\documentclass[twocolumn,twoside]{article}
\makeatletter\if@twocolumn\PassOptionsToPackage{switch}{lineno}\else\fi\makeatother

\usepackage{amsfonts,amssymb,amsbsy,latexsym,amsmath,tabulary,graphicx,times,xcolor}

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% Following additional macros are required to function some 
% functions which are not available in the class used.
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
\usepackage{url,multirow,morefloats,floatflt,cancel,tfrupee}
\makeatletter


\AtBeginDocument{\@ifpackageloaded{textcomp}{}{\usepackage{textcomp}}}
\makeatother
\usepackage{colortbl}
\usepackage{xcolor}
\usepackage{pifont}
\usepackage[nointegrals]{wasysym}
\urlstyle{rm}
\makeatletter

%%%For Table column width calculation.
\def\mcWidth#1{\csname TY@F#1\endcsname+\tabcolsep}

%%Hacking center and right align for table
\def\cAlignHack{\rightskip\@flushglue\leftskip\@flushglue\parindent\z@\parfillskip\z@skip}
\def\rAlignHack{\rightskip\z@skip\leftskip\@flushglue \parindent\z@\parfillskip\z@skip}

%Etal definition in references
\@ifundefined{etal}{\def\etal{\textit{et~al}}}{}


%\if@twocolumn\usepackage{dblfloatfix}\fi
\usepackage{ifxetex}
\ifxetex\else\if@twocolumn\@ifpackageloaded{stfloats}{}{\usepackage{dblfloatfix}}\fi\fi

\AtBeginDocument{
\expandafter\ifx\csname eqalign\endcsname\relax
\def\eqalign#1{\null\vcenter{\def\\{\cr}\openup\jot\m@th
  \ialign{\strut$\displaystyle{##}$\hfil&$\displaystyle{{}##}$\hfil
      \crcr#1\crcr}}\,}
\fi
}

%For fixing hardfail when unicode letters appear inside table with endfloat
\AtBeginDocument{%
  \@ifpackageloaded{endfloat}%
   {\renewcommand\efloat@iwrite[1]{\immediate\expandafter\protected@write\csname efloat@post#1\endcsname{}}}{\newif\ifefloat@tables}%
}%

\def\BreakURLText#1{\@tfor\brk@tempa:=#1\do{\brk@tempa\hskip0pt}}
\let\lt=<
\let\gt=>
\def\processVert{\ifmmode|\else\textbar\fi}
\let\processvert\processVert

\@ifundefined{subparagraph}{
\def\subparagraph{\@startsection{paragraph}{5}{2\parindent}{0ex plus 0.1ex minus 0.1ex}%
{0ex}{\normalfont\small\itshape}}%
}{}

% These are now gobbled, so won't appear in the PDF.
\newcommand\role[1]{\unskip}
\newcommand\aucollab[1]{\unskip}
  
\@ifundefined{tsGraphicsScaleX}{\gdef\tsGraphicsScaleX{1}}{}
\@ifundefined{tsGraphicsScaleY}{\gdef\tsGraphicsScaleY{.9}}{}
% To automatically resize figures to fit inside the text area
\def\checkGraphicsWidth{\ifdim\Gin@nat@width>\linewidth
	\tsGraphicsScaleX\linewidth\else\Gin@nat@width\fi}

\def\checkGraphicsHeight{\ifdim\Gin@nat@height>.9\textheight
	\tsGraphicsScaleY\textheight\else\Gin@nat@height\fi}

\def\fixFloatSize#1{}%\@ifundefined{processdelayedfloats}{\setbox0=\hbox{\includegraphics{#1}}\ifnum\wd0<\columnwidth\relax\renewenvironment{figure*}{\begin{figure}}{\end{figure}}\fi}{}}
\let\ts@includegraphics\includegraphics

\def\inlinegraphic[#1]#2{{\edef\@tempa{#1}\edef\baseline@shift{\ifx\@tempa\@empty0\else#1\fi}\edef\tempZ{\the\numexpr(\numexpr(\baseline@shift*\f@size/100))}\protect\raisebox{\tempZ pt}{\ts@includegraphics{#2}}}}

%\renewcommand{\includegraphics}[1]{\ts@includegraphics[width=\checkGraphicsWidth]{#1}}
\AtBeginDocument{\def\includegraphics{\@ifnextchar[{\ts@includegraphics}{\ts@includegraphics[width=\checkGraphicsWidth,height=\checkGraphicsHeight,keepaspectratio]}}}

\DeclareMathAlphabet{\mathpzc}{OT1}{pzc}{m}{it}

\def\URL#1#2{\@ifundefined{href}{#2}{\href{#1}{#2}}}

%%For url break
\def\UrlOrds{\do\*\do\-\do\~\do\'\do\"\do\-}%
\g@addto@macro{\UrlBreaks}{\UrlOrds}



\edef\fntEncoding{\f@encoding}
\def\EUoneEnc{EU1}
\makeatother
\def\floatpagefraction{0.8} 
\def\dblfloatpagefraction{0.8}
\def\style#1#2{#2}
\def\xxxguillemotleft{\fontencoding{T1}\selectfont\guillemotleft}
\def\xxxguillemotright{\fontencoding{T1}\selectfont\guillemotright}

\newif\ifmultipleabstract\multipleabstractfalse%
\newenvironment{typesetAbstractGroup}{}{}%

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

\usepackage[authoryear]{natbib}



\newif\ifnokeywords\nokeywordsfalse

\makeatletter\input{size10-pointfive.clo}\makeatother%
\definecolor{kwdboxcolor}{RGB}{242,242,242}
\usepackage[hidelinks,colorlinks=true,allcolors=blue]{hyperref}
\linespread{1}                 
\def\floatpagefraction{0.8}

