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\begin{document}


\def\RunningAuthor{Jaikumar S et al.}
\firstPage{5748}
\articleType{Original Article}
\receivedDate{19 Jun 2020}
\acceptedDate{20 Jul 2020}
\revisedDate{19 Jul 2020}
\journalVolume{2020, 11}
\journalIssue{4}
\journalDoi{ijrps.v11i4.3220}
\copyrightYear{2020}
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\title{\textit{\textbf{In-vitro}} \textbf{ Anti-Inflammatory activity of \textit{Digera muricata} Extracts}}
\author{Balaji~Subramaniyan~R\textsuperscript{1},  
            Jaikumar~S\textsuperscript{*}\textsuperscript{2}, 
            Sekkizhar~M\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Dentistry, Sri Lakshmi Narayana institute of medical sciences, Ossudu kudapakkam, Pondicherry, India}~\\{\textsuperscript{2}Department of Pharmacology, Sri Lakshmi Narayana institute of medical sciences,  Puducherry - 605502, India}~\\{\textsuperscript{3}Department of Pharmacology, Vinayaka missions medical college and hospital, Karaikal, Tamilnadu, India}}

\begin{abstract}
Inflammation is a process that is generally termed for pain and redness, which includes swelling. But it is defined as a heterogeneous group of processes which result in the pain and increase in vascular circulation and denaturation of protein that results in tissue degeneration. There are many synthetic drugs available to treat and relieve the inflammation. They are potent and have many side effects too. So there is an increased search for alternative means of treatment option. Thereby there was a high focus on the medicinal plants and herbs. \textit{Digera muricate}, which is used to treat many diseases like analgesic activity, antidiabetic activity and anticancer activity too, this plant has proven to possess the antioxidant potential also. So there is no enough work done to verify the inflammatory potential of the plant. This was the primary consideration for this research to select the plant to prove it has anti-inflammatory potential. Having in the account that the ethical problems in the usage of lab animals, the anti-inflammatory potential of the plant was evaluated \textit{in-vitro} using enzyme inhibition assays. The \textit{in-vitro} models of the inflammatory activity are used for testing the anti-inflammatory activity, and the results were not different from \textit{invivo} models. The extracts showed a dose based rise in the action, and the plant process a better anti-inflammatory activity than the standard drugs. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Invitro,\newline Inflammation,\newline Digera muricate
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Jaikumar~S~\\ Phone:\ 8825343635~\\  Email:\ jaipharma2007@gmail.com 
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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        \makeatother
        
    
\section{Introduction}
Inflammation is a process that is generally termed for pain and redness, which includes swelling. But it is defined as a heterogeneous group of processes which result in the pain and increase in vascular circulation and denaturation of protein that results in tissue degeneration. This inflammation may be due to micro-organisms, physiological stress, oxidative stress etc. and result in pain, swelling and redness. There may be seen as an inability to do the normal functioning of the body and its organs \unskip~\citep{848613:19971849}. When there is an inflammation in the body, tissue releases cytokines and Pg's and histamines which are called as pain mediators. This will increase blood flow to the site of the injury, which results in the production of chemotaxis. Therefore, tissue repair starts to work \unskip~\citep{848613:19971839}. 

There are many synthetic drugs available to treat and relieve the inflammation. They are potent and 


