of hemokinesis and the production of inflammatory factors (Shi and Hu, 2007), all of these aspects could lead to blood clots and serious threat to the health even life of human. To deal with the thrombus, a lot of researches were conducted with focus on anticoagulant drugs, antiplatelet drugs and thrombolytic drugs (Metharom et al., 2015; Tang et al., 2016; Zhang, 2012). There are abound in achievements were made to treat thrombus, but the effect of these therapies on reducing the mortality rates still remained small and the side effects such as bleeding, thrombocytopenia and resistances still confused researchers (Barrett et al., 2008). Study on anti-thrombus was on the way and development of the antithrombotic drugs from natural products is the hotspot nowadays (Chen et al., 2015). Flavonoids derived from fruits, vegetables and other plant have potential antithrombotic value (Wright et al., 2013). In this study, the antithrombotic activity and acting mechanism of myricetrin were investigated. Myricetrin was extracted from C. chinensis leaves and reported its extensive bioactivities such as anti-inflammatory, anti-tumor, antihyperlipidemic, antibacterial activity but no antithrombotic activity was reported (Wu et al., 2016; 18. Chen et al., 2016; Feng, 2016; Xu and Zhang, 2013; Nostro et al., 2016; He et al., 2016; Innocenti et al., 2010). The results of Plasma Coagulation Parameters in vitro and in vivo indicated that myricitrinon had antithrombotic effect of intrinsic and extrinsic coagulation pathways, and hindering fibrin formation in vivo. Plasma coagulation process is a series of plasma coagulation factors in successive digestion process of activation, which generally can be divided into endogenous clotting pathway, extrinsic coagulation pathway and common pathway of coagulation (Macfarlane, 1964). APTT, PT, TT and FIB are the main indexes to reflect the status of the blood clotting system in clinical setting (Zhang et al., 2013). As one of most importance indexes, PT is sensitive and commonly used to test and reflect the activities of plasma coagulation factors such as coagulation factors Ⅰ, Ⅱ, Ⅴ, Ⅷ and Ⅹ for extrinsic blood coagulation systems. APTT is sensitive and commonly used to reflect level of coagulation factors Ⅷ, Ⅸ, Ⅺ and Ⅻ for the intrinsic clotting system (Yang et al., 2007; Yoo et al., 2009; Wang, 2009; Zhang et al., 2017). TT was mainly used to indicate the activity of fibrinogen/fibrin and if there is coagulating substances or not. The FIB level is one of the important risk factors of thrombosis and cardiovascular diseases and FIB is synthesized by the liver and can be hydrolyzed by the clotting enzyme to form peptide A and peptide B, eventually form the insoluble fibrin to stop bleeding (Zhang et al., 2013). TXA2/PGI2 system was regulated by myricitrin to maintain
6. Conclusion The flavonoids derived from fruits, vegetables and other plant have potential value of antithrombotic. In this paper, the antithrombotic activity of myricetrin isolated from C. chinensis leaves was assayed and the mechanism was investigated in vivo. The results showed that myricitrin is capable of prolonging APTT, PT and TT, increasing the levels of 6-Keto-PGF1α, eNOS and reducing FIB, TXB2, ET - 1, ESR, PCV, WBV and PV to achieve its relatively strong antithrombotic effect. Ethic approval and consent to participate The study obtained ethical clearance from the Ethics Committee of 540
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College of Medical, Henan University (NO: 2016-36). The rabbits and rats were treated as per the guidelines on the care and use of animals for scientific purposes.