\usepackage[paperheight=11.69in,paperwidth=8.26in,top=1in,bottom=1in,left=1in,right=.75in,headsep=24pt]{geometry}

\usepackage{multirow-custom}

\makeatletter

\def\hlinewd#1{%
  \noalign{\ifnum0=`}\fi\hrule \@height #1%
  \futurelet\reserved@a\@xhline}
\def\tbltoprule{\hlinewd{1pt}\\[-14pt]}
\def\tblbottomrule{\noalign{\vspace*{6pt}}\hline\noalign{\vspace*{2pt}}}
\def\tblmidrule{\hline\noalign{\vspace*{2pt}}}



\let\@articleType\@empty
\let\@journalDoi\@empty
\let\@journalVolume\@empty
\let\@journalIssue\@empty
\let\@crossMarkLink\@empty
\let\@receivedDate\@empty
\let\@acceptedDate\@empty
\let\@revisedDate\@empty
\let\@publishedDate\@empty
\let\@copyrightYear\@empty
\let\@firstPage\@empty
\def\articleType#1{\gdef\@articleType{#1}}
\def\journalDoi#1{\gdef\@journalDoi{#1}}
\def\crossMarkLink#1{\gdef\@crossMarkLink{#1}}
\def\receivedDate#1{\gdef\@receivedDate{#1}}
\def\acceptedDate#1{\gdef\@acceptedDate{#1}}
\def\revisedDate#1{\gdef\@revisedDate{#1}}
\def\publishedDate#1{\gdef\@publishedDate{#1}}
\def\copyrightYear#1{\gdef\@copyrightYear{#1}}
\def\journalVolume#1{\gdef\@journalVolume{#1}}
\def\journalIssue#1{\gdef\@journalIssue{#1}}
\def\firstPage#1{\gdef\@firstPage{#1}}
\let\@issuelogo\@empty%
\let\@issuelogoex\@empty%
\def\issuelogo#1{\gdef\@issuelogo{#1}}
\def\issuelogoex#1{\gdef\@issuelogoex{#1}}


\def\author#1{%
  \gdef\@author{%
    \hskip-\dimexpr(\tabcolsep)\hskip5pt%
    \parbox{\dimexpr\textwidth-1pt}%
    {\fontsize{11}{13}\selectfont\raggedright #1}%
  }%
}


\usepackage{pharmascope-abs}
\usepackage{caption}
\usepackage{lastpage}
\usepackage{fancyhdr}
\usepackage[noindentafter,explicit]{titlesec}
\usepackage{fontspec}

\setmainfont[%
BoldFont=cambriab.ttf,%
ItalicFont=CAMBRIAI.TTF,%
BoldItalicFont=CAMBRIAZ.TTF]{Cambria.ttf}
\lefthyphenmin = 3

\def\title#1{%
  \gdef\@title{%
      \vspace*{-40pt}%
       \ifx\@articleType\@empty\else{\fontsize{10}{12}\scshape\selectfont\hspace{8pt}\@articleType\hfill\mbox{}\par\vspace{2pt}}\fi%
       \minipage{\linewidth}
         \hrulefill\\[-0.7pt]%%{\includegraphics[width=75pt,height=50pt]{ijrps_logo.png}}\hfill
         \mbox{~}\hspace{5pt}\parbox{.1\linewidth}{\IfFileExists{images/\@issuelogo.\@issuelogoex}{\includegraphics[width=79pt,height=61pt]{images/\@issuelogo.\@issuelogoex}}{\includegraphics[width=75pt,height=50pt]{ijrps_logo.png}}}\hfill
         \fcolorbox{kwdboxcolor}{kwdboxcolor}{\parbox{.792\linewidth}{%
          \begin{center}\fontsize{17}{17}\selectfont\scshape\vskip-7pt International Journal of Research in Pharmaceutical Sciences\hfill\end{center}%
          \vspace*{-10pt}\hspace*{4pt}{\fontsize{8}{9}\selectfont Published by JK Welfare \& Pharmascope Foundation\hfill  Journal Home Page: \href{www.ijrps.com}{\color{blue}\underline{\smash{www.ijrps.com}}}}\hspace*{4pt}\mbox{}}}%
       \par\vspace*{-1pt}\rule{\linewidth}{1.3pt}%
       \endminipage%
    \par\vspace*{9.2pt}\parbox{.98\linewidth}{\linespread{.9}\raggedright\fontsize{14}{17}\selectfont #1}%
    \vspace*{-8pt}%
  }
}


\setlength{\parindent}{0pt}
\setlength{\parskip}{0.4pc plus 1pt minus 1pt}

\def\abbrvJournalTitle{Int. J. Res. Pharm. Sci.}

\fancypagestyle{headings}{%
  \renewcommand{\headrulewidth}{0pt}%
  \renewcommand{\footrulewidth}{0.3pt}
  \fancyhf{}%
  \fancyhead[R]{%
    \fontsize{9.12}{11}\selectfont\RunningAuthor,\ \abbrvJournalTitle,\ \ifx\@journalVolume\@empty X\else\@journalVolume\fi%
    \ifx\@journalIssue\@empty\else(\@journalIssue)\fi%
    ,\ \ifx\@firstPage\@empty 1\else\@firstPage\fi-\pageref*{LastPage}%
  }%
  \fancyfoot[LO,RE]{\fontsize{9.12}{11}\selectfont\textcopyright\ International Journal of Research in Pharmaceutical Sciences}%
  \fancyfoot[RO,LE]{\fontsize{9.12}{11}\selectfont\thepage}
}\pagestyle{headings}