\begin{table*}[!htbp]
\caption{\boldmath {Anti-inflammatory activity invitro of \textit{Digera muricate}} }
\label{tw-e34a5dbf24e3}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.1312\linewidth-2\tabcolsep}p{\dimexpr.15649999999999997\linewidth-2\tabcolsep}p{\dimexpr.15229999999999999\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Treatment & \multicolumn{2}{p{\dimexpr(.28\linewidth-2\tabcolsep)}}{\cAlignHack Protein Denaturation Assay} & \multicolumn{2}{p{\dimexpr(.27119999999999997\linewidth-2\tabcolsep)}}{\cAlignHack Proteinase inhibition Assay} & \multicolumn{2}{p{\dimexpr(.30879999999999995\linewidth-2\tabcolsep)}}{\cAlignHack Lipoxygenase inhibiting Assay}\\
\rowcolor{kwdboxcolor} & \cAlignHack Absorbance at 660nm & \cAlignHack \% Activity & \cAlignHack Absorbance at 210nm & \cAlignHack \% Activity & \cAlignHack Absorbance at 234nm & \cAlignHack \% Activity\\
\tblmidrule 
Control &
  \cAlignHack 0.40\ensuremath{\pm}0.07 &
  \cAlignHack - &
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  \cAlignHack - &
  \cAlignHack 0.42\ensuremath{\pm}0.04 &
  \cAlignHack -\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.28\ensuremath{\pm}0.04\ensuremath{^{**}} &
  \cAlignHack 33.12 &
  \cAlignHack 0.32\ensuremath{\pm}0.07\ensuremath{^{**}} &
  \cAlignHack 22.48 &
  \cAlignHack 0.38\ensuremath{\pm}0.05\ensuremath{^{**}} &
  \cAlignHack 10.23\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.22\ensuremath{\pm}0.03\ensuremath{^{**}} &
  \cAlignHack 48.36 &
  \cAlignHack 0.29\ensuremath{\pm}0.02\ensuremath{^{**}} &
  \cAlignHack 28.01 &
  \cAlignHack 0.34\ensuremath{\pm}0.07\ensuremath{^{**}} &
  \cAlignHack 18.41\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.18\ensuremath{\pm}0.05\ensuremath{^{**}} &
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  \cAlignHack 0.26\ensuremath{\pm}0.09\ensuremath{^{*}} &
  \cAlignHack 37.39 &
  \cAlignHack 0.32\ensuremath{\pm}0.03\ensuremath{^{**}} &
  \cAlignHack 27.65\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.15\ensuremath{\pm}0.03\ensuremath{^{**}} &
  \cAlignHack 66.58 &
  \cAlignHack 0.24\ensuremath{\pm}0.03\ensuremath{^{**}} &
  \cAlignHack 43.54 &
  \cAlignHack 0.23\ensuremath{\pm}0.08\ensuremath{^{**}} &
  \cAlignHack 49.74\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.13\ensuremath{\pm}0.09\ensuremath{^{**}} &
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  \cAlignHack 54.63 &
  \cAlignHack 0.17\ensuremath{\pm}0.04\ensuremath{^{**}} &
  \cAlignHack 62.86\\
Standard &
  \cAlignHack 0.14\ensuremath{\pm}0.02\ensuremath{^{**}} &
  \cAlignHack 69.25 &
  \cAlignHack 0.19\ensuremath{\pm}0.03\ensuremath{^{**}} &
  \cAlignHack 56.82 &
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  \cAlignHack 87.09\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Anti-inflammatory activity \textit{invitro} of \textit{Digera muricate}} }
\label{tw-9fdf6c0a7f22}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2366\linewidth-2\tabcolsep}p{\dimexpr.1939\linewidth-2\tabcolsep}p{\dimexpr.1527\linewidth-2\tabcolsep}p{\dimexpr.2168\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Treatment & \multicolumn{2}{p{\dimexpr(.3466\linewidth-2\tabcolsep)}}{\cAlignHack Heat induced hemolysis} & \multicolumn{2}{p{\dimexpr(.4168\linewidth-2\tabcolsep)}}{\cAlignHack Hypotonicity induced Heamolysis}\\
\rowcolor{kwdboxcolor} & \cAlignHack Absorbance  & \cAlignHack \% Activity & \cAlignHack Absorbance & \cAlignHack \% Activity\\
\tblmidrule 
Control &
  \cAlignHack 0.32\ensuremath{\pm}0.05 &
  \cAlignHack - &
  \cAlignHack 0.33\ensuremath{\pm}0.04 &
  \cAlignHack -\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.26\ensuremath{\pm}0.10\ensuremath{^{**}} &
  \cAlignHack 22.67 &
  \cAlignHack 0.24\ensuremath{\pm}0.03\ensuremath{^{**}} &
  \cAlignHack 31.77\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.23\ensuremath{\pm}0.09\ensuremath{^{**}} &
  \cAlignHack 31.24 &
  \cAlignHack 0.18\ensuremath{\pm}0.11\ensuremath{^{**}} &
  \cAlignHack 49.90\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.21\ensuremath{\pm}0.07\ensuremath{^{**}} &
  \cAlignHack 37.51 &
  \cAlignHack 0.16\ensuremath{\pm}0.06\ensuremath{^{**}} &
  \cAlignHack 55.62\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.19\ensuremath{\pm}0.02\ensuremath{^{*}} &
  \cAlignHack 44.73 &
  \cAlignHack 0.13\ensuremath{\pm}0.09\ensuremath{^{**}} &
  \cAlignHack 62.45\\
DME \mbox{}\protect\newline 100\ensuremath{\mu}g/ml &
  \cAlignHack 0.16\ensuremath{\pm}0.05\ensuremath{^{*}} &
  \cAlignHack 52.06 &
  \cAlignHack 0.08\ensuremath{\pm}0.04\ensuremath{^{**}} &
  \cAlignHack 76.84\\
Standard &
  \cAlignHack 0.11\ensuremath{\pm}0.08\ensuremath{^{**}} &
  \cAlignHack 72.92 &
  \cAlignHack 0.17\ensuremath{\pm}0.02\ensuremath{^{**}} &
  \cAlignHack 52.03\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
 have many side effects too. So there is an increased search for alternative means of treatment option. Thereby there was a high focus on the medicinal plants and herbs. \unskip~\citep{848613:19971843}. They are serving humans as an alternative treatment option in many diseases like diabetes, hypertension, CV disorders etc. Many of those plants were used to treat inflammations also \unskip~\citep{848613:19971845}.