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Consent for publication Not applicable. Availability of data and materials All the materials are described within the manuscript. Moreover, the most relevant data are contained within the manuscript too. Competing interests The authors declare that there is no conflict of interests regarding the publication of this paper. Funding This work was supported by Henan Province University Science and Technology Innovation Team (16IRTSTHN019). Author contributions Nan He, Pengyu Wang, Yingying Niu and Wen-yi Kang conceived and designed the experiments. Nan He, Pengyu Wang and Yingying Niu performed the experiments. Nan He and Yingying Niu made substantial contributions to interpretation of data. Nan He and Changqin Li wrote the first draft of the manuscript. Wenyi Kang and Jinqiang Chen revised the draft and approved the version submitted. Acknowledgements We would like to thank all participants in the study. References Barrett, N.E., Stanley, R.G., Tucker, K.L., Wright, B., Gibbins, J.M., et al., 2008. Future innovations in anti-platelet therapies. Br. J. Pharmacol. 154 (5), 918–939 [View Article]. Bruno, R.M., et al., 2011. Interactions between sympathetic nervous system and endogenous endothelin in patients with essential hypertension. Hypertension 57 (1), 79–84 [View Article]. Chen, C., et al., 2015. Natural products for antithrombosis. Evid. Based Complement. Alternat. Med. 3, 1–17 [View Article]. Chen, Y.P., et al., 2016. Protective effects of myricitrin on ischemic/reperfusion injury in isolated rat hearts. China J. Comp. Med. 26 (05), 31–39 [View Article]. Chen, W.W., et al., 2017. Summary of Chinese cardiovascular disease report in 2016. China Circ. J. 32 (6), 521–530 [View Article]. Dawood, M.Y., Khan-Dawood, F.S., 2007. Differential suppression of menstrual fluidprostaglandin F2α, prostaglandin E2, 6-ket oprostaglandin F1αand thromboxane B2 by suprofen in women with primary dysmenorrhea. Prostaglandins. Other Lipid Mediat. 83 (1–2), 146–153 [View Article]. Editorial Commission of China Flora of Chinese Academy of Science, 1988. Flora of China 39. Science Press, Beijing, pp. 144. Feng, Z.Y., 2016. NOX2 Mediated Myricitrin Inhibition on LPS-Induced Inflammatory Response. Master’s Thesis. Wan Nan Medical College [View Article]. Forstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33 (829–837) 837a–837d. [View Article]. Gao, J., et al., 2017. Therapeutic effects of breviscapine in cardiovascular diseases: a review. Front. Pharmacol. 8, 1–3 [View Article]. He, K., et al., 2016. Hypolipidemic effects of Myrica rubra extracts and main compounds in C57BL/6j mice. Food Funct. 7 (8), 3505–3515 [View Article]. Innocenti, G., et al., 2010. Antioxidant activity and redox properties of flavonoids from Limonium narbonense. Plant Med. 76 (12), 1249 [View Article]. Jiangsu New Medical College, 1997. Dictionary of Traditional Chinese Medicine. Shanghai Science and Technology Publishing House, Shanghai, pp. 2364. Kawanabe, Y., Nauli, S.M., 2011. Endothelin. Cell Mol. Life Sci. 68 (2), 195 [View Article].
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Evaluation antithrombotic activity and action mechanism of myricitrin Nan He
a,b,1
, Pengyu Wang
a,b,1
a
d
, Yingying Niu , Jinqiang Chen , Changqin Li
a,b,⁎
, Wen-yi Kang
a,b,c,⁎
T
a
Joint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Kaifeng 475004, China National Center for Research and Development of Edible Fungus Processing Technology, Henan University, Kaifeng 475004, China Kaifeng Key Laboratory of Functional Components in Health Food, Kaifeng 475004, China d Exonano RNA LLC, Rev1 Ventures, 1275 Kinnear Road, Columbus, Ohio, 43212, USA b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Myricitrin Antithrombosis In vivo
Myricitrin was isolated from Cercis chinensis leaves and its antithrombotic activity was evaluated by activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB) in vitro and rat acute blood stasis model in vivo. APTT, PT and TT were significantly prolonged and plasma FIB was significantly shortened in myricitrin group compared with model group in vitro. In rat acute blood stasis model, myricitrin (5 mg/mL and 2.5 mg/mL) could extend APTT, PT and TT, increase the level of 6-keto prostaglandin F1α (6-keto-PGF1α) and nitric oxide synthase (eNOS), decrease the level of plasma FIB, thromboxane B2 (TXB2), endothelin-1 (ET-1), blood sedimentation (ESR), hematocrit (PCV), whole blood viscosity (WBV) and plasma viscosity (PV). All of above revealed that myricitrin had good antithrombotic effects.