                                                         
\fancypagestyle{plain}{%
  \renewcommand{\headrulewidth}{0pt}%
  \renewcommand{\footrulewidth}{0.3pt}%
  \fancyhf{}%
  \fancyhead[R]{%
    \fontsize{9.12}{11}\selectfont\RunningAuthor,\ \abbrvJournalTitle,\ \ifx\@journalVolume\@empty X\else\@journalVolume\fi%
    \ifx\@journalIssue\@empty\else(\@journalIssue)\fi%
    ,\ \ifx\@firstPage\@empty 1\else\@firstPage\fi-\pageref*{LastPage}%
  }%
  \fancyfoot[LO,RE]{\fontsize{9.12}{11}\selectfont\textcopyright\ International Journal of Research in Pharmaceutical Sciences}%
  \fancyfoot[RO,LE]{\fontsize{9.12}{11}\selectfont\thepage}
  \ifx\@firstPage\@empty\else\setcounter{page}{\@firstPage}\fi
}


\def\NormalBaseline{\def\baselinestretch{1.1}}
\usepackage{textcase}
\setcounter{secnumdepth}{0}

\titleformat{\section}[block]{\bfseries\boldmath\NormalBaseline\filright\fontsize{10.5}{13}\selectfont}
{\thesection}
{6pt}
{\MakeTextUppercase{#1}}
[]
\titleformat{\subsection}[block]{\bfseries\boldmath\NormalBaseline\filright\fontsize{10.5}{12}\selectfont}
{\thesubsection}
{6pt}
{#1}
[]
\titleformat{\subsubsection}[block]{\NormalBaseline\filright\fontsize{10.5}{12}\selectfont}
{\thesubsubsection}
{6pt}
{#1}
[]
\titleformat{\paragraph}[block]{\NormalBaseline\filright\fontsize{10.5}{10}\selectfont}
{\theparagraph}
{6pt}
{#1}
[]
\titleformat{\subparagraph}[block]{\NormalBaseline\filright\fontsize{10.5}{12}\selectfont}
{\thesubparagraph}
{6pt}
{#1}
[]

\titlespacing{\section}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\subsection}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\subsubsection}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\paragraph}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\subparagraph}{0pt}{.5\baselineskip}{.5\baselineskip}

\captionsetup[figure]{skip=1.4pt,font=bf,labelsep=colon,justification=raggedright,singlelinecheck=false}
\captionsetup[table]{skip=1.4pt,font=bf,labelsep=colon,justification=raggedright,singlelinecheck=false}

\def\bibyear#1{#1}
%\def\bibjtitle#1{#1}   %%Publisher request
\def\bibauand{}
\setlength\bibsep{3pt}
\setlength\bibhang{8pt}

\makeatother
\date{}


\usepackage[flushleft]{threeparttablex}

\usepackage{float}

\begin{document}


\def\RunningAuthor{Arulmozhi S et al.}
\firstPage{1283}
\articleType{Original Article}
\receivedDate{20 Feb 2021}
\acceptedDate{29 Mar 2021}
\revisedDate{20 Mar 2021}
\journalVolume{2021, 12}
\journalIssue{2}
\journalDoi{ijrps.v12i2.4678}
\copyrightYear{2021}
\def\authorCount{5}
\def\affCount{3}

\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{Formulation of wedelolactone enriched extract with enhanced potential to inhibit cytokines in experimental arthritis}
\author{Atre~B~N\textsuperscript{1},  
            Arulmozhi~S\textsuperscript{*}\textsuperscript{1},  
            Sathiyanarayanan~L\textsuperscript{2},  
            Dhapte-Pawar~V~V\textsuperscript{3}, 
            Mahadik~K~R\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Erandwane, Pune, Maharashtra-411038, India}~\\{\textsuperscript{2}Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Erandwane, Pune, Maharashtra-411038, India}~\\{\textsuperscript{3}Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Erandwane, Pune, Maharashtra-411038, India}}

\begin{abstract}
\textit{Eclipta alba} Linn. (Asteraceae) is traditionally claimed and reported to possess antarthritic and anti-inflammatory activity. Wedelolactone, an active constituent of \textit{Eclipta alba} Linn responsible for its therapeutic effectiveness, is poorly water soluble and less bioavailable. Hence in the present study, we proposed a SNEDDS formulation of wedelolactone rich extracts of \textit{Eclipta alba} Linn. (EAF) and evaluated its effect in Freund's complete adjuvant (FCA) induced arthritis in rats.EAF reduced increase in paw volume and hyperalgesia in 50, 100 and 200 mg/kg doses significantly. It restored all cytokines (Tumor Necrosis Factor-\ensuremath{\alpha }, Interleukin-1\ensuremath{\beta } and Interleukin-6); hematological and biochemical parameters altered due to FCA induction. The histopathology further supported the potential of EAF in arresting the progress of the disease. It protected cartilage destruction and exudation of inflammatory cells dose dependently. In conclusion, the EAF exhibited potent antarthritic potential due to the increased bioavailability of wedelolactone and other constituents. The mechanism of action is mainly mediated through inhibition of cytokines, anti-inflammatory and antioxidant potential of wedelolactone.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Eclipta alba,\newline wedelolactone,\newline cytokines,\newline rheumatoid arthritis
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Arulmozhi~S~\\ Phone:\ 9371085077~\\  Email:\ pharmarul@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
        \textsc{DOI:}\ \href{https://doi.org/10.26452/\@journalDoi}{\textcolor{blue}{\underline{\smash{https://doi.org/10.26452/\@journalDoi}}}}\par%
        \vspace*{-11pt}\hrulefill\\{\fontsize{9.12}{10.12}\selectfont Production and Hosted by}\par{IJRPS |~\href{www.ijrps.com}{www.ijrps.com}}\par%
        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
        \vspace*{-11pt}\rule{\linewidth}{1.2pt}
        \makeatother
        
    
\section{Introduction}
Rheumatoid arthritis is an autoimmune chronic inflammatory condition manifested as pain and stiffness joints due to cartilage destruction. The age group of 35 to 50 years is at higher risk but can occur at any age and affects 1\% of the population globally  \unskip~\citep{1056244:21841716}. The current treatment goals are pain management, prevention of joint inflammation, and suppression of cartilage destruction \unskip~\citep{1056244:21841718}. Research over the years and the discovery of newer agents have optimized the therapeutic approach for rheumatoid arthritis. 

