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\def\citeauthor{~Swathi~Krishna~K~V,
              ~Likitha~E,
              ~Benzawada~Yaswantha,
              ~Chiramana~Umakavya,
              ~Komaragiri~Usha~Sri,
              ~Mulumudi~Suneel,
              ~Sumanth~T,
              ~Vemuru~Vamsi}
\def\citetitle{Evaluation of Anti-Inflammatory Activity on Whole Plant of \textit{Aerva lanata} (L.) by \textit{In-Vitro} Methods}
\def\copyrightstmt{This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial- ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.}
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\def\RunningAuthor{Likitha E et al.}
\firstPage{134}
\articleType{Original Article}
\receivedDate{20 Jun 2021}
\acceptedDate{01 Jul 2021}
\revisedDate{29 Jun 2021}
\journalVolume{1}
\journalIssue{3}
\journalDoi{fjphs.v1i3.222}
\copyrightYear{2021}
\eISSN{2583-116X}
\journalDoi{fjphs.v1i3.222}
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\def\journalTitle{Future Journal of Pharmaceuticals and Health Sciences}

\title{Evaluation of Anti-Inflammatory Activity on Whole Plant of \textit{Aerva lanata} (L.) by \textit{In-Vitro} Methods}
\author{Swathi~Krishna~K~V\textsuperscript{1},  
            Likitha~E\textsuperscript{*}\textsuperscript{2},  
            Benzawada~Yaswantha\textsuperscript{3},  
            Chiramana~Umakavya\textsuperscript{3},  
            Komaragiri~Usha~Sri\textsuperscript{3},  
            Mulumudi~Suneel\textsuperscript{3},  
            Sumanth~T\textsuperscript{3}, 
            Vemuru~Vamsi\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Pharmaceutics, Saastra College of Pharmaceutical Education \& Research, Near Varigonda Jwalamukhi Temple, Muthukur Raod, Kakupalli, Nellore-524 311 Andhra Pradesh, India}~\\{\textsuperscript{2}Department of Pharmacy practice, Saastra College of Pharmaceutical Education \& Research, Near Varigonda Jwalamukhi Temple, Muthukur Raod, Kakupalli, Nellore-524 311, Andhra Pradesh, India}~\\{\textsuperscript{3}Saastra College of Pharmaceutical Education \& Research, Near Varigonda Jwalamukhi Temple, Muthukur Raod, Kakupalli, Nellore-524 311, Andhra Pradesh, India}}

\begin{abstract}
To evaluate the anti-inflammatory events of plant extracts going from \textit{A.lanata} by \textit{In-vitro} strategies\textbf{. } The main objectives of the proposed work is to evaluate anti-inflammatory utilization of aqueous (AQEAL) and alcoholic (ALEAL) extracts containing plant of \textit{Aerva lanata} plant in different models of experimental. The Whole plant of \textit{A.lanata} plant powder was made and extracted with 95\% alcohol and water (maceration process) to get alcoholic (ALEAL) and aqueous (AQEAL). Preliminary phyto-chemical studies with AQEAL and ALEAL revealed sensational ubiquity of phytoconstitutents of both extracts. Both the extracts of whole plant of \textit{A.lanata } had prevented protein denaturation against heat induced proteolysis. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Anti-Inflammatory,\newline Whole Plant,\newline Aerva lanata,\newline In-vitro Methods
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright{\Large$^\ast$}Corresponding Author\par Name:\ Likitha~E~\\ Phone:\ +91 70324 69879~\\  Email:\ likkymenen711@gmail.com
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\section{Introduction}
Inflammation whatever need the general treatment as chronic time period and the use of medicine can cause numerous unwanted effects, usually with damage of significant organs \unskip~\cite{1138204:22527080}. But in herbal/traditional medicines, there are several preparations prescribed to treat hyperacidity and arthritics. One of the most vital outgrowth thinking about the general defense reaction opposed to local injury plus diseases, but it frequently develops to painful or pathologically harmful infections involving pharmacologic treatment. 

