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\begin{document}


\def\RunningAuthor{Norhaida Che Azmi et al.}
\firstPage{241}
\articleType{Original Article}
\receivedDate{19.08.2019}
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\title{Evaluation of \textit{Clinacanthus nutans }leaves extract on \textit{Prothrombin} Time and Activated Partial \textit{Prothrombin} Time test}
\author{Nur~Farahhim~Abd~Rahman,  
            Azlina~Muhsin, 
            Norhaida~Che~Azmi\textsuperscript{*}~\\[5pt]{Universiti Kuala Lumpur, Institute of Medical Science Technology (UniKL MESTECH), 43000 Kajang, Selangor, Malaysia}}

\begin{abstract}
Limitations in existing anticoagulants have prompted a search for the natural origin of anticoagulants. Since there is an increasing need to source for pharmacological and medicinal materials from a plant source, thus an exploratory effort towards identifying and characterizing new anticoagulants from plants is worthwhile. \textit{Clinacanthus nutans} (\textit{C. nutans}) commonly known as Sabah snake grass belongs to Acanthaceae family was chosen since it has therapeutic and medicinal benefits. This plant possessed anti-oxidant, anti-microbial, anti-inflammatory, and anti-diabetic activities. This study was carried out to identify the anticoagulant activities of \textit{C. nutans} leaves in aqueous and methanol extracts at different concentrations based on prothrombin time (PT) and activated partial thromboplastin time (aPTT). The dried leaves of \textit{C. nutans} were ground into a fine powder and extracted using aqueous and methanol. Anticoagulant assays of PT and aPTT were done on three different concentrations of \textit{C. nutans} aqueous and methanol extracts (10 mg/mL, 20 mg/mL, and 30 mg/mL) and control group respectively. PT and aPTT of plasma samples were prolonged in both types of extracts (p{\textless}0.05). This study highlights that the anticoagulant activity of aqueous and methanol extracts of \textit{C.nutans} affects the intrinsic and extrinsic pathway, possibly due to the inhibitory action of the clotting factors. These results suggested that aqueous and methanol extracts of \textit{C. nutans} exhibited anticoagulant activity at certain concentrations.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Clinacanthus nutans,\newline PT,\newline aPTT
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Norhaida~Che~Azmi~\\ Phone:\ +60 17 437 1138~\\  Email:\ norhaida@unikl.edu.my
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Thrombosis or formation of blood clotting is commonly related to disruption and blockage of blood flow. The impact of this blood clotting contributes to the development of many diseases such as stroke, atherosclerosis, and heart attack. Blood coagulation is one of the components of haemostasis \unskip~\citep{649781:15267327}. The indicators of blood coagulation are PT and aPTT \unskip~\citep{649781:15267320}. PT is used to evaluate the coagulation factors V, VII, and X in the extrinsic pathway, while aPTT is used to evaluate the coagulation factors such as VIII, IX, XI, XII, and prekalikrein in the intrinsic coagulation pathway of the coagulation cascade. Current available treatment, such as heparin and warfarin, acts as an anticoagulant in order to treat blood clotting disease \unskip~\citep{649781:15267323}. However, these drugs can cause adverse effects such as bleeding, especially in the elderly, patient with antiplatelet therapy, and patient with other diseases such as cancer, renal, and liver problem \unskip~\citep{649781:15267317}. Meanwhile, to overcome these problems, plants can become natural remedies and alternative sources to treat blood coagulation \unskip~\citep{649781:15267323}. Thus, many studies have been explored on the anticoagulant activity of various medicinal plants with the hope to find new and more effective anticoagulant agents \unskip~\citep{649781:15267322,649781:15267321}. In this study, \textit{Clinacanthus nutans }(hereafter referred to as \textit{C. nutans}) belongs to Acanthaceae family was chosen since it has potential therapeutic and medicinal benefits. According to \unskip~\cite{649781:15267318}, \textit{C. nutans} is known as 'Sabah snake grass'or 'belalai gajah' in Malay and 'Phaya Yo' in Thai. In Thailand, \textit{C. nutans} is useful in treating skin rashes, insect and snake biting, herpes simplex virus (HSV) and varicella-zoster virus (VZV) lesions \unskip~\citep{649781:15267329}. The phytochemical studies demonstrated that \textit{C. nutans} plant has rich sources of active compounds such as flavonoids, glycosides, glycoglycerolipids, cerebrosides, and monoacylmonogalatosyl glycerol that serve as anti-inflammatory, antiviral, antioxidant and anti-diabetic activities \unskip~\citep{649781:15267318}. As of today, there is no study reported on C.nutans anticoagulant activities. Therefore, this study was conducted to evaluate the effect of aqueous and methanol extraction of \textit{C. nutans} leaves extract on prothrombin time (PT) and activated partial thromboplastin time (aPTT) test on platelet-poor plasma samples.
    
