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\def\citeauthor{~Suchitra~M,
              ~Harinadha~Baba~K,
              ~Yamini~R,
              ~Sri~Harshitha~P,
              ~Surekha~G,
              ~Pavani~T,
              ~Sakshi~Jain}
\def\citetitle{To develop and evaluate the \textit{in-vitro} anti oxidant and anti-inflammatory activity of polyherbal Gel}
\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{Monapati Suchitra et al.}
\firstPage{377}
\articleType{Original Article}
\receivedDate{10 Jul 2023}
\acceptedDate{27 Jul 2023}
\revisedDate{26 Jul 2023}
\journalVolume{3}
\journalIssue{3}
\journalDoi{fjphs.v3i3.496}
\copyrightYear{2023}
\eISSN{2583-116X}
\journalDoi{fjphs.v3i3.496}
\def\authorCount{7}
\def\affCount{3}

\def\journalTitle{Future Journal of Pharmaceuticals and Health Sciences}

\title{To develop and evaluate the \textit{in-vitro} anti oxidant and anti-inflammatory activity of polyherbal Gel}
\author{Suchitra~M\textsuperscript{*}\textsuperscript{1}\textsuperscript{\href{https://orcid.org/0000-0003-4792-6700}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}},  
            Harinadha~Baba~K\textsuperscript{2},  
            Yamini~R\textsuperscript{3},  
            Sri~Harshitha~P\textsuperscript{3},  
            Surekha~G\textsuperscript{3},  
            Pavani~T\textsuperscript{3}, 
            Sakshi~Jain\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Pharmaceutical chemistry, Narayana Pharmacy College, Chinthareddy Palem, Nellore - 524003, Andhra Pradesh, India}~\\{\textsuperscript{2}Department of Pharmaceutical Analysis, Narayana Pharmacy College, Chinthareddy Palem, Nellore {\textendash} 524003, Andhra Pradesh, India}~\\{\textsuperscript{3}Narayana Pharmacy College, Chinthareddy Palem, Nellore - 524003, Andhra Pradesh, India}}

\begin{abstract}
Herbal medicines are used for their safety, efficacy, cultural acceptability and lesser side effects. The chemical constituents present in plants are a part of the physiological functions of living system and hence they are believed to have better compatibility with the human body. These drugs are made from renewable resources of raw materials by eco-friendly processes and will bring economic prosperity. An herb is a plant or plant part used for its scent, flavor, or therapeutic properties. They are sold as tablets, capsules, powders, teas, extracts, and fresh or dried plants. People use herbal medicines to try to maintain or improve their health. Products made from botanicals, or plants, that are used to treat diseases or to maintain health are called herbal products, botanical products, or phytomedicines. A product made from plants and used solely for internal use is called an herbal supplement. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Herbal treatments,\newline in-vitro,\newline World Health Organisation,\newline Curcuma longa
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright{\Large$^\ast$}Corresponding Author\par Name:\ Suchitra~M~\\ Phone:\ 90525 82816~\\  Email:\ jajulasuchitra@gmail.com
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\section{Introduction}
Herbal treatments are employed because they are safe, effective, culturally acceptable, and have fewer negative effects. Because plant chemical ingredients are part of the physiological operations of living systems, they are thought to be more compatible with the human body \unskip~\cite{2012982:28578071}. These pharmaceuticals will create economic prosperity because they are made from renewable raw ingredients using eco-friendly procedures \unskip~\cite{2012982:28578072}. Herbs are plants or plant parts that are utilised for their smell, flavour, or therapeutic effects. Dietary supplements include herbal medicines \unskip~\cite{2012982:28578073}. They are available in the form of tablets, capsules, powders, teas, extracts, and fresh or dried plants. Herbal remedies are used by people in an attempt to preserve or enhance their health \unskip~\cite{2012982:28578074}. Herbal products, botanical products, or phytomedicines are products made from botanical or plants that are used to treat illnesses or maintain health \unskip~\cite{2012982:28578075}. An herbal supplement is a plant-derived substance that is only used internally. Medicinal plants, commonly referred to as medicinal herbs, have been identified and utilised in traditional therapeutic practises since prehistoric times. Plants generate hundreds of chemical substances for a number of purposes, including resistance and protection from insects, fungi, illnesses, as well as herbivorous mammals. Whether in modern or traditional medicine, medicinal plants are used to preserve health, to treat a specific illness, or both. In 2002, the Food and Agriculture Organisation estimated that over 50,000 medicinal plants were utilised globally. In 2016, the Royal Botanic Gardens, Kew estimated that 17,810 plant species have a therapeutic application, out of around 30,000 plants for which usage of any type is documented. Plants have been the foundation of various traditional medicinal systems around the world, and they continue to supply humans with novel treatments. The World Health Organisation (WHO) describes traditional medicine (including herbal medications) as therapeutic practises that existed before the establishment and spread of modern medicine, frequently for hundreds of years, and are still in use today. Herbal medicine is a combination of the therapeutic experience of a generation of indigenous medicine practitioners. Traditional remedies include medicinal herbs, minerals, organic substances, and so on. Herbal medications have  been  in  use in Indian, Chinese, Syrian, Roman, Egyptian, Greek texts for thousands of years as per  recorded  evidences. 
    