One of those plants is \textit{Digera muricate}, which is used to treat many diseases like analgesic activity, antidiabetic activity and anticancer activity too \unskip~\citep{848613:19971839}. This plant had been proven to possess the antioxidant potential also. So there is no enough work done to verify the inflammatory potential of the plant. This was the primary consideration for this research to select the plant to prove it has anti-inflammatory potential. There were many folklore claims of the plant had used to treat many other diseases like diarrhoea, and it as has a cooling effect on the body. Having in the account that the ethical problems in the usage of lab animals, the anti-inflammatory potential of the plant was evaluated \textit{in-vitro} using enzyme inhibition assays.
    
\section{\textbf{METHODS}}
\textbf{Extraction}

The aerial plant parts were collected in November and dried under shade and then ground into a fine powder with a blender. This powder is stored in room temperature for a day to use for further extraction. This powder was used for extraction using methanol using Soxhlet extractor, and the filtrate is reduced to get the desired consistency of extract, which is like paste consistency using an evaporator and stored the extract in a desiccator. The plant extract was dissolved in distilled water, that the concentration of extracts was maintained at 100, 200, 300, 400 and 500 microgram/ml. This was used for further Assay.

\textbf{Activity}

The anti-inflammatory activity of the plant was assayed \textit{invitro} using enzyme analysis as follows

\textbf{Protein denaturation inhibition assay}

In this method, the heat was used to denaturate the protein as per the method described by \unskip~\cite{848613:19971842}. The UV absorption was measured for all the concentrations at 660nm.

\textbf{Antagonizing proteinase }

This method was adopted to test the activity according to the method described by \unskip~\cite{848613:19971848}. The UV absorption was measured at 210nm. The percentage inhibition and absorbance were noted according to the method.