1. Introduction Thrombotic disease, which causes high risk of morbidity and mortality, is a serious threat to human health and is gradually increasing in recent years worldwide (Chen et al., 2017; Song et al., 2017). Thrombosis disease is caused by both thrombosis and thromboembolism. There are three types of vascular thrombosis: arterial thromboembolism, venous thromboembolism and microvascular thromboembolism (Pang and Wang, 2011). Many clinical events are caused by thrombotic disease, such as acute myocardial infarction, ischemic stroke, pulmonary embolism and disseminated intravascular coagulation (Li, 2006). The treatments of thrombotic diseases include antithrombotic treatment, thrombolysis, interventional therapy, surgery, antiplatelet and anticoagulant drugs, used alone or in combination, are a cornerstone of clinical treatment for human diseases associated with increased thrombotic risks (Metha et al., 2009; Metharom et al., 2015). However, the lower cure rate and side effects such as higher bleeding risk and gastrointestinal dysfunctions have puzzled researchers (Barrett et al., 2008). Therefore, it is still a difficult and important task to find new antithrombotic drugs, and much more researches focus on natural active ingredient due to its significant effect and lower side effects (Chen
et al., 2015), and had found some natural products with effective antithrombotic (Wang et al., 2017; Gao et al., 2017; Park et al., 2016; Pomin, 2012; Xie et al., 2017). In order to find active components that have antithrombotic effect from natural products, our group isolated and identified active components from leaves of Cercis chinensis. C. chinensis, belonging to family Leguminosae, is distributed to southeast of China and its compounds are mainly flavonoids (Flora of China, 1988; Zhang et al., 2014). Bark and wood of C. chinensis have been used for promoting blood circulation, clearing heat and detoxicating, relieving swelling and pain. The flower of C. chinensis can be used to treat rheumatism. The fruit of C. chinensis can be used to treat cough (Jiangsu New Medical College, 1997). Compounds were isolated from leaves of C. chinensis by our group. Myricitrin was one of the highly abundant compounds isolated from leaves of C. chinensis. There have been many reports about the pharmacology of myricitrin, such as anti-inflammatory, anti-tumor, antihyperlipidemic, antibacterial activity (Wu et al., 2016; Chen et al., 2016; Feng, 2016; Xu and Zhang, 2013; Nostro et al., 2016; He et al., 2016; Innocenti et al., 2010). However, no researches on antithrombotic activity of myricitrin have been reported. This study aimed to investigate the antithrombotic activity and acting mechanism of myricitrin.
Abbreviations: APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time; FIB, plasma fibrinogen; 6-keto-PGF1α, 6-keto prostaglandin F1α; eNOS, nitric oxide synthase; TXB2, thromboxane B2; ET-1, endothelin-1; ESR, blood sedimentation; PCV, hematocrit; WBV, whole blood viscosity; PV, plasma viscosity; TXA2, thromboxane A2; AA, arachidonic acid; TP, TXA2 receptor; PGI2, prostacyclin ⁎ Corresponding authors at: Joint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Kaifeng 475004, China. E-mail addresses: [email protected] (N. He), [email protected] (P. Wang), [email protected] (Y. Niu), [email protected] (J. Chen), [email protected] (C. Li), [email protected] (W.-y. Kang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.indcrop.2018.12.036 Received 14 October 2018; Received in revised form 24 November 2018; Accepted 10 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
3.2. Coagulation time test in vitro (Wang et al., 2017)
2.1. Materials
Blood sample (3.6 mL) was drawn from auricular vein of the male New Zealand white rabbits and placed in a centrifuge tube containing 40 μL of 0.109 mol/L sodium citrate, mixed lightly, and centrifuged at 3000 rpm for 15 min and obtained plasma. Different concentration samples of myricitrin (0.70, 0.35, 0.18, 0.09 and 0.04 mg/mL) were tested.
The leaves of Cercis chinensis were collected in Aug, 2016 in the KaiFeng (Henan, China) and was identified by Professor Changqin Li of Henan University (Kaifeng, Henan, China). A voucher specimen was deposited in the Institute of Chinese Materia Medica, Henan University, KaiFeng, Henan, China. Activated Partial Thrimboplastin Time Reagent (1121781), Thrombin Time Reagent (121119), Prothrombin time Reagent (105262), Fibrinogen Assay Reagent (132079) were purchased from Shanghai Sunbio limited company. 6-Keto-PGF1α ELISA Kit (201707), TXB2 ELISA Kit (201707), eNOS ELISA Kit (201707) and ET-1 ELISA Kit (201707) were purchased from NanJing Jiancheng Bioengineering Institute.