\section{Materials  and  Methods}
\textbf{Materials}

Curcuma  longa,  Zingiber  officinale,  Ocimum  tenuiflorum, ethanol,  ammonium  chloride,  silica  gel-G,  dihydrogen  phosphate,  sodium  chloride,  iodine,  hexane,  ethyl  acetate,  Carbopol,  triethanolamine,  benzoic  acid,  sodium  lauryl  sulphate,  glycerin,  dragondroff's  reagent,  fehling's  reagent,  ferric  chloride,  sodium  acetate,  hydrogen  peroxide,  ascorbic  acid,  phosphate  buffer, diclofenac  sodium,  fresh  hen's  egg  albumin,  methylparaben  eucalyptus  oil.

\textbf{Plant  materials:}

Rhizomes  of  Curcuma  longa,  rhizomes  of  Zingiber  officinale  and  leaves  of  Ocimum  tenuiflorum was collected from Gudur, Nellore district, Andhra Pradesh, India. The plant  specimen was verified to be of the correct species by Dr.K. Vishnu Vardhan, a botanist from  the  Department  of  Botany,  S.K.R.  Government  junior  college,  Gudur.

\textbf{Principle of soxhlation:}

The components are extracted utilising the condensed vapours of the solvent by the Soxhlet extractor. The sample powder comes into touch with the condensed vapours, and the soluble part of the powder is combined with the solvent.

\textbf{Preparation  of  extract:}

The Soxhlet extraction process was used to prepare aqueous-ethanolic (30:70) extract of  polyherbal formulation for 4 extracts (1:1:1). The collected plant materials were shade dried  until the moisture was removed from the plant materials. After drying the plant materials was  grinded to a coarse powder and stored in a sealed container for further experiments. The  polyherbal formulation of (1:1:1) were taken 10 g of tulsi, turmeric and ginger powders. After  preparing thimble pour ethanol onto the thimble and macerate for 2 hours. After maceration  starts  the  soxhlation apparatus.

After  completion  of  soxhlation  process  keeps  the  reflex  condenser  for  recollecting  the  solvent  used  in  the  soxhlation.

\textbf{Evaluation  of  polyherbal  powders}

\textbf{Detection  of  total  ash:}

In  an  ignited  and  weighted  silica  crucible,  polyherbal  powder  (1:1:1)  2grms  was  placed.  It was burnt at a low temperature until it was after being rid of carbon, it was cooled and weighed. The percentage of ash was estimated using the  air-dried.

\textbf{Detection  of  water-soluble  ash:}

Total ash was added to 25ml of water and heated for 5 min then filtered through ash-free  filter  paper  (Whatman  filter  paper)  the  water  insoluble  substance  was  weighed  after  the filter paper was rinsed in hot water and burned in the silica crucible. The water soluble ash was determined by removing the water-insoluble  materials from  total ash.

\textbf{Detection  of  extractive  values:}

\textbf{Alcoholic  soluble  extractive:}

Sample(5g) was weighed and macerated in 100ml of alcohol in a closed flask for  24hrs. to prevent alcohol loss, the contents of the flask were rapidly filtered, then 25ml of the filtrate was evaporated until it was dry at  105\ensuremath{^{o}}c  in  a  tarred  shallow  dish  and then  weighed.  Using  the  air-dried  powder,  the  proportion  of  alcohol-soluble  extractive  was  calculated.

\textbf{Moisture  content:}

To determine moisture content, a 5g pre-drying weight sample was placed in the oven. The pre-drying weight was compared to the post-drying weight to assess how much moisture was removed. The  moisture  content  was  calculated as a  percentage.