\textit{Aerva lanata} (L) is a tropical plant which is grown extensively in India, Arabia, Africa, Sri Lanka, Phillipines and Java. It's a perennial weed associated with sensational family Amaranthaceae. \textit{Aerva lanata} may be a medicine that justify remedy a diverse disorder specified helminthic, diabetes, inflammation. 

Therein study we have a tendency to aim to research the aqueous extract going from \textit{A. lanata} whole plant for phytochemical composition, membrane stabilization method, protein denaturation method, Heat induced hemolysis \unskip~\cite{1138204:22527086}.
    
\section{Materials and methods }
Diclofenac sodium used to be buying from Sigma-Aldrich Chemical Company USA. Egg albumin, Phosphate buffered pH 6.8, Normal saline was purchased from Himedia Laboratories Private Limited Mumbai, India. Almost all chemical substances used have an analytical grade.


\begin{table*}[!htbp]
\caption{\boldmath {Nature and Percentage yield of the extracts} }
\label{tw-c62ab8b3d435}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10719999999999999\linewidth-2\tabcolsep}p{\dimexpr.2629\linewidth-2\tabcolsep}p{\dimexpr.12040000000000003\linewidth-2\tabcolsep}p{\dimexpr.17349999999999998\linewidth-2\tabcolsep}p{\dimexpr.33599999999999994\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S. No. & Name of the extract & Nature & Color & \cAlignHack \% yield in g (\% w/w)\\
\tblmidrule 
1. &
  AQEAL &
  Sticky &
  Dark brown &
  \cAlignHack 12.5 \%\\
2. &
  ALEAL &
  Sticky &
  Dark green &
  \cAlignHack 10\%\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Plant material}

\textit{A. lanata} used to be self-collected of the natural universal growing in the Seshachalam Forest Area, Chittoor district, Andhra Pradesh, India \unskip~\cite{1138204:22527084}.

\textbf{Processing of plant }

The whole plant of \textit{A. lanata }was collected and washed thoroughly in distilled water and cut into small pieces. The whole plant was shade dried at room temperature. The powder was extracted in distilled water using a Soxhlet apparatus \unskip~\cite{1138204:22527079}. These extracts were concentrated with a rotary evaporator and dried using lyophilizer. 

\textbf{Phytochemical screening }

The powder was screened for the presence of sterols, glycosides, Saponins, Alkaloids, flavonoids, proteins, amino acids, gums and mucilage \unskip~\cite{1138204:22527081}.

\textbf{Methodology}

\textbf{Preparation of aqueous extract}

About 200g of powder of \textit{A.lanata} was taken into a TLC chamber and macerated with 2 liters of distilled water plus 20ml going from ethyl alcohol (preservatives) as 7 days as well as infrequent shaking for 3 times in a day. The marc used to be taken away by way of tracking the extract and the aqueous extract (AQEAL) evaporated at room temperature until the solvent was evaporated \unskip~\cite{1138204:22527085}.

\textbf{Preparation of alcoholic extract}

Almost 100 grams consisting of powder of \textit{A.lanata} was taken into a conical flask and macerated with 400 ml of ethanol for 7 days with occasional shaking for 3 times in a day. The marc was once got rid of by filtering the squeeze its alcohol squeeze (ALEAL) was distilled, alcohol was recollected under distillation, until to obtain a residue of alcohol and that was dried at room temperature \unskip~\cite{1138204:22527082}. 

\textbf{Inhibition of albumin denaturation\ensuremath{^{}}}

The reaction mixture (5 ml) consisted of 0.2 ml of egg albumin, 2.8 ml of phosphate buffered pH 6.4 and 2 ml of varying concentrations of extract so that final concentrations. Similar volume of double distilled water served as control. Then the mixtures were incubated at 37\ensuremath{^{0}}C\ensuremath{\pm}2\ensuremath{^{0}}C in a BOD incubator for 15mins and then heated at 70\ensuremath{^{0}}C for 5mins \unskip~\cite{1138204:22527088}. Diclofenac Sodium at the final concentration of was used as reference drug and treated similarly for determination of absorbance. The percentage inhibition of protein denaturation was calculated by using the following formula:
\begin{eqnarray*}\style{font-family:'Times New Roman'}{\style{font-size:8px}{\%\;of\;Inhibition\;=100\;X\;\lbrack V_t/V_c-1\rbrack}} \end{eqnarray*}
Where, 

V\ensuremath{_{t}} = absorbance of test sample, 

V\ensuremath{_{c}} = absorbance of control.