3.2.1. APTT assay In briefly, sample of myricitrin (25 μL), plasma (100 μL) and APTT reagents (100 μL) were added successively and incubated at 37℃ for 5 min, and followed by adding 25 mM CaCl2 (100 μL). The clotting time was taken as the value of APTT. Solvent without myricitrin (DMSO: Tween 80: physiological saline in the ratio of 5%: 3%: 92%) was used as control group. Myricitrin milt in above-mentioned solvent was used as medicated group. Breviscapine was used as positive group.
2.2. Animals 3.2.2. PT assay Sample of myricitrin (25 μL) was mixed with 100 μL of plasma and incubated at 37℃ for 3 min, followed by adding 200 μL of PT reagent that was pre-incubated at 37℃. The clotting time was recorded.
Male and female Sprague–Dawley (SD) rats (6–8 weeks, 200–250 g) and male New Zealand white rabbits (6 months, 2.0–2.5 kg) were obtained from the Experimental Animal Center of Henan Province (Zhengzhou, Henan, China). The animals were maintained in a 12 h light/12 h dark cycle, at 25 °C and 45–65% humidity, and fed with standard rodent diet and water adlibitum. All the animal procedures were approved by the Ethical Committee in accordance with "Institute ethical committee guidelines" for Animal Experimentation and Care. Animals were housed in standard cages. The experiment was carried out according to the guidelines of the National Institutes of Health for Care and Use of Laboratory Animals and was approved by the Bioethics Committee of Henan University.
3.2.3. TT assay Sample of myricitrin (50 μL) was mixed with 200 μL of plasma and incubated for 3 min at 37℃, then 200 μL of reagent of TT was added and the clotting time was recorded. 3.2.4. FIB assay Plasma (200 μL) was mixed with sample of myricitrin (100 μL), then buffer (700 μL) was added to obtain mixed solution. The mixed solution (200 μL) was taken and incubated at 37℃ for 3 min after blending, then 100 μL of all the enzyme solution was added and the content of FIB was recorded. PT, APTT, TT, and FIB assays were conducted by Semiautomated Coagulation Analyzer.
3. Experimental 3.1. Extraction and isolation Dried and powered leaf (4.5 kg) of C. chinensis was extracted by 70% ethanol under 50℃ three times for 9 h totally. Then, solution was concentrated under reduced pressure to yield total extract. After total extract subjected to D101 macroporous resin column with gradient ethanol, 4 fractions (20%, 40%, 60% and 95% ethanol) were obtained. 20% fraction was subjected to silica gel column and eluted with CH2Cl2MeOH (30:1-3:1) gradient to obtain raw myricitrin and separated through Sephadex LH-20 column with methanol to obtain myricitrin. Myricitrin, EI-MS m/z: 464.38[M]+, 1H-NMR (DMSO-d6, 400 MHz) δ: 12.68 (1H, s), 6.88 (2H, s, H-2′, H-6′), 6.37 (1H, d, J = 2.4 Hz, H-8), 6.20 (1H, d, J = 2.4 Hz, H-6), 5.19 (1H, brs, J = 1.2, H-1′'), 0.84 (3H, d, J = 6.2 Hz, H-6′'); 13C-NMR (DMSO-d6,100 MHz) δ: 177.79 (C-4), 164.27 (C-7), 161.33 (C-5), 157.51 (C-2), 155.44 (C-9), 145.80 (C-3′, C5′), 135.49 (C-4′), 134.29 (C-3), 119.61(C-1′), 107.91 (C-2′, C-6′), 104.04 (C-10), 101.95 (C-1′'), 98.71 (C-6), 93.56 (C-8), 71.29 (C-4′'), 70.59 (C-2′'), 70.40 (C-3′'), 70.04 (C-5”), 17.57 (C-6′') (Zhang et al., 2006) (Fig. 1).