Currently, available therapy includes analgesics, anti-inflammatory, glucocorticoids and disease-modifying anti-rheumatic drugs. Recently, the practice of biological agents has transformed the therapeutic outline of RA and improved the efficacy \unskip~\citep{1056244:21841746}. Still, the various side effects associated with biologicals and high cost drive many researchers to discover cost effective newer agents with more efficacy and fewer side effects. 

\textit{Eclipta alba} Linn. (Asteraceae) is one of the potential medicinal plants reported to have anti-inflammatory activity. The chemical composition of \textit{E. alba}, includes alkenynes, alkaloids, flavonoids, steroids, saponins, coumestans, lipids, polyacetylene, phytosterol and triterpenes \unskip~\citep{1056244:21841715}. One of the constituents, wedelolactone found to possess a variety of activities like antiarthritic \unskip~\citep{1056244:21841711,1056244:21841742}, anti-inflammatory \unskip~\citep{1056244:21841719,1056244:21841741}and antioxidant \unskip~\citep{1056244:21841744}. However, wedelolactone is a poorly water soluble compound and it's reported to have poor absorption through the intestine with poor bioavailability \unskip~\citep{1056244:21841747}, which may influence the pharmacological activity of the extract. Hence it is worthwhile to design a formulation to increase the bioavailability of wedelolactone in the extract and, ultimately, the pharmacological activities.  

Self nano emulsifying drug delivery system (SNEDDS) is a novel formulation comprising of an isotropic mixture of oil surfactant and co surfactant. It is specially designed to enhance the aqueous solubility and eventually absorption of poorly water soluble lipophilic drugs through the oral route. It is a preconcentrate when added in aqueous media, readily disperses with simple agitation to form a thermostable nanoemulsion \unskip~\citep{1056244:21841749}. Prompted by the above fact, this study was aimed to prepare a SNEDDS of \textit{Eclipta alba} Linn enriched with wedelolactone and evaluate preclinically in Freund's complete adjuvant (FCA) induced experimental arthritis. 
    
\section{Materials and Methods}
\textbf{Materials}

Labrasol (Caprylcaproyl Macrogol 8-Glycerides), labrafil M2125CS (Linoleoyl Polyoxyl-6 glycerides) and Labrafac WL 1349 were obtained as gift samples from Gattefosse Inc., Canada, tween 60 (Polyoxyethylene sorbitan monostearate), tween 80 (Polyoxyethylene sorbitan monooleate), tween 20 (Polyoxyethylene sorbitan monolaurate), span 80 (Sorbitan monooleate), span 20 (Sorbitan monolaurate) and propylene glycol were purchased from S.D. Fine Chem. Ltd., Mumbai, India. FCA (Sigma Aldrich, USA), etoricoxib (Zydus Cadila, Gujarat), Wedelolactone (Natural Remedies, India), biochemical diagnostic kits (Accurex Biomedical Pvt. Ltd.), ELISA kits (Ray Biotech Lexington, KY). 

\textbf{Animals }

Animals were maintained with free access to feed and water at 12: 12 light: dark cycle. The approval of protocol was obtained (Approval No CPCSEA/28/2014) from Institutional Animal Ethics Committee (Registration Number: 1703/PO/c/13/CPCSEA).

\textbf{Methods} \textbf{\space }

\textbf{Extraction and Enrichment of extract with active constituents}

The ethanolic extract (EEA) and wedelolactone rich extract of \textit{Eclipta alba} Linn (WEA) was prepared as per previously reported methods \unskip~\citep{1056244:21841711}. 

\textbf{Quantification of Wedelolactone by High Performance Liquid Chromatography (HPLC)}

The solutions of wedelolactone (50\ensuremath{\mu}g/ml) and WEA (1000\ensuremath{\mu}g/ml) were prepared in methanol and quantification of wedelolactone in EEA and WEA were determined using a validated HPLC system (Jasco Corporation, Japan) \unskip~\citep{1056244:21841711}.  

\textbf{Formulation }

\textbf{Selection of lipid}

200 mg WEA was added to 1 ml of lipid viz labrafac, labrafil and labrasol followed vertexing (Remi Mumbai, India) for 20 min to aid proper mixing and extraction in the lipids. The mixtures were kept stable at room temperature for 2 days. Subsequently centrifuged at 12000 rpm for 20 min and the supernatant was collected. The supernatant solution collected, which was then filtered through a Millipore membrane filter (0.45 \ensuremath{\mu}m) followed by dilution with methanol. The solubility was checked visually to get a clear solution. 

\textbf{Selection of surfactant}

The surfactants Tween 20 (HLB=16.7), tween 60 (HLB=14.9) and tween 80 (HLB=15) were selected based on their efficiency to emulsify the specific lipid. To 1 ml of the selected lipid, surfactant at various volumes viz. 0.2,0.4,0.6 ml was added and mixed thoroughly. To study the ease of emulsification, 25 ml of distilled water was added to 25 \ensuremath{\mu}l of the mixture and the number of inversions of the volumetric flask needed to produce a uniform emulsion was recorded. The emulsions were kept for 2 h and checked for stability.

\textbf{Selection of co-surfactant}

Various co-surfactants such as span 20 (HLB=8.6), span 80 (HLB=4.3), and propylene glycol (HLB=3.4) were selected in accordance with emulsification ability. To 1 ml of selected lipid, 0.6 ml surfactant was added and co-surfactants were mixed at various volumes viz. 0.2ml 0.4 ml and 0.6 ml. Distilled water was added to 25 \ensuremath{\mu}l of this mixture to make the final volume 25 ml. The ease of emulsion formation and stability were studied. 