\textbf{Membrane stabilization test \ensuremath{^{}}}

\textbf{Preparation}\textbf{ of }\textbf{Red }\textbf{Blood cells } \textbf{(RBCs) suspension}

Fresh blood (10 ml) used to be self collected and reassigned to with the heparinzed centrifuged tubes. The tubes have been centrifuged at 3000 rpm for 10 min and have been scrubbed 3 times with equivalent volume of regular saline \unskip~\cite{1138204:22527083}. The volume of the blood was measured and reconstituted as 10\%v/v suspension with normal saline.

\textbf{Heat induced hemolysis}

The reaction mixture (2 ml) consisted of ml of 10\% RBCs suspension and 1 ml of varying concentrations of extract and standard drug so that final concentrations. Diclofenac Sodium was taken as a standard drug. All the centrifuge tubes containing reaction mixture were incubated in a water bath at 56\ensuremath{^{o}}C for 30 min. At the end of the incubation, the tubes were cooled under running tap water \unskip~\cite{1138204:22527087}. The reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm.
    
\section{RESULTS \& DISCUSSION }
\textbf{Qualitative Phytochemical analysis}

Preliminary phyto-chemical analysis of the AQEAL and ALEAL have revealed the presence of sterols, alkaloids, glycosides, flavanoids, tannins and carbohydrates, saponins, proteins and amino acids in both the extracts [Table~\ref{tw-dc2830a59168}].

\textbf{Preparation of alcoholic extract}

The AQEAL and ALEAL were examined for his colour and consistency as well as their percent yield used to be calculated with regards to the amount used extraction, stored in air-tight plastic containers in a refrigerator beneath 4\ensuremath{^{o}}C [Table~\ref{tw-c62ab8b3d435}].