3.3. Assays of the antithrombotic effect of myricitrin in vivo Rats were randomly divided into 6 groups as follows: blank control group, the model group (rats in the group were built in acute blood stasis model), the positive control group (XiangDan injection, which was a traditional Chinese medicine injection used for treating thrombotic diseases), high dose myricitrin (10 mg/kg), moderate dose (5 mg/ kg) and low dose group (2.5 mg/kg). Each group included 8 rats. Rats in blank control and model group were given corresponding blank solvent (DMSO: Tween 80: physiological saline in the ratio of 5%: 3%: 92%), rats in the positive control group were given XiangDan injection (3.6 mL/kg) and experimental groups were given blank solvent with corresponding drugs, respectively. Once one day for 7 days totally. Acute blood stasis model (Li et al., 2011) was built 6 days later as follows: Rats were putted to ice-water (0∼2℃) for 5 min. Adrenaline hydrochloride (0. 8 mg/kg) was injected 2 h later to build acute blood stasis rat model. The second injection of Adrenaline hydrochloride was administered 4 h later and the rats began to fast with free access to water with administration was continued. Myricitrin was injected 16 h later after the second injection of adrenaline hydrochloride. Rats were injected with 10% chloral hydrate anesthesia (300 mg/kg) and the blood samples were taken from the abdominal aorta 30 min later. 1 mL of blood was kept in tube at room temperature for 20 min, then serums was collected after being centrifuged at 3000 r/min for 20 min. The eNOS, ET, 6-Keto-PGF1α, TXB2 in supernatant were measured. Blood sample (2 mL) was used for measuring WBV and PV. A part of blood sample (3 mL) was collected in the vacuum tube with sodium citrate, followed by centrifuging at
Fig. 1. The structure of myricitrin. 537
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Fig. 2. Anticoagulation activity of different concentrations of myricetrin. Data represent mean ± SD, n = 4. Compare with control group: ***P < 0.001, **P < 0.01, *P < 0.05; Compare with Breviscapine group: ### P < 0.001, ##P < 0.01.
Fig. 3. Effect of myricitrinon plasma coagulation parameters in vivo test. Data represent the mean ± SD, n = 8. Compared with control group: *** P < 0.001, or, **P < 0.01; Compared with model group: ### P < 0.001; Compared with Xiangdan injection: &&&P < 0.001.
3000 r/min for 15 min to get plasma. APTT, PT, TT and FIB in plasma were measured. Other blood (2 mL) was used to measure ESR and PCV.
were not as good as those in Breviscapine group.
3.4. Statistical analysis
4.2. Effects on plasma coagulation parameters in vivo
All the experimental results were expressed as mean ± standard deviation (SD). Statistical analysis was performed with the SPSS19.0. Comparison between any two groups was evaluated using one-way analysis of variance (ANOVA).
In Fig. 3, APTT, PT, and TT of model group were significantly shortened (P < 0.01, P < 0.001, P < 0.001, respectively) and the level of FIB was significantly elevated (P < 0.01) compared with those of control group, which suggested that acute blood stasis model was successfully established. Xiangdan injection group, myricitrin (10 mg/ kg) and myricitrin (5 mg/kg) could significantly prolong APTT, PT, TT (P < 0.001) and elevate the level of FIB (P < 0.001) compared with model group. However, effects of myricitrin (10 mg/kg) and myricitrin (5 mg/kg) on Plasma Coagulation Parameters were not as good as those of the Xiangdan injection group in vivo.
4. Results 4.1. Effects on plasma coagulation parameters in vitro In Fig. 2, all the concentrations of myricitrin significantly (P < 0.001) elevated the level of FIB, suggesting an extremely significant anticoagulant activity compared with the control group. All of treatments but myricitrin (0.7 mg/mL) significantly prolonged APTT, PT and TT (P < 0.001). No significant differences in APTT assay were found between myricitrin (0.09 mg/mL) and control group. However, the effect of all the experimental groups in APTT, PT, TT, FIB assay
4.3. Effect on TXB2 and 6-Keto-PGF1α In Fig. 4, the level of TXB2 was significantly increased (P < 0.001) whereas that of 6-Keto-PGF1α was significantly reduced (P < 0.001) 538
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Fig. 5. Effects of myricitrinon ET-1 and eNOS. Data represent mean ± SD, n = 8. Compared with control group: **P < 0.01; Compared with model group: ###P < 0.001; Compare with positive group: △ P < 0.05.
Fig. 4. Effects of myricitrinon TXB2 and 6-Keto-PGF1α. Data represent mean ± SD, n = 8. Compared with control group: ***P < 0.001; Compared with model group: ###P < 0.001.
compared with control group. The rate of TXB2/6-Keto-PGF1α was significantly elevated, which indicated that acute blood stasis model was successfully established. Compared with model group, myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) significantly reduced the level of TXB2 (P < 0.001) and the rate of TXB2/6-Keto-PGF1α (P < 0.001) whereas increased the level of 6-Keto-PGF1α (P < 0.001). There were no differences in TXB2 and 6-Keto-PGF1α assay between myricertrin (10 mg/kg) and model groups. In addition, the effects of myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) on TXB2 and 6-Keto-PGF1α were similar to those of Xiangdan injection.