\textbf{Preparation of SNEDDS}

The finalized ratio 1:0.6:0.4 of labrafil: tween80: span 80 was selected for preparation of SNEDDS. To the tube containing 200 mg WEA, 1 ml of labrafil, 0.6 ml of tween 80 and 0.4 ml of span 80 were added, the mixture was vortexed. The prepared EAF (SNEDDS formulation WEA) was stored in a tightly sealed glass bottle at 25\ensuremath{^\circ}C. The stable formulation (EAF) was characterized in terms of dilution studies and self-emulsification time.

\textbf{Characterization of formulations}

\textbf{Self emulsification time}

 The 900 ml of distilled water was taken in the standard USP II dissolution apparatus (37.0 \ensuremath{\pm} 0.5\ensuremath{^\circ}C, 100rpm paddle speed). The formulation (1 ml) was added to this in a dropwise manner. The time taken for the complete disappearance of the formulation was noted. Further stability was assessed for the next 2h. 

\textbf{Robustness to dilution}

SNEDDS was diluted serially to 50, 100 and 1000 times with water in volumetric flask. These flasks were kept aside for 12h and observed for phase separation or precipitation. 

\textbf{Pharmacological Activity}

\textbf{Acute toxicity studies}

The overnight fasted female rats (n=3) were administered 5mg/kg of EAF orally (OECD guidelines 425). Any behavioral change and autonomic profile of each animal was recorded after dosing with special care for the first 4 h and observed daily for 14 days. The same test was performed for 50, 300and 2000 mg/kg of body weight.

\textbf{FCA induced arthritis }

The female wistar rats were assigned into six groups (n=6). Group I~was vehicle control (Blank formulation without extracts), Group II was arthritic control (treated with vehicle), Group III as standard was administered with 10 mg/kg etoricoxib (p.o.), Group IV, V and VI received 50, 100 and 200 mg/kg EAF (p.o.) respectively. On day 1, FCA (0.1 ml) was injected in the left hind paw (subplantar) of all animals of Group II to VI. The respective treatment (p. o) started from day 12 after the appearance of signs of arthritis.

Body weight, paw volume and mechanical hyperalgesia were measured on days 0, 1, 4, 8, 12, 16, 20, 24 and 28\ensuremath{^{th}}. Blood was withdrawn on day 28 under anesthesia by retro orbital puncture and hematology was determined. The serum was separated for estimation of various biochemical and cytokine levels \unskip~\citep{1056244:21841711}. For isolation of liver and ankle joints, animals were sacrificed by CO\ensuremath{_{2}} euthanasia. The liver homogenate was used for estimation of the antioxidant level, while ankle joints were sent for histopathology study \unskip~\citep{1056244:21841711}.