\begingroup
\makeatletter\if@twocolumn\@ifundefined{theposttbl}{\gdef\TwoColDocument{true}\onecolumn\onecolumn}{}\fi\makeatother \setlength\LTcapwidth{\textwidth}
\begin{longtable}{p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.362\linewidth-2\tabcolsep}p{\dimexpr.165\linewidth-2\tabcolsep}p{\dimexpr.223\linewidth-2\tabcolsep}}
\caption{\boldmath {Details of Qualitative Phyto-chemical tests} }
\label{tw-dc2830a59168}
\def\arraystretch{1.1}\\\endfirsthead \hline \noalign{\vskip3pt} \noalign{\textit{Table \thetable\ continued}} \noalign{\vskip3pt} 
\tbltoprule \rowcolor{kwdboxcolor}Test & Indication & AQEAL & ALEAL\\
\tblmidrule \endhead \hline \noalign{\vskip3pt} \noalign{\textit{\hfill Continued on next page}} \noalign{\vskip3pt} \endfoot \endlastfoot 
\tbltoprule \rowcolor{kwdboxcolor}Test & Indication & AQEAL & ALEAL\\
\tblmidrule 
\textbf{a)Tests for sterols} &
   &
   &
  \\
1.Salkowski's test: CHCL3+Conc.H2SO4from sidelong retaining wall the test tube. &
  Presence of yellow colored ring between junctions of 2 solutions observed which turned into red colored ring after 2 mins &
  + &
  +\\
2.Libermann Burchard's test: \mbox{}\protect\newline CHCL3  + (CH3CO)2O + Conc.H2SO4  from side long retaining wall the test tube.  &
  Presence of violet to blue colored ring between junctions of 2 solutions observed &
  + &
  +\\
\textbf{b)Tests for Glycosides} &
   &
   &
  \\
1.Baljet's test : \mbox{}\protect\newline Solution A:1g picric acid in 100ml ethyl alcohol \mbox{}\protect\newline Solution B:10gs NaOH in 100ml distilled water \mbox{}\protect\newline Both solutions combined and 2 to 3 drops of combined solution in 2 to 3 mg of test sample &
  Formation of orange to deep red color  &
  + &
  +\\
2.Keller-killiani test:  Extract 50mg was dissolved in 2ml Chloroform. \mbox{}\protect\newline H2SO4 was added to form a layer and the color at interphase recorded  &
  Presence of brown ring at interphase  &
  + &
  +\\
3.Borntrager's test: Extract +Dil. H2SO4, boil and filter.  \mbox{}\protect\newline filtrate + CHCL3. \mbox{}\protect\newline Separate CHCL3 layer and add dil.NH3 &
  Formation of pink to red color. &
  + &
  +\\
\textbf{c)Test for Saponins} &
   &
   &
  \\
Foam test: Extract shaken with water &
  Presence of foam observed &
  + &
  +\\
\multicolumn{4}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\textbf{d)Test for Carbohydrates} \mbox{}\protect\newline The extract (50mg) dissolved in 3ml of water and filtered. The filtrate was subjected to the following tests. }\\
1.Molish's Test: 2ml filterate + \ensuremath{\alpha }-napthol+1ml H2SO4 &
  Presence of violet ring &
  + &
  +\\
2.Barfoed's test: 1ml filtrate +Barfoed's reagent (heated for 2 mins) &
  Presence of red precipitate &
  + &
  +\\
3.Benedict' test: 0.5ml filtrate + Benedict's reagent (heated for 2 mins) &
  Characteristic colored precipitate &
  + &
  +\\
4.Fehling's test: 0.5ml of extract+0.5ml of both Fehling's A and B solutions heated for 2 mins. &
  Characteristic colored precipitate &
  + &
  +\\
\multicolumn{4}{p{\dimexpr(1\linewidth-2\tabcolsep)}}{\textbf{E)Test For Alkaloids} \mbox{}\protect\newline The Extract 50mg stirred up along with some milliliter going from HCl and screened, the filtrate used to be well-tired as well tried as well as varied alkaloid testing agent as follows }\\
1.Mayer's test: \mbox{}\protect\newline Mayer's reagent +Filtrate &
  Creamy precipitate &
  + &
  +\\
2.Wagner's test: \mbox{}\protect\newline Wagner's reagent + Filtrate &
  Reddish {\textendash} Brown precipitate &
  + &
  +\\
3.Hager's test: \mbox{}\protect\newline Hager's reagent + Filtrate &
  Prominent yellow precipitate &
  + &
  +\\
\textbf{f)Test for Flavonoids} &
   &
   &
  \\
1.Ferric chloride test: \mbox{}\protect\newline Extract + FeCl3 &
  Formation of deep green color &
  + &
  +\\
2.Shinoda test : \mbox{}\protect\newline Extract + dil. HCl and Magnesium turning  &
  Presence of red color &
  + &
  +\\
3.Lead acetate test: &
   &
  + &
  +\\
\textbf{g)Test for Tannins} &
   &
   &
  \\
1.Ferric chloride test: Extract + FeCl3 &
  Formation of deep green color &
  + &
  +\\
2.Gelatin test &
   &
  + &
  +\\
 &
   &
   &
  \\
\textbf{h)Test for Proteins and amino acids } &
   &
   &
  \\
1.Millon's test : Filtrate + Millon's reagent &
  Presence of white precipitate &
  + &
  +\\
 &
   &
   &
  \\
2.Ninhydyin test : Filtrate + Ninhydrine solution  &
  Formation of purple color &
  + &
  +\\
 &
   &
   &
  \\
\textbf{i)Tests for Gum, Mucilage} &
   &
   &
  \\
1.Swelling test : Dry powder + water &
  Powder swells &
  + &
  +\\
 &
   &
   &
  \\
2.Molisch's test: 2 ml filtrate + \ensuremath{\alpha }-Naphthol + 1ml H2SO4 &
  Presence of Violet ring &
  + &
  +\\
\tblbottomrule 
\end{longtable}
\endgroup
\makeatletter\@ifundefined{TwoColDocument}{}{\twocolumn}\makeatother 