Fig. 6. Effects of myricitrinon ESR and PCV. Data represent mean ± SD, n = 8. Compare with control group: *P < 0.05; Compare with model group: ###P < 0.001, or, ##P < 0.01, or, #P < 0.05.
4.4. Effects on ET-1 and eNOS
4.6. Hemorheology parameters
In Fig. 5, ET-1 was significantly increased (P < 0.001) and eNOS (U/mL) was significantly decreased (P < 0.001) compared with that of control group. Except myricitrin (10 mg/kg) that showed no difference in ET-1 with model group, all the other groups of myricitrin significantly decreased the level of ET-1(P < 0.001) and increased the level of eNOS (P < 0.001) compared with the model group. Besides, myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) could significantly decrease the level of ET-1(P < 0.05) compared with positive group.
In Fig. 7, WBV at all shear rates (P < 0.001) as well as PV (P < 0.001) were significantly increased in model group compared with control group, which suggested that acute blood stasis model was established. Myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) significantly decreased WBV at all shear rates (P < 0.001 and P < 0.01, respectively) and PV (P < 0.05) while myricitrin (10 mg/kg) has no significant effect on WVB at all shares (P > 0.05) compared with model group.
4.5. Effects on ESR and PCV
5. Discussion
In Fig. 6, ESR and PCV of model groups were significantly improved (P < 0.01 and P < 0.05, respectively) compare with control group. There was no significant difference between myricitrin (10 mg/kg) and model group (P > 0.05). Compared with model group, myricitrin (5 mg/kg) and myricitrin (2.5 mg/kg) significantly decreased ESR (P < 0.05) and PCV (P < 0.01, P < 0.001, respectively). The effects of myricitrin (5 and 2.5 mg/g) were similar to positive group.
The balance of coagulation system and anticoagulation system plays an important role in maintaining the normal physiological function of body. Under normal circumstances, a combination of coagulation system and anticoagulation system can guarantee hemostasis effectively when the hemorrhage occurred, prevent formatting the thrombus and 539
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hemokinesis. Thromboxane A2 (TXA2) is potent vasoconstriction and platelet aggregation (Sekiya et al., 1991) and is synthesized form arachidonic acid (AA) to provide in exogenous or endogenous phospholipids by the activated platelets. TXA2 is released from the cell and bines with TXA2 receptor (TP) to stimulate platelet deformation and aggregation, causing the contraction of vascular smooth muscle. The halflife of TXA2 is short because it is rapidly metabolized into stable inert TXB2 (Tian et al., 2010). Contrary to TXA2, prostacyclin (PGI2) can effectively inhibit platelet aggregation and blood vessel expansion. PGI2 is produced by endothelial cells and metabolized to 6-Keto-PGF1α speedy after being released by endothelial cells (Liang et al., 2014; Oberti et al., 1993). The dynamic balance of TXA2 and PGI2 in blood plays an important role in maintaining Cardiovascular homeostasis (Li et al., 2001; Dawood and Khan-Dawood, 2007). By detecting the contents of TXB2, 6-Keto-PGF1α and TXB2/6-Keto-PGF1α, we can indirectly estimate the contents of TXA2, PGI2 and ratio of TXA2/PGI2 in blood. Nitrite oxide (NO) is a powerful signal molecule synthesized by the endothelial nitric oxide synthase (eNOS) in endothelial cells and has various biological functions, such as inhibition of regulating blood pressure, platelet aggregation, dilate blood vessels, inhibition of smooth muscle proliferation and prevention of thrombosis (Forstermann and Sessa, 2012; Li and Förstermann, 2000; Li et al., 2002). Dysregulation of NO/eNOS system will cause a variety of cardiovascular diseases such as hypertension, atherosis, and heart failure, etc. (Bruno et al., 2011). The content of NO could be detected indirectly by testing its rate-limiting enzymes eNOS because NO is metabolized rapidly. ET is a polypeptide composed of 23 amino acid residues synthesized by endothelial cells. The function of ET is the opposite of NO, it can strongly inhibit vessel contraction (Li et al., 2014). ET/NO system plays an important role in maintaining the homeostasis of the cardiovascular system (Kawanabe and Nauli, 2011). In our research, myricitrin could significantly down-regulate ET-1 and up-regulate eNOS, indicating that myricitrin is able to maintain the balanced cardiovascular homeostasis and to protect vascular endothelium by regulating ratio of ET/NO. In addition, hemorheology was measured to study the mechanism underlying the action of myricetrin. Hemorheologyis is related to blood flow and pressure, flow volume, and resistance of blood vessels, including WBV, PV, ESR and PCV and used for diagnosis cardiovascular diseases in clinic (Xie et al., 2017; Tian et al., 2017). Results