In vivo and in vitro hemostatic activity of Chromolaena odorata leaf extract

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 (<2.5 min) in rats but did not induce platelet aggregation or blood clotting in the in vitro study. The in vitro blood clotting times of all extracts were > 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

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 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; 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 (Sigma-Aldrich, 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.

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).

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 < 0.05). The same *symbol represents the same value of each group (normal saline was a control for lyophilized aqueous extract while 70% ethanol was a control for ethanol extracts).

In vitro hemostatic activity

Platelet aggregation test

The % transmission was calculated using the optical density method. By means of setting the % transmission of PPP and PRP as 100% and 0%, respectively, the amount of optical density that passed through the PRP containing extracts was calculated. The % transmission was varied directly with the increasing of platelet aggregation. All extracts could not stimulate the platelet aggregation compared to the controls. But when compared between extracts, all alcoholic extracts showed slightly higher % transmission than the aqueous extract while the 70% ethanol dried leaf extract showed the highest % transmission. The dried leaf extract could also stimulate the platelet aggregation better than the fresh leaf extract. This finding was in accordance with the coagulogram study (Table 2).

Table 2.  Effect of different C. odorata leaf extracts on blood coagulation and platelet aggregation in in vitro tests (n = 3) (p < 0.05).

Samples	Coagulogram study		%Transmission after adding aggregating agent (ADP 0.1 M 20 µl)
	PT (min)	APTT (min)
Normal saline	0.15 ± 0.01	0.38 ± 0.01	82.50 ± 2.50
1% Tween 80	0.16 ± 0.01	0.41 ± 0.01	72.50 ± 0.00
Lyophylized aqueous extract	1.89 ± 0.01	X	70.83 ± 1.44
50% Ethanol dried leaf extract	0.70 ± 0.02	X	72.50 ± 2.50
70% Ethanol fresh leaf extract	1.45 ± 0.01	X	71.67 ± 1.91
70% Ethanol dried leaf extract	0.64 ± 0.01	X	75.00 ± 5.00
95% Ethanol dried leaf extract	0.89 ± 0.01	X	72.50 ± 0.00

PT = prothrombin time, APTT = activated partial thromboplastin time.

X refers to non-clotted blood within 4 min (normal saline was a control for lyophilized aqueous extract while 1% Tween 80 was a control for ethanol extracts).

Blood coagulation test

All extracts prolonged blood clot formation after being tested as shown by the coagulogram study. The blood clotting times of all extracts in PT test were more than those of the controls. The clot formation did not occurred in APTT test. Nevertheless, when compared between extracts, all ethanol extracts demonstrated shorter bleeding time than aqueous extract in the PT test, whereas the 70% ethanol extract of dried leaves showed the shortest bleeding time. Furthermore, when compared 70% ethanol fresh leaf extract with 70% ethanol dried leaf extract, dried leaf extract could accelerate blood coagulation better than the fresh leaf extract (Table 2).

Discussion

Herein, the bleeding time in rats, in vitro platelet aggregation and coagulogram study tests confirmed that most alcoholic extracts enhanced hemostatic activity better than the lyophilized aqueous extract. The bleeding time in rat demonstrated that all alcoholic extracts, except 50% ethanol extract, significantly decreased bleeding time better than the lyophilized aqueous extract. The 70% ethanol dried leaf extract was found to be the extract with the most potent hemostatic activity. This potential activity of C. odorata extracts possibly occurred via platelet and/or vascular function. We further confirmed the hemostatic mechanisms of C. odorata leaf extracts evaluated in platelet aggregation and blood coagulation in vitro tests. Addition of ADP (0.1 M) as an aggregating agent into the sheep plasma did not stimulate the platelet aggregation in all aqueous and alcoholic extracts. The results were different from a previous study (Wongkrajang et al., 1990). Our experiment on blood clotted pathway conducted in coagulogram study indicated no coagulation in both PT and APTT in all extracts, confirming Soogarun’s report (Soogarun et al., 2005). It was likely that an ineffectiveness of the platelet aggregation test and also prolongation of PT and APTT occurred from the interference of clotting system such as plasma pH or some chemical compounds in the extracts (Wongkrajang et al., 1994). C. odorata extracts contain many phytochemical constituents including volatile oil, phenolic compounds, tannins, saponins, terpenoids, alkaloids, and flavonoids which are the main component (Thongpraditchot et al., 1994; Pisutthanan et al., 2005; Umukoro & Ashorobi, 2006). Several flavonoids such as kaempferol-4′-methylether, naringenin-4′-methylether (isosakuranetin), 4′,5,6,7-tetramethoxy-flavone, etc., were isolated from C. odorata extracts. 4′,5,6,7-Tetramethoxy-flavone was found to be a blood coagulant, accelerating blood coagulation through the intrinsic pathway detected by APTT test (Naengchomnong, 1989; Triratana et al., 1991). Besides this active compound, calcium is also the important aggregating and clotting agent (Wongkrajang et al., 1990, 1994). In vitro negative results in this study might be due to an insufficient amount of calcium or active compound to induce blood coagulation and platelet aggregation. Therefore, the amount of calcium and active compound in the extracts should be further investigated.

Conclusions

This experiment indicated that 70% ethanol extract from the dried leaves of C. odorata was the appropriate extract promoting the highest hemostatic activity both in vivo and in vitro. The in vivo study in rats confirmed the significant ability of this plant extracts to stop bleeding. However, the sufficient amount of calcium and active compounds to enhance blood coagulation and platelet aggregation in in vitro tests should be further studied.

Acknowledgements

The authors would like to thank Mrs. Kanchana Muangklum for in vitro test techniques and to Mr. Panupon Khumsupan for proofreading the manuscript.

Declaration of interest

This work is a part of the project on Ph.D. Sandwich Program in the Strategic Scholarships Fellowships Frontier (CHE-PhD-SW) No. CHE510780, which is financial supported by Office of The Higher Education Commission, Thailand. The authors declare no conflict of interest.