In vivo and in vitro hemostatic activity of Chromolaena

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areas of Thailand, Vietnam, Malaysia, Nigeria and Ivory. Coast (Akah ..... Thesis (Organic Chemistry). Faculty of Graduate Studies. Mahidol. University. Bangkok.
Pharmaceutical Biology, 2012; 50(9): 1073–1077 © 2012 Informa Healthcare USA, Inc. ISSN 1388-0209 print/ISSN 1744-5116 online DOI: 10.3109/13880209.2012.656849

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In vivo and in vitro hemostatic activity of Chromolaena odorata leaf extract Hataichanok Pandith1, Suchitra Thongpraditchote2, Yuvadee Wongkrajang2, and Wandee Gritsanapan1 Department of Pharmacognosy and 2Department of Physiology, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudthaya Road, Ratchathevi, Bangkok, Thailand 1

Abstract Context: Chromolaena odorata (L.) R.M.King & H.Rob. (Asteraceae) or Siam weed has long been used to stop bleeding in Thailand and many countries. Only the aqueous leaf extract was investigated in in vivo and there have been conflicting results of in vitro hemostatic mechanisms of this plant. Objective: The most appropriate C. odorata leaf extract that promoted the highest hemostatic activity and the hemostatic mechanisms of these plant extracts will be investigated. Materials and methods: The lyophilized aqueous leaf extract and alcoholic (50, 70, and 95% ethanol) extracts from the fresh and dried leaves were investigated both in vivo and in vitro. The bleeding time in male Wistar rats was measured to investigate the hemostatic effect. The hemostatic mechanisms were tested using in vitro platelet aggregation and blood coagulation tests in sheep plasma. Results: All extracts displayed significantly reducing bleeding time ( 0.6 min. Ethanol extract (70%) from the dried leaves proved to be the extract producing the highest hemostatic activity in vivo with the bleeding time of 1.85 min. Discussion and conclusion: The in vivo study with rats confirmed the significant ability of this plant extract to stop bleeding. However, the sufficient amount of calcium and active compounds which are aggregating and clotting agents to enhance blood coagulation and platelet aggregation in in vitro tests should be further studied. Keywords:  Chromolaena odorata, Siam weed, hemostatic activity, bleeding time, Wistar rats

Introduction

cause hemostasis, a process by which the body spontaneously stops bleeding and maintains blood in the fluid state within the vascular compartment (Rosado et al., 2009). It can be achieved by three mechanisms, i.e. the vascular constriction, the formation of platelet plug and the blood coagulation (Wongkrajang et  al., 1990; DeMoranville, 2006). The effectiveness of this plant promoting hemostatic effect was investigated in Wistar rats. The aqueous extract and fresh crushed leaves decreased the bleeding time and blood volume of wounds in rats (Wongkrajang et al., 1990). C. odorata extracts were reported to stimulate vasoconstriction in rat and guinea pig vasa deferentia, rabbit arterial strips and pig coronary artery (Akah, 1990;

Chromolaena odorata (L.) R.M.King & H.Rob. (Asteraceae) (formerly known as Eupatorium odoratum L.) or Siam weed is a perennial scandent or semi-woody shrub. It is native to Central and South America and spreads throughout the tropical and subtropical areas of the world (Muniappan & Marutani, 1991). The fresh cut leaves of C. odorata are widely used as a hemostatic agent to stop bleeding in many countries, such as in rural areas of Thailand, Vietnam, Malaysia, Nigeria and Ivory Coast (Akah, 1990; Wongkrajang et al., 1990; Bamba et al., 1993; Phan et al., 2001a,b; Thang et al., 2001; Umukoro & Ashorobi, 2006; Ling et al., 2007a,b,c). Hemostatic agents

Address for Correspondence:  Wandee Gritsanapan, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudthaya Road, Ratchathevi, Bangkok, Thailand (Received 19 September 2011; revised 27 November 2011; accepted 08 January 2012)

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1074  H. Pandith et al. Narongsanti et  al., 1991; Thongpraditchot et  al., 1994), but they show no effect on isolated guinea pig ileum, rat stomach strip preparations and rabbit aortic strip tone (Akah, 1990; Thongpraditchot et  al., 1994). The lyophilized aqueous extract from the fresh leaves of C. odorata stimulated platelet aggregation by adding ADP (2 µM) as an aggregating agent in human plasma (Wongkrajang et al., 1990). In former reports, the blood coagulation test of this plant was evaluated in human/cow plasma. Some reports (Phithanchort et al., 2006; Khengraeng, 2007) proposed that the aqueous and 95% ethanol extracts could stimulate blood coagulation with/without exogenous calcium via prothrombin time (PT) and activated partial thromboplastin time (APTT). It could be deduced that C. odorata enhanced blood coagulation via both extrinsic (PT) and intrinsic (APTT) pathways. However, some other reports proposed that it stimulated either PT or APTT test (Rawiwong et al., 1988; Wongkrajang et al., 1990; Triratana et al., 1991) whereas Soogarun et al. (2005) found that it could not stimulate any of these pathways. These conflicting results could be due to the variability over the collected locations of this plant and the solvents used for the preparation of the extracts. Therefore, our first objective was to investigate the appropriate solvent that promoted the highest in vivo hemostatic activity. Leaves collected from a single location were used to complete this objective. The hemostatic activity study was determined by bleeding time in rats. Another objective was to determine the in vitro hemostatic mechanisms of this plant extracts via platelet aggregation and blood coagulation using sheep plasma. The in vitro test was done in sheep plasma, which closely resembled human plasma (Osbaldiston & Hoffman, 1971). The European Pharmacopeia monograph (EP) (2009) also recommended using sheep plasma to determine the activated partial thromboplastin time (APTT) (Alban et al., 2011).