of our research showed that the plasma viscosity and whole blood viscosity of myricitrin (5 mg/kg and 2.5 mg/kg) were significantly down-regulated compared with the model group, indicating that myricitrin can protect cardiovascular system by regulating WBV and PV. As the important factor influencing blood viscosity, packed cell volume (PCV) refers to the volume of red blood cells. It has been shown that PCV is the main factor influencing the plasma viscosity and that there is a positive correlation between PCV and plasma viscosity (Tian et al., 2017). The erythrocyte sedimentation rate (ESR) is the rate of sedimentation of red blood cells under certain conditions. Our research indicated that myricitrin (5 mg/kg and 2.5 mg/kg) could down-regulate PCV and ESR compared with model group, which revealed that myricitrin had an anticoagulant effect by regulating PCV and ESR.
Fig. 7. Effects of myricitrin on WBV and PV. Data represent mean ± SD, n=8. Compared with control group: ***P < 0.001; Compared with model group: ###P < 0.001, or, ##P < 0.01, #P < 0.05.
block blood vessels to maintain the physiological hemokinesis (Lv et al., 2015). However, the damage of vessel walls, the change of hemokinesis and the production of inflammatory factors (Shi and Hu, 2007), all of these aspects could lead to blood clots and serious threat to the health even life of human. To deal with the thrombus, a lot of researches were conducted with focus on anticoagulant drugs, antiplatelet drugs and thrombolytic drugs (Metharom et al., 2015; Tang et al., 2016; Zhang, 2012). There are abound in achievements were made to treat thrombus, but the effect of these therapies on reducing the mortality rates still remained small and the side effects such as bleeding, thrombocytopenia and resistances still confused researchers (Barrett et al., 2008). Study on anti-thrombus was on the way and development of the antithrombotic drugs from natural products is the hotspot nowadays (Chen et al., 2015). Flavonoids derived from fruits, vegetables and other plant have potential antithrombotic value (Wright et al., 2013). In this study, the antithrombotic activity and acting mechanism of myricetrin were investigated. Myricetrin was extracted from C. chinensis leaves and reported its extensive bioactivities such as anti-inflammatory, anti-tumor, antihyperlipidemic, antibacterial activity but no antithrombotic activity was reported (Wu et al., 2016; 18. Chen et al., 2016; Feng, 2016; Xu and Zhang, 2013; Nostro et al., 2016; He et al., 2016; Innocenti et al., 2010). The results of Plasma Coagulation Parameters in vitro and in vivo indicated that myricitrinon had antithrombotic effect of intrinsic and extrinsic coagulation pathways, and hindering fibrin formation in vivo. Plasma coagulation process is a series of plasma coagulation factors in successive digestion process of activation, which generally can be divided into endogenous clotting pathway, extrinsic coagulation pathway and common pathway of coagulation (Macfarlane, 1964). APTT, PT, TT and FIB are the main indexes to reflect the status of the blood clotting system in clinical setting (Zhang et al., 2013). As one of most importance indexes, PT is sensitive and commonly used to test and reflect the activities of plasma coagulation factors such as coagulation factors Ⅰ, Ⅱ, Ⅴ, Ⅷ and Ⅹ for extrinsic blood coagulation systems. APTT is sensitive and commonly used to reflect level of coagulation factors Ⅷ, Ⅸ, Ⅺ and Ⅻ for the intrinsic clotting system (Yang et al., 2007; Yoo et al., 2009; Wang, 2009; Zhang et al., 2017). TT was mainly used to indicate the activity of fibrinogen/fibrin and if there is coagulating substances or not. The FIB level is one of the important risk factors of thrombosis and cardiovascular diseases and FIB is synthesized by the liver and can be hydrolyzed by the clotting enzyme to form peptide A and peptide B, eventually form the insoluble fibrin to stop bleeding (Zhang et al., 2013). TXA2/PGI2 system was regulated by myricitrin to maintain
6. Conclusion The flavonoids derived from fruits, vegetables and other plant have potential value of antithrombotic. In this paper, the antithrombotic activity of myricetrin isolated from C. chinensis leaves was assayed and the mechanism was investigated in vivo. The results showed that myricitrin is capable of prolonging APTT, PT and TT, increasing the levels of 6-Keto-PGF1α, eNOS and reducing FIB, TXB2, ET - 1, ESR, PCV, WBV and PV to achieve its relatively strong antithrombotic effect. Ethic approval and consent to participate The study obtained ethical clearance from the Ethics Committee of 540
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College of Medical, Henan University (NO: 2016-36). The rabbits and rats were treated as per the guidelines on the care and use of animals for scientific purposes.