\textbf{Qualitative  phytochemical  screening  of  ethanolic  extract}

The  4  Ethanolic  extract  of  poly  herbal  formulation  were  screened  for  their  chemical  constituents. A little amount of dried extract was utilised to determine the alkaloids, carbohydrates, and other constituents,  phenols. The following procedures are used to isolate flavonoids, steroids, as well as triterpenoids. The  total  flavonoid  content of the ethanolic  extract  of CZO.  was  estimated  by  reported  method. Aluminum  chloride  method  used  for  flavonoid  content determination \unskip~\cite{2012982:28657469,2012982:28711899}.

\textbf{In vitro anti-oxidant  activity}

\textbf{Hydrogen  peroxide  scavenging  activity}

Hydrogen peroxide (H2O2) belongs to a non-radical reactive species that is produced in the body by various oxidising enzymes such as SOD. It is the least reactive ROS yet has the greatest ability to cross biological membranes. 

1  ml  of  sample  (1  mg/ml)  was  mixed  with  3  ml  of methanol,  0.2  ml  of  10\%  aluminium  chloride, 0.2 ml of 1 M potassium acetate and 5.6 ml of distilled water and remains at  room  temperature  for  30  min.

The absorbance of the reaction mixture was measured at 420nm with UV-Visible  spectrophotometer \unskip~\cite{2012982:28657470}.

The content was determined from extrapolation of calibration curve which was made  by  preparing gallic acid  solution (50-150 \ensuremath{\mu}g/ml) in  distilled water. Calibration curve for gallic acid was obtained by plotting absorbance on Y-axis and  their  corresponding concentration on X-axis. The  concentration  of  flavonoids  was expressed  in  terms of  \ensuremath{\mu}g/ml.

\textbf{In  Vitro Anti-Inflammatory  Activity}

\textbf{Protein  Denaturation  Method:}

Protein  denaturation  method  was  used  for  the  estimation  of  anti-inflammatory  activity

\textbf{In Vitro}

In a beaker mix 0.2 ml of egg albumin (collected from fresh hen's egg), 2.8 mL of phosphate buffer saline (PBS pH-6.4) as well as 2mL of diclofenac sodium at various concentrations (10, 20, 30, 40, 50 g/ml). As a control, prepare the same volume of double-distilled water (Figures~\ref{f-d9916140e1d5} and~\ref{f-f9150df00ced}).

Incubate the mixtures at 370\ensuremath{^\circ}C for 15 minutes in a BOD incubator, then heat at 700\ensuremath{^\circ}C for 5 minutes, chill the solution, and measure the absorbance at 660nm. Assume the vehicle is empty (Figures~\ref{f-126fb1e8aabc}, \ref{f-1e97f90f6dfb} and~\ref{f-244989a4bc25}).

\textbf{HRBC  Membrane  Stabilization  Method}

HRBC  method  was  used  for  the  estimation  of  anti-inflammatory  activity INVITRO. Blood  was  collected  from  healthy  volunteers  and  was  mixed  with  equal  volume  of  sterilized  Alsevers solution. This  blood  solution  was  centrifuged  at  3  000  rpm  and the  packed  cells  were  separated. The  packed  cells  were  washed  with  isosaline  solution  and  a  10\%  v/v  suspension  was  made  with isosaline. This  HRBC  suspension  was used  for  the  estimation of  anti-inflammatory property. Different  concentrations  of  extract,  reference  sample  and  control  were  separately  mixed  with  1mL of  phosphate buffer,  2 mL  of hypo saline  and 0.5  mL of  HRBC suspension. All  the  assay  mixtures  were  incubated  at  37  \ensuremath{^\circ}C  for  30  minutes  and  centrifuged  at  3  000  rpm. The  supernatant  liquid  was  decanted  and  the  hemoglobin  content  was  estimated  by  a  spectrophotometer  at 560 nm \unskip~\cite{2012982:28657467}. 

The  percentage  hemolysis  was  estimated  by  assuming  the  hemolysis  produced  in  the  control  as 100\%.

Herbal gel was made using a mechanical stirrer and the gelling chemical Carbopol 934 in a 1\% w/w concentration with deionized water. 

The skin pH (6.8-7) was then maintained by adding tri-ethanolamine dropwise with continuous stirring. 

Various concentrations 100mg, 250mg,  500mg, 750mg, 1000mg polyherbal ethanolic extract of (1:1:1) has been added to the gel and swirled for a sufficient amount of time to ensure homogenous mixing of the extract in the gel base. 

These formulas were kept in a cool, dry place. The formulation was tested for the following criteria. The formulation fo Polyherbal gel according to the [Table~\ref{tw-2752fafc974e}].

\textbf{Evaluation studies for gel:}

\textbf{Organoleptic evaluation}

Colour and appearance were recorded as physical parameters.