\section{Materials and Methods}
\textbf{Preparation of plant material}

\textit{C. nutans} leaves were collected and identified from Alor Setar, Kedah, and used freshly for extraction. The leaves were washed with fresh water and dried under shade at room temperature. The leaves were ground into a powder and stored in a dry, dark place prior to anticoagulant testing.

\textbf{Preparation of plant extracts}

\textbf{\textit{C. nutans }aqueous extraction}

The aqueous extract of\textit{ C. nutans} was prepared according to the method by \unskip~\cite{649781:15267330} with some modification. 800 g of \textit{C. nutans} leaves were cut into small pieces and let to be dried under the sunshade. The complete dried of the leaves pieces were ground into a fine powder. 100 g of the fine powder was weighed on a balance scale (AY220, Shimadzu, Japan) and soaked in 1 L of distilled water (1:10 w/v) at room temperature. Then the mixture was stirred by using magnetic stirrer (IKA C-Mag HS7) in a conical flask and left overnight. After that, the mixture was filtered by using Whatman No.1 filter paper (Whatman, England), and the supernatant was collected. In order to produce a thick syrupy mass crude extract, rotary evaporator (R-215, Butchi rotavaporator, Switzerland) was used to evaporate the filtrates under vacuum reduced pressure at 60\ensuremath{^\circ}C. The crude extract was kept at -20\ensuremath{^\circ}C in Scott's bottle prior to further analysis

\textbf{\textit{C. nutans} methanol extraction}

The methanol extract of Clinacanthus nutans was prepared according to the method by \unskip~\citep{649781:15267328} with some modification. 500 g of \textit{C. nutans }leaves were cut into small pieces and let to be dried at room temperature. The complete dried of the leaves pieces were ground into a fine powder. 100 g of the fine powder was weighed on a balance scale (AY220, Shimadzu, Japan). The fine powder was soaked in 1 L of 90\% methanol at room temperature. Then the mixture was stirred by using magnetic stirrer (IKA C-Mag HS7) in a conical flask and left overnight. After that, the mixture was filtered by using Whatman No.1 filter paper (Whatman, England), and the supernatant was collected. In order to produce a thick syrupy mass crude extract, rotary evaporator (R-215, Butchi rotavaporator, Switzerland) was used to evaporate the filtrates under vacuum reduced pressure at 60\ensuremath{^\circ}C. The crude extract was kept at -20\ensuremath{^\circ}C in Scott's bottle prior to further analysis.

\textbf{\textit{C. nutans} solution preparation of Aqueous and Methanol Extraction}

The stock of aqueous and methanol extraction of C.nutans were prepared and diluted with sterile distilled water and normal saline, respectively, to a final concentration of 10, 20, and 30mg/ml.

\textbf{Platelet Poor Plasma Preparation }

The platelet-poor plasma (PPP) was obtained by purchasing the normal control plasma (Diagnostica Stago, France). The samples of normal PPP were stored at -20\ensuremath{^\circ}C prior to further analysis. Different concentrations of 10, 20 and 30 mg/mL of \textit{C. nutans} extracts were used to mix with the plasma and incubated in water bath (Memmert, USA) at 37\ensuremath{^\circ}C for 7 minutes in order to measure the prothrombin time (PT) and activated partial thromboplastin time (aPTT) within 3 hours of sample preparation.

\textbf{In vitro Anticoagulant Activity}

The sample of normal platelet-poor plasma (PPP) (Diagnostica Stago, France) was divided control group and experimental group. For the negative control group of normal prothrombin time (PT) and normal activated partial thromboplastin time (aPTT), there is no anticoagulant such as heparin or extracts was added, whereas, for positive control, the tube contained heparin. For the experimental group, the PPP were spiked with different concentrations of C.nutans (10, 20, and 30 mg/mL). The sample was tested in a water bath at 37\ensuremath{^\circ}C.

The PT test was carried out using STA-Neoplastine{\textsuperscript{\textregistered}} CI Plus (Diagnostica Stago, France) that consists of calcium thromboplastin while the aPTT test using thromboplastin reagent C.K. Prest{\textsuperscript{\textregistered}} 2 (Diagnostica Stago, France) containing a standardized amount of cephalin and silica. Both reagents were reconstituted by adding activator and was mixed gently. Coagulation assay was then performed manually according to the manufacturer's instruction. 