\begin{table}[!htbp]
\caption{\boldmath {\textit{In{\textendash}vitro} Anti-inflammatory activity of AQEAL \& ALEAL by Membrane stabilization method} }
\label{tw-356e7a9bcd0b}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3155\linewidth-2\tabcolsep}p{\dimexpr.32790000000000006\linewidth-2\tabcolsep}p{\dimexpr.3566\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Test sample & \cAlignHack Conc.(\ensuremath{\mu}g/ml) & \cAlignHack \% Protection\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.3155\linewidth-2\tabcolsep)}}{\multirow{5}{\linewidth}{\cAlignHack Aqueous extract of \textit{Aerva Lanata}}} &
  \cAlignHack 50 &
  \cAlignHack 23.8\\
 &
  \cAlignHack 100 &
  \cAlignHack 35.6\\
 &
  \cAlignHack 200 &
  \cAlignHack 56.3\\
 &
  \cAlignHack 400 &
  \cAlignHack 78.6\\
 &
  \cAlignHack 800 &
  \cAlignHack 104.4\\
\multicolumn{1}{p{\dimexpr(.3155\linewidth-2\tabcolsep)}}{\multirow{5}{\linewidth}{\cAlignHack Alcoholic extract of \textit{Aerva Lanata}}} &
  \cAlignHack 50 &
  \cAlignHack 27.07\\
 &
  \cAlignHack 100 &
  \cAlignHack 42.9\\
 &
  \cAlignHack 200 &
  \cAlignHack 71.3\\
 &
  \cAlignHack 400 &
  \cAlignHack 94.9\\
 &
  \cAlignHack 800 &
  \cAlignHack 119.4\\
\multicolumn{1}{p{\dimexpr(.3155\linewidth-2\tabcolsep)}}{\multirow{5}{\linewidth}{\cAlignHack Effect of Diclofenac sodium \mbox{}\protect\newline (std .drug)}} &
  \cAlignHack 50 &
  \cAlignHack 22.9\\
 &
  \cAlignHack 100 &
  \cAlignHack 31.2\\
 &
  \cAlignHack 200 &
  \cAlignHack 96.4\\
 &
  \cAlignHack 400 &
  \cAlignHack 119.7\\
 &
  \cAlignHack 800 &
  \cAlignHack 132.8\\
\tblbottomrule 
\end{tabulary}\par 
\end{table}

\begin{table}[!htbp]
\caption{\boldmath {AQEAL \& ALEAL by Protein denaturation method} }
\label{tw-a3513e924874}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3321\linewidth-2\tabcolsep}p{\dimexpr.3279\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Sample & \cAlignHack Conc.(\ensuremath{\mu}g/ml) & \cAlignHack \% Protection\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.3321\linewidth-2\tabcolsep)}}{\multirow{5}{\linewidth}{\cAlignHack Aqueous extract of \textit{Aerva Lanata}}} &
  \cAlignHack 50 &
  \cAlignHack 15.8\\
 &
  \cAlignHack 100 &
  \cAlignHack 31.08\\
 &
  \cAlignHack 200 &
  \cAlignHack 75.3\\
 &
  \cAlignHack 400 &
  \cAlignHack 82.9\\
 &
  \cAlignHack 800 &
  \cAlignHack 129.3\\
\multicolumn{1}{p{\dimexpr(.3321\linewidth-2\tabcolsep)}}{\multirow{5}{\linewidth}{\cAlignHack Alcoholic extract of \textit{Aerva Lanata}}} &
  \cAlignHack 50 &
  \cAlignHack 17.5\\
 &
  \cAlignHack 100 &
  \cAlignHack 45.1\\
 &
  \cAlignHack 200 &
  \cAlignHack 76.5\\
 &
  \cAlignHack 400 &
  \cAlignHack 104.9\\
 &
  \cAlignHack 800 &
  \cAlignHack 130.2\\
\multicolumn{1}{p{\dimexpr(.3321\linewidth-2\tabcolsep)}}{\multirow{5}{\linewidth}{\cAlignHack Effect of Diclofenac sodium \mbox{}\protect\newline (std .drug)}} &
  \cAlignHack 50 &
  \cAlignHack 5.2\\
 &
  \cAlignHack 100 &
  \cAlignHack 13.1\\
 &
  \cAlignHack 200 &
  \cAlignHack 31.6\\
 &
  \cAlignHack 400 &
  \cAlignHack 63.9\\
 &
  \cAlignHack 800 &
  \cAlignHack 128.1\\
\tblbottomrule 
\end{tabulary}\par 
\end{table}
Anti-inflammatory effect of AQEAL and ALEAL have been planned considerably by testing varied \textit{in-vitro} parameters. The consequence of AQEAL on membrane stabilization at abundant dose ranges 50, 100, 200, 400 and 800\ensuremath{\mu}g/ml showed significant protective cover against RBC membrane damage found to be 23.8\%, 35.6\%, 56.3\%, 78.6\% and 104.4 \% respectively.