\begin{table*}[!htbp]
\caption{\boldmath {Effect of EAF on hematological, biochemical, cytokine and antioxidant parameters in FCA induced arthritis in rats} }
\label{tw-b838e8ed28b3}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.153\linewidth-2\tabcolsep}p{\dimexpr.127\linewidth-2\tabcolsep}p{\dimexpr.1439\linewidth-2\tabcolsep}p{\dimexpr.1361\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Groups & Vehicle control & Arthritic Control & Etoricoxib 10 mg/kg & EAF 50 mg/kg & EAF 100 mg/kg & EAF 200 mg/kg\\
\tblmidrule 
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Hematological parameters}\\
ESR \mbox{}\protect\newline (mm/h) &
  8.33\ensuremath{\pm} \mbox{}\protect\newline 0.29 &
  15.45\ensuremath{\pm} \mbox{}\protect\newline 0.53\ensuremath{^{\#}} &
  9.93\ensuremath{\pm} \mbox{}\protect\newline 0.54*** &
  14.00\ensuremath{\pm} \mbox{}\protect\newline 0.20* &
  13.13\ensuremath{\pm} \mbox{}\protect\newline 0.20*** &
  11.25\ensuremath{\pm} \mbox{}\protect\newline 0.56***\\
CRP \mbox{}\protect\newline (mg/lit) &
  1.60\ensuremath{\pm} \mbox{}\protect\newline 0.03 &
  7.18\ensuremath{\pm} \mbox{}\protect\newline 0.17\ensuremath{^{\#}} &
  3.68\ensuremath{\pm} \mbox{}\protect\newline 0.14*** &
  6.55\ensuremath{\pm} \mbox{}\protect\newline 0.15*** &
  5.72\ensuremath{\pm} \mbox{}\protect\newline 0.18*** &
  4.35\ensuremath{\pm} \mbox{}\protect\newline 0.05 ***\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Biochemical parameters}\\
TP (gm/dL) &
  7.45\ensuremath{\pm} \mbox{}\protect\newline 0.0646 &
  5.625\ensuremath{\pm} \mbox{}\protect\newline 0.229\ensuremath{^{\#}} &
  6.525\ensuremath{\pm} \mbox{}\protect\newline 0.1652*** &
  6.25\ensuremath{\pm} \mbox{}\protect\newline 0.0.065* &
  6.625\ensuremath{\pm} \mbox{}\protect\newline 0.085*** &
  6.60\ensuremath{\pm} \mbox{}\protect\newline 0.280***\\
AST (U/L) &
  41.17\ensuremath{\pm} \mbox{}\protect\newline 1.457 &
  126.9\ensuremath{\pm} \mbox{}\protect\newline 1.557\ensuremath{^{\#}} &
  106.1\ensuremath{\pm} \mbox{}\protect\newline 2.349*** &
  118.5\ensuremath{\pm} \mbox{}\protect\newline 3.141** &
  116.8\ensuremath{\pm} \mbox{}\protect\newline 1.211*** &
  113.0\ensuremath{\pm} \mbox{}\protect\newline 0.609***\\
ALT (U/L) &
  52.34\ensuremath{\pm} \mbox{}\protect\newline 1.534 &
  188.6\ensuremath{\pm} \mbox{}\protect\newline 1.73\ensuremath{^{\#}} &
  172.2\ensuremath{\pm} \mbox{}\protect\newline 2.923*** &
  180.6\ensuremath{\pm} \mbox{}\protect\newline 0.632** &
  164.1\ensuremath{\pm} \mbox{}\protect\newline 2.558*** &
  155.9\ensuremath{\pm} \mbox{}\protect\newline 1.55***\\
ALP (U/L) &
  74.94\ensuremath{\pm} \mbox{}\protect\newline 2.076 &
  451.6\ensuremath{\pm} \mbox{}\protect\newline 2.514\ensuremath{^{\#}} &
  438.8\ensuremath{\pm} \mbox{}\protect\newline 1.438*** &
  441.0\ensuremath{\pm} \mbox{}\protect\newline 0.713** &
  422.2\ensuremath{\pm} \mbox{}\protect\newline 2.247*** &
  400.3\ensuremath{\pm} \mbox{}\protect\newline 2.205***\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Cytokine levels}\\
TNF- \ensuremath{\alpha }(pg/ml) &
  38.33 \ensuremath{\pm}  \mbox{}\protect\newline 0.98 &
  121.2 \ensuremath{\pm} \mbox{}\protect\newline  1.56\ensuremath{^{\#}} &
  115.8\ensuremath{\pm} \mbox{}\protect\newline  0.70** &
  112.5 \ensuremath{\pm} \mbox{}\protect\newline  0.76*** &
  100.2 \ensuremath{\pm} \mbox{}\protect\newline  1.56*** &
  89.67 \ensuremath{\pm}  \mbox{}\protect\newline 2.319***\\
IL - 1\ensuremath{\beta }(pg/ml) &
  120.5\ensuremath{\pm} \mbox{}\protect\newline 0.78 &
  428.9\ensuremath{\pm}  \mbox{}\protect\newline 1.66\ensuremath{^{\#}} &
  410.5\ensuremath{\pm} \mbox{}\protect\newline  0.97* &
  392.1\ensuremath{\pm} 3.19*** &
  314.1\ensuremath{\pm}  \mbox{}\protect\newline 1.660*** &
  240.1\ensuremath{\pm}  \mbox{}\protect\newline 2.092***\\
IL {\textendash} 6(pg/ml) &
  142.2\ensuremath{\pm}  \mbox{}\protect\newline 1.18 &
  407.3\ensuremath{\pm}  \mbox{}\protect\newline 1.89\ensuremath{^{\#}} &
  397.2 \ensuremath{\pm}  \mbox{}\protect\newline 2.13** &
  397.3 \ensuremath{\pm}  \mbox{}\protect\newline 2.02** &
  380.8 \ensuremath{\pm} \mbox{}\protect\newline 1.77*** &
  354.6\ensuremath{\pm} \mbox{}\protect\newline 1.83***\\
\multicolumn{7}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\cAlignHack Antioxidant parameters}\\
SOD (mU/mg protein) &
  4.508\ensuremath{\pm} \mbox{}\protect\newline 0.025 &
  2.392\ensuremath{\pm} \mbox{}\protect\newline 0.03\ensuremath{^{\#}} &
  2.532\ensuremath{\pm} \mbox{}\protect\newline 0.01** &
  2.543\ensuremath{\pm} \mbox{}\protect\newline 0.01** &
  3.038\ensuremath{\pm} \mbox{}\protect\newline 0.04*** &
  3.188\ensuremath{\pm} \mbox{}\protect\newline 0.03***\\
MDA (nmole MDA/mg protein) &
  1.998\ensuremath{\pm} \mbox{}\protect\newline 0.03 &
  3.447\ensuremath{\pm} \mbox{}\protect\newline 0.016\ensuremath{^{\#}} &
  2.922\ensuremath{\pm} \mbox{}\protect\newline 0.023*** &
  3.327\ensuremath{\pm} \mbox{}\protect\newline 0.02* &
  3.29\ensuremath{\pm} \mbox{}\protect\newline 0.03*** &
  2.793\ensuremath{\pm} \mbox{}\protect\newline 0.03***\\
GSH (\ensuremath{\mu}mol/mg protein) &
  71.01\ensuremath{\pm} \mbox{}\protect\newline 0.42 &
  45.16\ensuremath{\pm} \mbox{}\protect\newline 0.75\ensuremath{^{\#}} &
  50.23\ensuremath{\pm} \mbox{}\protect\newline 0.94*** &
  48.81\ensuremath{\pm} \mbox{}\protect\newline 0.35** &
  51.81\ensuremath{\pm} \mbox{}\protect\newline 0.46*** &
  61.96\ensuremath{\pm} \mbox{}\protect\newline 1.2***\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{EAF: formulation of WEA; TP: Totalprotein, }
    
\item{Represented as mean\ensuremath{\pm} S.E.M.; n=6. ANOVA followed by Dunnett's test was used for analysis. \ensuremath{^{*}}P{\textless}0.05, \ensuremath{^{**}}P{\textless}0.01, \ensuremath{^{***}}P{\textless}0.001compared with Arthritic control and \# P{\textless}0.001 when arthritic control compared to vehicle control.}
\end{tablenotes}
\end{threeparttable}

\end{table*}
\textbf{Statistical Analysis}

One way ANOVA followed by Dunnett's test was used to analyze single day parameter while two way ANOVA followed by Bonferroni's post hoc test to analyze \textit{in-vivo } parameters in Graph Pad Prism software (San Diego, CA). The significance was set at P{\textless}0.05.