Materials and methods Plant materials C. odorata mature leaves were collected from Samut Sakhon province, Thailand in December 2009. The plant samples were identified by Dr. Wandee Gritsanapan, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. The voucher specimen (No.CO-003) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University. A portion of fresh leaves was used for extraction while another portion was dried in a hot air oven at 60°C for 24 h. The dried sample was ground into moderate powder.

Animals and blood sample Eighteen male Wistar rats, weighing 80-120 g and citrated sheep whole blood were purchased from the National Laboratory Animal Center, Mahidol University Salaya, Thailand. The rats were divided into 7 groups. The first and second groups were regarded as control groups. The 

other groups were studied for the effect of C. odorata extracts on bleeding time.

Chemical and reagents High purity grade of all chemicals and reagents; Tween 80 (Sigma-Aldrich, USA), calcium chloride 0.025 M (SigmaAldrich, USA), high ADP 0.1 M (Sigma-Aldrich, USA), human thromboplastin containing calcium and activated partial thromboplastin reagents (Dade Behring Marburg GmbH, Germany) and ethanol (Merck, Germany) were used.

Preparation of plant extracts Lyophilyzed extract Fresh mature leaves (200 g) were mixed with 200 ml of distilled water and crushed in a blender. The mixture was filtered through muslin cloth and the aqueous extract was lyophilized. The dried extract was weighed and kept in a seal vial at 4°C until used. Organic solvent extracts The fresh leaves (100 g) were minced by a blender. The minced fresh leaves were exhaustively extracted with 70% ethanol (1:10 w/v). The dried powder (100 g each) was separately extracted with 50, 70, and 95% v/v ethanol (1:10 w/v) on a shaker at 25°C, 120 rpm for 12 h. The mixture was filtered through Whatman filter paper No.1 and the filtrate was concentrated using a rotary evaporator. The dark green viscous extracts were separately kept in a seal vial at 4°C until used.

In vivo hemostatic activity test The 10% w/v freshly prepared solutions of all extracts (lyophilized, 70% ethanol fresh leaf, 50, 70, and 95% ethanol dried leaf extracts) were used. The lyophilized extract was dissolved in normal saline while all ethanol extracts were dissolved in 70% ethanol. The in vivo protocol (proof no. PYT 010/2552) was approved by the Mahidol University Animal Care and Use Committee (PY-ACUC). The bleeding time of 4 weeks old male Wistar rats was examined for hemostatic activity. The incision was made at a foot pad of each rat using a No.11 blade. Length and depth of a wound was 1 × 0.1 cm. The 20 µl of each leaf extract solution was applied to the wound. The normal saline and 70% ethanol were used as controls for lyophilized aqueous extract and ethanol extracts, respectively. Bleeding time was recorded immediately after making the wound until the blood stop. The halt of bleeding was indicated by no blood strain on a filter paper when blotting the wound (Wongkrajang et al., 1990).

In vitro hemostatic activity test Preparation of blood samples and test samples Platelet-poor plasma (PPP) and platelet-rich plasma (PRP) were separated from citrated sheep whole blood. The PPP was centrifuged at 3,000 rpm for 15 min while the PRP was centrifuged at 800 rpm for 10 min. Both plasmas were stored at −20°C until used. Pharmaceutical Biology

Hemostatic activity of Chromolaena odorata 1075

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The 10% w/v of all extracts were freshly prepared. The lyophilized extract was dissolved in normal saline while all ethanol extracts were dissolved in 1% Tween 80. In vitro platelet aggregation test The PRP and PPP were used in this study. The PRP was mixed with each extract solution at a ratio of 90:1 v/v. ADP 0.1 M (20 µl) was added as an aggregating agent to stimulate the platelet aggregation. The results were recorded as % transmission. The % transmission of PPP and PRP were set at 100% and 0%, respectively. The normal saline and 1% Tween 80 were used as controls for lyophilized aqueous extract and ethanol extracts, respectively. In vitro blood coagulation test The PPP was mixed with each leaf extract solution at a ratio of 1:1 v/v before evaluating for the hemostatic mechanism by the coagulogram study − the activated partial thromboplastin time (APTT) and prothrombin time (PT) (Quick, 1935; Langdell et  al., 1953). The normal saline and 1% Tween 80 were used as controls for lyophilized aqueous extract and ethanol extracts, respectively.

Statistics Analysis of all results was performed using ANOVA with Tukey’s Multiple Comparison Test. P values for significance were set at 0.05. Values for all measurements are expressed as the mean ± SEM. Statistical analysis was conducted using GraphPad Prism 4.

Results In vivo hemostatic activity test All extracts could significantly shorten the bleeding time in rats. All alcoholic extracts could stop bleeding better than the aqueous extract. The 70% ethanol dried leaf extract yielded the shortest bleeding time (Table 1).

In vitro hemostatic activity Platelet aggregation test The % transmission was calculated using the optical density method. By means of setting the % transmission of Table 1.  Effect of different C. odorata leaf extracts on bleeding time in Wistar rats (n = 5). Samples Bleeding time (min) Normal saline 3.30 ± 0.03 70% Ethanol 3.34 ± 0.06 Lyophylized aqueous extract 2.47 ± 0.02* 50% Ethanol dried leaf extract 2.39 ± 0.02*,** 70% Ethanol fresh leaf extract 2.04 ± 0.05*** 70% Ethanol dried leaf extract 1.85 ± 0.05**** 95% Ethanol dried leaf extract 2.30 ± 0.01** *, **, ***, **** represent significant difference compared to the values of controls and compared between groups (p