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Consent for publication Not applicable. Availability of data and materials All the materials are described within the manuscript. Moreover, the most relevant data are contained within the manuscript too. Competing interests The authors declare that there is no conflict of interests regarding the publication of this paper. Funding This work was supported by Henan Province University Science and Technology Innovation Team (16IRTSTHN019). Author contributions Nan He, Pengyu Wang, Yingying Niu and Wen-yi Kang conceived and designed the experiments. Nan He, Pengyu Wang and Yingying Niu performed the experiments. Nan He and Yingying Niu made substantial contributions to interpretation of data. Nan He and Changqin Li wrote the first draft of the manuscript. Wenyi Kang and Jinqiang Chen revised the draft and approved the version submitted. Acknowledgements We would like to thank all participants in the study. References Barrett, N.E., Stanley, R.G., Tucker, K.L., Wright, B., Gibbins, J.M., et al., 2008. Future innovations in anti-platelet therapies. Br. J. Pharmacol. 154 (5), 918–939 [View Article]. Bruno, R.M., et al., 2011. Interactions between sympathetic nervous system and endogenous endothelin in patients with essential hypertension. Hypertension 57 (1), 79–84 [View Article]. Chen, C., et al., 2015. Natural products for antithrombosis. Evid. Based Complement. Alternat. Med. 3, 1–17 [View Article]. Chen, Y.P., et al., 2016. Protective effects of myricitrin on ischemic/reperfusion injury in isolated rat hearts. China J. Comp. Med. 26 (05), 31–39 [View Article]. Chen, W.W., et al., 2017. Summary of Chinese cardiovascular disease report in 2016. China Circ. J. 32 (6), 521–530 [View Article]. Dawood, M.Y., Khan-Dawood, F.S., 2007. Differential suppression of menstrual fluidprostaglandin F2α, prostaglandin E2, 6-ket oprostaglandin F1αand thromboxane B2 by suprofen in women with primary dysmenorrhea. Prostaglandins. Other Lipid Mediat. 83 (1–2), 146–153 [View Article]. Editorial Commission of China Flora of Chinese Academy of Science, 1988. Flora of China 39. Science Press, Beijing, pp. 144. Feng, Z.Y., 2016. NOX2 Mediated Myricitrin Inhibition on LPS-Induced Inflammatory Response. Master’s Thesis. Wan Nan Medical College [View Article]. Forstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33 (829–837) 837a–837d. [View Article]. Gao, J., et al., 2017. Therapeutic effects of breviscapine in cardiovascular diseases: a review. Front. Pharmacol. 8, 1–3 [View Article]. He, K., et al., 2016. Hypolipidemic effects of Myrica rubra extracts and main compounds in C57BL/6j mice. Food Funct. 7 (8), 3505–3515 [View Article]. Innocenti, G., et al., 2010. Antioxidant activity and redox properties of flavonoids from Limonium narbonense. Plant Med. 76 (12), 1249 [View Article]. Jiangsu New Medical College, 1997. Dictionary of Traditional Chinese Medicine. Shanghai Science and Technology Publishing House, Shanghai, pp. 2364. Kawanabe, Y., Nauli, S.M., 2011. Endothelin. Cell Mol. Life Sci. 68 (2), 195 [View Article].
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