\textbf{Viscosity}

A Brookfield viscometer with a spindle number was used to determine the viscosity of the gel. 

\textbf{Spreadability:}

The equipment, which consists of a wooden block with a pulley at one end, was used to determine spreadability. Spreadability was determined using this method based on the slip and drag characteristics of gels. On the ground slide, an excess of gel (approximately 2 g) was placed under investigation. The gel was then Utilising a hook and a glass slide of the same size as the fixed ground slide, they were positioned between one another. A 1 kg weight was set on top of the two slides for 5 minutes in order to let air out and create a uniform gel coating between the slides. The borders were scraped clean of extra gel. The top plate was then pulled with an 80-gram weight using a thread that was attached to the hook, and the amount of time (in seconds) that it took for the top slide to move 7.5 cm was noted. The spreadability improves with decreasing interval length.

The following formula was used to determine spreadability, 

S=M\ensuremath{\times}L/T

Where

S  =  Spreadability; M  = (Tied to the upper slide) Pan weight; L  = The length that the glass slide moved; T  = The amount of time (in seconds) needed to separate the top slide from the bottom slide \unskip~\cite{2012982:28657468}.

\textbf{Extrudability:}

The gel compositions were placed in conventional capped collapsible aluminium tubes and crimped shut. The weights of the tubes were taken down \unskip~\cite{2012982:28657466}. Between two glass slides, the tubes were secured. When 500 g were positioned over the slides, the cover was taken off. The amount of gel that was extruded was measured and gathered. Calculated extruded gel percentages include {\textgreater}90\% excellent, {\textgreater}80\% good, and {\textgreater}70\% fair (extrudability {\textgreater}80\%).

\textbf{Homogeneity:}

All created gels were placed in containers and visually inspected for uniformity \unskip~\cite{2012982:28657473}. They were examined for the existence of aggregates and their appearance. 

Inhibition of Protein Denaturation Reaction mixtures were incubated in a water bath at 37\ensuremath{\circ }C 2\ensuremath{\circ }C for 15-20 minutes before being heated to 70\ensuremath{\circ }C and maintained for 5 minutes \unskip~\cite{2012982:28657471}. 

The reaction mixture was then allowed to cool for 15 minutes at room temperature. A colorimeter was used to measure the absorbance 206 of the reaction mixture before and after denaturation at 660 nm for each concentration (10 g/ml, 20 g/ml, 40 g/ml, 60 g/ml, 80 g/ml, and 100 g/ml). 

Each test was repeated three times, and the mean absorbance was recorded each time. Using the following formula, the percentage of protein inhibition was calculated in relation to the control.
    
\section{Result and Discussion}
\textbf{Physical  appearance  and  \%  yield  of  extracts:}

The Curcuma longa, Zingiber officinale, Ocimum tenuiflorum  extracts were prepared using  ethanol. 

The prepared extract was coded as CZO.  The curcuma longa extract was prepared  using ethanol, The prepared extract was coded as cl.  

The zingiber officinale extract was prepared using ethanol, the  prepared  extract  was  coded  as zo.  

The  Ocimum  tenuiflorum  extract  was  prepared using ethanol, the  prepared  extract was  coded as not (Tables~\ref{tw-2752fafc974e}, \ref{tw-abcc9bda779a} and~\ref{tw-345afa6f6cf9}).