\bgroup
\fixFloatSize{images/995cbfa6-30a3-48fe-89b9-ff90b024f85e-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/995cbfa6-30a3-48fe-89b9-ff90b024f85e-upicture1.png}{\includegraphics{images/995cbfa6-30a3-48fe-89b9-ff90b024f85e-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Effect of EAF on body weight in FCA induced arthritis in rats}}
\label{f-1781b87eaf91}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/e5238ebb-6cc8-4e39-859c-098c3be59690-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/e5238ebb-6cc8-4e39-859c-098c3be59690-upicture2.png}{\includegraphics{images/e5238ebb-6cc8-4e39-859c-098c3be59690-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Effect of EAF on paw volume in FCA induced arthritis in rats}}
\label{f-1c6a07ff8ffa}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/3b098b23-2b74-4619-b5cc-01dda5e687c0-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/3b098b23-2b74-4619-b5cc-01dda5e687c0-upicture3.png}{\includegraphics{images/3b098b23-2b74-4619-b5cc-01dda5e687c0-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Effect of EAF on Mechanical hyperalgesia (Paw withdrawal threshold) in FCA induced arthritis in rats }}
\label{f-8eb9f55e15ac}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/7583af93-1532-4968-b961-a34fc4892daa-upicture4.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/7583af93-1532-4968-b961-a34fc4892daa-upicture4.png}{\includegraphics{images/7583af93-1532-4968-b961-a34fc4892daa-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Histopathology of ankle joint}}
\label{f-cf6904f380a6}
\end{figure*}
\egroup

    
\section{Results }
\textbf{Standardization of extracts }

The concentration of wedelolactone was found to be 6.62\% w/w in ethanolic extract and 25.3 \% w/w in WEA by HPLC analysis. This confirmed there was a remarkable enrichment achieved \unskip~\citep{1056244:21841711}. 

\textbf{Formulation Studies}

Based on the solubility profile of extract and emulsifying property, labrafil, tween-80 and span 80 were selected as lipid, surfactant and cosurfactant, respectively for the development of optimal SNEDDS using extract. The final composition of the formulation consisted of 200 mg WEA, which was added to a mixture of 1ml labrafil: 0.6 ml of tween 80:0.4 ml of span 80. 

\textbf{Characterization of formulations}

\textbf{Self emulsification time}

Emulsification ability is a crucial parameter for the evaluation of SNEDDS and can be assessed by the time taken to disperse completely when subjected to dilution under slight agitation using standard USP II dissolution apparatus. The time taken to form a uniform emulsion was 45 sec. The formulation showed uniform and stable emulsion even after 2 h when observed visually.  

\textbf{Robustness to dilution} \textbf{\space }

Even after 24 h, there was no precipitation, cloudiness and phase separation which confirm the stability of emulsion upon reconstitution. 

\textbf{Preclinical evaluation in FCA induced arthritis in rats.}

\textbf{Acute toxicity study}

The autonomic profile and behavior of test animal remained unchanged during the test period. No mortality was observed with 2000 mg/kg of EAF. Hence, EAF was found to be safe till the dose of 2000 mg/kg.

\textbf{Effect of EAF on body weight in FCA induced arthritis in rats}

The body weight decreased gradually and significantly in arthritic animals since day 8. Treatment with etoricoxib significantly (P{\textless}0.05) prevented body weight loss only on day 28 compared to arthritic animals. Furthermore, treatment with EAF 200 mg/kg significantly (P{\textless}0.01, P{\textless}0.001) prevented body weight loss on day 24 and 28, respectively, when compared to arthritic animals (Figure~\ref{f-1781b87eaf91}). While EAF 100 mg/kg had a significant (P{\textless}0.001) effect in arresting the loss of body weight on day 28 compared to arthritic animals. 

\textbf{Effect of EAF on change in paw volume in FCA induced arthritis in rats}

Significant (P{\textless}0.001) increase in paw volume of all FCA injected rats was evident on day 1 and was found to be increased progressively till day 28 in arthritic animals. Treatment with etoricoxib (10 mg/kg) produced a significant (P{\textless}0.05) decrease in the paw volume on day 16, which continued till day 28. After receiving 200 mg/kg of EAF, the paw volume decreased significantly (P{\textless}0.01) on day 16. In contrast to arthritic control animals, treatment with EAF 100 and 50 mg/kg resulted in a significant (P{\textless}0.001) reduction in paw volume from day 20 (Figure~\ref{f-1c6a07ff8ffa}).

\textbf{Effect of EAF on Mechanical hyperalgesia in FCA induced arthritis in rats }

In the arthritic control group, the paw withdrawal threshold decreased significantly (P{\textless}0.001) till day 28. Administration of etoricoxib (10 mg/kg) significantly improved the paw withdrawal threshold (P{\textless}0.01) on day 16 and (P{\textless}0.001) from day 20 onwards. Administration of EAF at dose 200 mg/kg showed significant (P{\textless}0.05, P{\textless}0.001, P{\textless}0.001, P{\textless}0.001 respectively) improvement in paw withdrawal threshold on 16, 20, 24 and 28 days, respectively, when compared to arthritic animals. Treatment with EAF 100 mg/kg caused a significant paw withdrawal threshold on days 16, 20, 24 and 28. However, from day 20 onwards, EAF 50 mg/kg caused significant (P{\textless}0.001) improvement in the paw withdrawal threshold (Figure~\ref{f-8eb9f55e15ac}).

\textbf{Effect of EAF on hematology, biochemical, cytokine and antioxidant parameters in FCA induced arthritis in rats}

Treatment with EAF at various doses significantly decreased ESR in comparison to arthritic animals. FCA induction resulted in significant increase in CRP level, EAF significantly decreased the CRP level dose dependently when compared to arthritic control animals. 