\textbf{Lipoxygenase inhibiting activity}

In this method, the extracts were tested from anti-inflammatory activity in different concentrations as planned above. The process was followed as per the procedure by \unskip~\cite{848613:19971840}. Lipoxidase enzyme was used as a marker for the inflammatory mediator, and the activity was measured in UV at 234 nm. The percentage of inhibition and absorbance was noted and tabulated. 

\textbf{Membrane stabilization assay}

In this method, RBC cells are used to study the stabilization of membrane of cells as there is a similarity of membranes of RBC and cytokines. In this method, a healthy human volunteer was selected who did not use any NSAID in near history for seven days, and his blood sample was withdrawn and centrifuged to separate RBC. The cells were collected and suspended in saline solution. 

In this method, hemolysis was measured in two ways.

\textbf{Haemolysis induced by heating.}

The method was adopted to estimate the amount of anti-inflammatory activity by preventing the haemolysis of the RBC \textit{invitro}, which is induced by heat according to the process detailed by \unskip~\cite{848613:19971850}. The standard drug used in this method is aspirin.

\textbf{Haemolysis induced by hypotonicity.}

In this method, the extracts were tested by different concentrations, and the haemolysis was induced using a hypotonic saline solution. The technique was adopted as given by procured by \unskip~\cite{848613:19971844}. The percentage inhibition was measured and compared with the standard, Diclofenac sodium.
    
\section{\textbf{RESULTS AND DISCUSSION}}
The anti-inflammatory activity of the plant was investigated \textit{in-vitro} using various models, and the results were tabulated in Table~\ref{tw-e34a5dbf24e3} \& Table~\ref{tw-9fdf6c0a7f22}. In protein denaturation assay, the extracts showed better activity than the standard drug, aspirin with almost 3-4 per cent better activity. During the inflammation process, the denaturation of protein occurs by the application of heat. It is the most notable causes of inflammation, and the extract showed an increase in activity in a dose based manner \unskip~\citep{848613:19971847}. 

In the lipoxygenase method, the extracts at different doses showed a dose-dependent increase in the activity so at 500microgm extracts showed the highest activity, which is less than that of the standard. Lipoxygenase induces the oxidative free radicals and initiates the stress-based inflammation in the body. The extracts, when successfully inhibited the enzymes, means the antioxidant activity of the extract was an added advantage \unskip~\citep{848613:19971841}.

Proteinase is an enzyme responsible for the inflammation mediation at the lysosomal level. Neutrophils had rich content of the enzyme. Therefore the enzyme inhibition is said to lower the inflammation via the mechanism of the enzyme. The extracts a better activity compared to the standard drug Table~\ref{tw-e34a5dbf24e3}. The antioxidant activity of the extract might have added advantage in the anti-inflammatory activity in this Assay. 

As discussed earlier, membrane stabilization assay was used on RBC as there is a similarity between the lysosome membrane and the RBC. These lysosome breakdowns and releases corrosive enzymes that digest the tissue and cell, which leads to inflammation. The extracts were used to prevent the breakage of the membrane and to stabilize it. So RBC hemolysis was assayed in two methods. In the heat-induced method and hypotonicity method, there was a significant reduction in membrane breakage. The activity was similar and higher than the standard drugs, Aspirin and Diclofenac sodium Table~\ref{tw-9fdf6c0a7f22}\unskip~\citep{848613:19971846}.
    
\section{Conclusion}
The \textit{in-vitro} models of the inflammatory activity are used for testing the anti-inflammatory action, and the results were not different from \textit{invivo} models. The extracts showed a dose based rise in the activity, and the plant possess a better anti-inflammatory activity than the standard drugs. 
    
\section{Acknowledgement}
The authors are thankful to all who have extended their constant support for the completion of the work.

\textbf{Conflict of Interest }

The authors declare that they have no conflict of interest for this study.

\textbf{Funding Support}

The authors declare that they have no funding support for this study.
    

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