The effect of ALEAL on membrane stabilization at dose intervals 50, 100, 200, 400 and 800 \ensuremath{\mu}g/ml had shown vital protective covering against RBC membrane damage found to be 27.07\%, 42.9\%, 71.3\%, 94.9\%, and 119.4\% respectively.

The effect of Diclofenac sodium on membrane stabilization at abundant recall dose levels 50, 100, 200, 400 and 800\ensuremath{\mu}g/ml had shown vital protection against RBC membrane damage found to be 22.9\%, 31.2\%, 96.4\%, 119.7\% and 132.8\% respectively [Table~\ref{tw-356e7a9bcd0b}].

The effect going from AQEAL on hold up of protein denaturation at poles apart dose levels 50, 100, 200, 400 and 800 \ensuremath{\mu}g/ml had shown significant protection against denaturation of proteins found to be 15.8\%, 31.08\%, 75.3\%, 82.9\% and 129.3\% respectively.

The effect going from ALEAL on hold up of protein denaturation abundant dose intervals 50, 100, 200, 400 and 800 \ensuremath{\mu}g/ml had shown significant protective covering opposed to denaturing of proteins found to be 17.5\%, 45.1\%, 76.5\%, 104.9\% and 130.2\%respectively [Table~\ref{tw-a3513e924874}].

The results of Diclofenac Sodium on delay of proein denaturing at abundant dose levels 50, 100, 200, 400 and 800 \ensuremath{\mu}g/ml had shown vital protective cover opposed to denaturing going from protein found to be 2.2\%, 13.1\%, 31.6\%, 63.9\% and 128.1\% respectively.
    
\section{CONCLUSION}
Therefore, it can be concluded that AQEAL \& ALEAL of whole plant possessed with anti-inflammatory activity. Preliminary phytochemical analysis the AQEAL plus ALEAL submit to unconcealed sensational ubiquity of sterols, alkaloids, glycosides, flavanoids, tannins and carbohydrates, saponins, proteins and amino acids in both the extracts. Both the extracts of whole plant of \textit{A.lanata } had prevented protein denaturation against heat induced proteolysis. Both the extracts of whole plant of \textit{A.lanata } had prevented membrane stabilization against heat induced hemolysis. Above two inferences strongly suggested that Anti-inflammatory activity of both the extracts exhibited by \textit{In-vitro } methods. However, AQEAL of whole plant has produced slightly better anti-inflammatory activity than the ALEAL of whole plant.
    
\section{ACKNOWLEDGEMENT}
I would like to thank Dr G. H. Srinivaasa Rao Sir (Founder and Manager) Saastra College of Pharmaceutical Education \& Research, Near Varigonda Jwalamuhi Temple, Muthukur Raod, Kakupalli, Nellore-524 311 Andhra Pradesh, India.

\textbf{Funding Support}

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

\textbf{Conflict of Interest}

The authors declare that there is no conflict of interest for this study.


    

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