\textbf{Statistical Analysis}

The data of PT and aPTT are shown as mean \ensuremath{\pm} S.E.M.(\ensuremath{n}=3), and analysis was performed using a one-way analysis of variance (ANOVA), followed by post hoc Dunnett's multiple range tests to determine the means of significance from the control (SPSS version 20.0). The student t-test was used for independent samples to determine the significant difference of clotting time among different extraction methods. The results with a p-value less than or equal to 0.05 were considered significant (*), p \ensuremath{\leq } 0.01 is highly significant (**), and p \ensuremath{\leq } 0.001 is very highly significant (***).
    
\section{Results and Discussion}
\textbf{PT and aPTT Clotting time of \textit{C. nutans} extraction}

Table~\ref{tw-2c4943c6f22e} showed the PT (in seconds) for aqueous and methanolic extract at three different concentrations (10, 20 and 30 mg/mL) were significantly higher in comparison to positive control (29.0 \ensuremath{\pm} 0.0) and negative control (12.0 \ensuremath{\pm} 0.0) with p{\textless}0.001. While the aPTT (in seconds) for aqueous and methanol extraction at three different concentrations were also significantly higher in comparison to a positive control (34.0 \ensuremath{\pm} 0.0) and negative control (30.0 \ensuremath{\pm} 0.0) with p{\textless}0.001.


\begin{table*}[!htbp]
\caption{\boldmath {PT and aPTT of aquoeus and methanol extraction of \textit{C. nutans} at three different concentrations} }
\label{tw-2c4943c6f22e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2956\linewidth-2\tabcolsep}p{\dimexpr.1751\linewidth-2\tabcolsep}p{\dimexpr.1833\linewidth-2\tabcolsep}p{\dimexpr.1682\linewidth-2\tabcolsep}p{\dimexpr.1778\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{}} & \multicolumn{2}{p{\dimexpr(.3584\linewidth-2\tabcolsep)}}{\cAlignHack Aqueous} & \multicolumn{2}{p{\dimexpr(.346\linewidth-2\tabcolsep)}}{\cAlignHack Methanol}\\
\rowcolor{kwdboxcolor} & PT & aPTT & PT & aPTT\\
\tblmidrule 
10 mg/mL &
  34.7 \ensuremath{\pm} 2.1 &
  40.3 \ensuremath{\pm} 1.5 &
  37.0 \ensuremath{\pm} 3.0 &
  50.3 \ensuremath{\pm} 1.5\\
20 mg/mL &
  40.7 \ensuremath{\pm} 1.2 &
  45.7 \ensuremath{\pm} 1.2 &
  48.0 \ensuremath{\pm} 3.6 &
  60.0 \ensuremath{\pm} 5.0\\
30 mg/mL &
  45.3 \ensuremath{\pm} 1.5 &
  50.0 \ensuremath{\pm} 2.0 &
  52.3 \ensuremath{\pm} 2.5 &
  62.0 \ensuremath{\pm} 6.2\\
Positive control (heparin) &
  29.0 \ensuremath{\pm} 0.0 &
  34.0 \ensuremath{\pm} 0.0 &
  29.0 \ensuremath{\pm} 0.0 &
  34.0 \ensuremath{\pm} 0.0\\
Negative control &
  12.0 \ensuremath{\pm} 0.0 &
  30.0 \ensuremath{\pm} 0.0 &
  12.0 \ensuremath{\pm} 0.0 &
  30.0 \ensuremath{\pm} 0.0\\
p-value &
  {\textless}0.001*** &
  {\textless}0.001*** &
  {\textless}0.001*** &
  {\textless}0.001***\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
Table~\ref{tw-2c4943c6f22e} Depicts the aqueous and methanol extract of PT and aPTT at three different concentrations of 10, 20, and 30 mg/mL, respectively. By using the ANOVA and post hoc analysis with Dunnet multiple comparisons test, both aqueos and methanol extract were significantly prolonged (p{\textless}0.001) the PT and APTT in a dose-dependent manner in comparison to the normal control plasma.


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\caption{\boldmath {PT clotting time of plasma on methanol and aqueous extraction of \textit{C. nutans}. Significance differences of PT test for both aqueous and methanol extraction at three different concentrations (10, 20, and 30 mg/mL) were determined by the Student t-test, whereby p{\textless}0.05 was considered significant}}
\label{f-c09ee84cef55}
\end{figure*}
\egroup
\textbf{Prothrombin Time Between Aqueous And Methanolic Extract}

Figure~\ref{f-c09ee84cef55} shows the comparison between aqueous and methanol extract of \textit{C. nutans} on PT (in seconds) at three different concentrations (10, 20, and 30 mg/mL). There was no significant difference of \textit{C. nutans} extract at 10 mg/mL with p-value 0.3.\textit{\space }C. nutans methanolic extract at 20 mg/mL and 30 mg/mL showed a significant prolongation of PT compared to aqueous extract at similar concentrations with a p-value of 0.03 and p-value 0.02, respectively.