FCA induction produced significant increase in serum AST, ALT and ALP levels in arthritic group animals when compared to vehicle control animals. Etoricoxib and EAF treatment significantly attenuated AST, ALT and ALP levels in comparison with the arthritic control group. FCA induction significantly declined TP level. While animals who received etoricoxib, EAF 100 and 200 mg/kg exhibited a significant increase in TP level. However, EAF 50 mg/kg was found to be less significant in restoring the TP level.

The challenge with FCA produced a significant rise in serum cytokine levels such as TNF-\ensuremath{\alpha }, IL-1\ensuremath{\beta } and IL-6 when contrary to the vehicle control group. Treatment with EAF (100 and 200 mg/kg) caused a significant decline in the serum TNF-\ensuremath{\alpha }, IL-1\ensuremath{\beta }and IL-6 levels in contrast to FCA challenged arthritic animals.

In the arthritic control group, there was a significant decline in GSH and SOD levels and a significant rise in MDA was observed compared with vehicle control animals. A significant increase in GSH and SOD levels and decreased MDA level was observed on treatment with EAF dose dependently (Table~\ref{tw-b838e8ed28b3}).

\textbf{Effect of EAF on histopathological analysis of ankle joint in FCA induced arthritis in rats}

The intact synovium and no signs of inflammation were observed in the ankle joint of vehicle control rats. The ankle joint of the arthritic control animal had a large influx of inflammatory cells, extreme inflammation, and a disrupted synovial lining. EAF 200 mg/kg, as well as etoricoxib treatment, resulted in significant cartilage protection and a low influx of inflammatory cells. EAF (100 mg/kg) treatment caused a moderate disturbance in synovial lining, with very few inflammatory cells, while EAF (50 mg/kg) treated rats showed evidence of disturbed synovial lining and influx of inflammatory cells (Figure~\ref{f-cf6904f380a6}).

\textbf{Discussion}

RA is a chronic autoimmune inflammatory disease with systemic polyarthritis of unknown etiology. It leads to a progressive disability affecting daily activities and quality of life with increased mortality \unskip~\citep{1056244:21841714,1056244:21841748}. Induction of arthritis using FCA is a validated preclinical model for evaluating antiarthritic potency as it mimics clinical features and histopathology of human RA in rats \unskip~\citep{1056244:21841720}. Body weight is an important parameter that signify disease recovery and health status in many diseases. In RA, inflammation reduces the absorption of nutrients from the intestine \unskip~\citep{1056244:21841720}. 

In the present study, EAF treated rats exhibited progressive inhibition in body weight loss compared to arthritic animals. Increased paw volume of FCA injected paw is one of the important signs of inflammation of RA in rats \unskip~\citep{1056244:21841717}. The EAF treatment showed progressive inhibition of inflammation when evaluated at regular intervals. EAF was found to possess potent anti-inflammatory activity. 

RA associated mechanical hyperalgesia is caused mainly due to the increase in various cytokines in the pathogenesis of RA \unskip~\citep{1056244:21841738}. EAF exhibited significant analgesic activity.

An increase in ESR is seen as a result of the presence of acute phase proteins, an important indicator of inflammation. CRP is also a vital biomarker of inflammatory disease with tissue degeneration like RA. On treatment with EAF, ESR and CRP were significantly restored \unskip~\citep{1056244:21841713}.

Measurement of the enzymes like AST, ALT and ALP is a straightforward and effective tool to assess liver and kidney functions which are one of the prime features of RA. EAF significantly reduced these enzyme levels. FCA challenge resulted in decreased total protein level, which was restored on treatment with EAF \unskip~\citep{1056244:21841739}. 

Cytokines play a major role in RA progression. Cytokines are proinflammatory in action, causes chemotaxis and worsen the disease pathology by the destruction of articular cartilage \unskip~\citep{1056244:21841721}. ELISA test results for TNF-\ensuremath{\alpha }, IL-1\ensuremath{\beta } and IL-6 showed a marked increase in the levels while EAF treatment significantly reduced cytokine levels indicating its mechanism in arresting the disease progression.

Injury in RA is due to oxidative damage by lipid peroxidation. The increased level of MDA was significantly decreased by treatment with EAF. While GSH and SOD levels were increased on treatment with EAF. EAF was found to possess significant and potent antioxidant activity \unskip~\citep{1056244:21841745}. 

Furthermore, histopathology of ankle joint revealed that EAF dose dependently protected joint destruction and exudation of inflammatory cells. EAF showed good protection against cartilage destruction due to arthritis.

In comparison to our previous study of wedelolactone enriched extracts of \textit{Eclipta alba}, the antiarthritic potential has improved to the extent of 14.89\% for anti-inflammatory activity and for cytokines inhibition TNF-\ensuremath{\alpha }-16.81\%, IL-1\ensuremath{\beta }- 24.87\%, and IL-6-11.28\% respectively. Thus, the present formulation has increased the antiarthritic potential of extract even at a submaximal dose of extract. 
    
\section{Conclusions}
The results obtained revealed the potent antiarthritic potential of EAF SNEDDS, which is attributed to, formulation strategy as well as Enrichment of wedelolactone active moiety. The antiarthritic potential of EAF is attributed to its strong anti-inflammatory activity as well as cytokine inhibitory activity of wedelolactone extracts.

\textbf{Acknowledgement}

The authors acknowledge the grant received from University Grants Commission (UGC) for the project grant under Major Research Project (MRP) (File No. 42-701/2013 (SR) dated 25/03/2013).

\textbf{Conflict of Interest }

The authors declare that they have no conflict of interest.

\textbf{Funding Support }

University Grants Commission (UGC) for the project grant under Major Research Project (MRP) (File No. 409 42-701/2013 (SR) dated 25/03/2013).
    

\bibliographystyle{pharmascope_apa-custom}

\bibliography{\jobname}
\end{document}