\textbf{Activated partial thromboplastin time of aqueous and methanolic extract}

Figure~\ref{f-2e65eca73632} shows the comparison between aqueous and methanol extract of \textit{C. nutans} on aPTT (in seconds) at three different concentrations. There was a significant prolonged aPTT of \textit{C. nutans }methanolic extract in comparison with aqueous extract; where at 10 mg/mL p-value was 0.001. For concentration 20 mg/mL the p-value was 0.008. At concentration 30 mg/mL the p-value was 0.03.  


\bgroup
\fixFloatSize{images/058ff51a-24f3-4e36-bb24-d44cfa987ca5-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/058ff51a-24f3-4e36-bb24-d44cfa987ca5-upicture2.png}{\includegraphics{images/058ff51a-24f3-4e36-bb24-d44cfa987ca5-upicture2.png}}{}
\makeatother 
\caption{\boldmath {aPTT clotting time of plasma on methanol and aqueous extraction of \textit{C. nutans}. Significance differences of aPTT test for both aqueous and methanol extraction at three different concentrations (10, 20 and 30 mg/mL) were determined by Student t-test where by p{\textless}0.05 was considered significant, *p{\textless}0.05, **p{\textless}0.01 and ***p{\textless}0.001}}
\label{f-2e65eca73632}
\end{figure*}
\egroup
In the present study, the aqueous and methanol extract of \textit{C. nutans} leaves exhibited a prolonged PT and aPTT at concentrations of 10, 20, and 30 mg/ml. This finding could be associated with interference in the one or more clotting factors of intrinsic and extrinsic coagulation pathways, respectively \unskip~\citep{649781:15267324,649781:15267316,649781:15267320}. The efficiency of the extraction and the anticoagulant activity obtained from the extracts were affected by the polarity of the solvents \unskip~\citep{649781:15267319}. The study also mentioned that solvents have characteristics such as low viscosity, low density, and high diffusivity allows them to diffuse easily into the pores of plant materials, thus causing the bioactive components to leach out. The extraction of plants depends on the type of solvent used in the extraction procedure. In addition, methanol has a lower boiling point, which is 64.7 \ensuremath{^\circ}C compare to water that has a high boiling point, which is 100 \ensuremath{^\circ}C \unskip~\citep{649781:15267319}. Methanol is a polar solvent that easily dissolved bioactive compounds in plants. Based on the results obtained, both aqueous and methanol solvents exhibited anticoagulant activities, but methanol exhibited a longer anticoagulation time than aqueous solvent. Thus, methanol is a better solvent to identify the anticoagulant activities of \textit{C. nutans} compare to the aqueous solution.

According to \unskip~\cite{649781:15267326}, bioactive compounds such as hexuronic acids, polysaccharides, and polyphenolics contribute to anticoagulant activities. Meanwhile, \unskip~\cite{649781:15267325} reported that tannins also displayed anticoagulant and antithrombotic effects. Besides, saponins, starch, alkaloids, and flavonoid also contribute to anticoagulant activities \unskip~\citep{649781:15267332}. Previous study by \unskip~\cite{649781:15267331} revealed that steroids, triterpenoids, cerebrosides, glycoglycero-lipids, glycerides, sulfur-containing glycosides were isolated from \textit{C. nutans}, whereas \unskip~\citet{649781:15267330} mentioned that \textit{C. nutans} contains phenolic, saponins, flavonoid and tannin which exhibited anticoagulant effects.
    
\section{Conclusions}
The anticoagulant activities of \textit{C. nutans} were shown in both aqueous and methanol extraction. In this study, the methanol extraction showed higher clotting time compared to aqueous extraction. The PT and aPTT for both extracts exceeded the reference range, thus indicates this plant has an effective anticoagulant effect. In conclusion, \textit{C. nutans} may possess as alternative anticoagulants properties in the future. However, further works such as isolation of bioactive compounds that responsible for the anticoagulation activity need to be done to validate this observation.
\section*{Acknowledgement}The authors would like to express their gratitude to the Clinical and Biomedical Science Section, Universiti Kuala Lumpur, and FYP research grants for funding this project.
    

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