\begin{table*}[!htbp]
\caption{\boldmath {Formulation of Polyherbal Gel} }
\label{tw-2752fafc974e}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.19709999999999997\linewidth-2\tabcolsep}p{\dimexpr.4596000000000001\linewidth-2\tabcolsep}p{\dimexpr.3433\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Ingredients & Quantity\\
\tblmidrule 
1. &
  Carbopol &
  1g\\
2. &
  Polyherbal  extract &
  1g\\
3. &
  Methyl  paraben &
  0.02g\\
4. &
  Distilled  water &
  100ml\\
5. &
  Triethanolamine &
  Qs\\
6. &
  Eucalyptus  oil &
  qs\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Percentage Yield of Extracts} }
\label{tw-abcc9bda779a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10400000000000002\linewidth-2\tabcolsep}p{\dimexpr.1703\linewidth-2\tabcolsep}p{\dimexpr.1457\linewidth-2\tabcolsep}p{\dimexpr.1552\linewidth-2\tabcolsep}p{\dimexpr.1248\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.no & Extract & \%Yield & \multicolumn{2}{p{\dimexpr(.28\linewidth-2\tabcolsep)}}{Physical  appearance  of the  extract} & Odour & Taste\\
\rowcolor{kwdboxcolor} &  &  & colour & Consistency &  & \\
\tblmidrule 
1 &
  CZO &
  10.5\% &
  Dark greenish  brown &
  Oily  viscous &
  Characteristic &
  Sour\\
2 &
  Curcuma longa &
  5.05\% &
  Dark \mbox{}\protect\newline yellowish  brown &
  Light  viscous &
  Characteristic &
  Pleasantly  bitter\\
3 &
  Zingiber  officinale &
  4.57\% &
  Brownish  yellow &
  Oily  viscous &
  Characteristic &
  Pungent\\
4 &
  Ocimum  tenuiflorum &
  4.91\% &
  Dark green &
  Viscous &
  Characteristic &
  mint\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Determination of Total Ash} }
\label{tw-345afa6f6cf9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.18710000000000004\linewidth-2\tabcolsep}p{\dimexpr.53940000000000005\linewidth-2\tabcolsep}p{\dimexpr.27349999999999998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.NO & INGREDIENTS & \% VALUE\\
\tblmidrule 
1 &
  CZO &
  9.1 \%\\
2 &
  Curcumin  longa &
  16.6 \%\\
3 &
  Zingiber  officinale &
  7.6\%\\
4 &
  Ocimum  tenuiflorum &
  3.1\%\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Water-soluble ash} }
\label{tw-2dcd2a146841}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1971\linewidth-2\tabcolsep}p{\dimexpr.5294000000000002\linewidth-2\tabcolsep}p{\dimexpr.27349999999999998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.NO & INGREDIENTS & \% VALUE\\
\tblmidrule 
1 &
  CZO &
  2.99 \%\\
2 &
  Curcumin longa &
  3.93 \%\\
3 &
  Zingiber officinale &
  3.366\%\\
4 &
  Ocimum tenuiflorum &
  1.7 \%\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Moisture content} }
\label{tw-34f2ffe3e433}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.17699999999999996\linewidth-2\tabcolsep}p{\dimexpr.29310000000000002\linewidth-2\tabcolsep}p{\dimexpr.27990000000000002\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.NO & Ingredients & Moisture  content & Alcohol  Soluble  Extractive  value\\
\tblmidrule 
1 &
  CZO &
  30.75\% &
  22.71 \%\\
2 &
  Curcumin  longa &
  3.14 \% &
  0.4 \%\\
3 &
  Zingiber  officinale &
  8.13\% &
  8.55\%\\
4 &
  Ocimum  tenuiflorum &
  81.02 \% &
  59.2\%\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Phytochemical Tests} }
\label{tw-5cdb729f973f}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.1689\linewidth-2\tabcolsep}p{\dimexpr.1334\linewidth-2\tabcolsep}p{\dimexpr.1622\linewidth-2\tabcolsep}p{\dimexpr.17549999999999997\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Chemical constituents & Tests & Czo & Curcuma longa & Zingiber officinale & Ocimum \mbox{}\protect\newline tenuiflorum\\
\tblmidrule 
Alkaloids &
  Dragendroff's test &
  + &
  + &
  + &
  +\\
 &
  Mayer's test &
  - &
  + &
  - &
  +\\
 &
  Wagner's test &
  - &
  - &
  + &
  +\\
 &
  Hager's test &
  + &
  + &
  + &
  +\\
Carbohydrates &
  Molisch's test &
  + &
  + &
  + &
  +\\
 &
  Fehling's test &
  + &
  + &
  + &
  +\\
 &
  Benedict's test &
  - &
  + &
  - &
  +\\
Amino acids \& proteins &
  Biuret test &
  + &
  - &
  + &
  +\\
 &
  Ninhydrin test &
  - &
  - &
  - &
  -\\
Flavonoids &
  Lead acetate test &
  - &
  + &
  - &
  -\\
 &
  Shinoda test &
  + &
  + &
  + &
  +\\
 &
  Alkaline test &
  + &
  + &
  + &
  +\\
Phenols &
  FeCl3 &
  - &
  + &
  + &
  -\\
Tannins &
  Bromine water test &
  + &
  + &
  + &
  +\\
 &
  Gelatin test &
  + &
  + &
  + &
  +\\
Steroids \& triterpenoids &
  Salkowski test &
  - &
  - &
  - &
  -\\
 &
  Libermann {\textendash} buchard test &
  - &
  - &
  + &
  +\\
Saponins &
  Froth test &
  - &
  + &
  - &
  +\\
 &
  Foam test &
  + &
  - &
  + &
  -\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Total Flavonoid Content} }
\label{tw-9667f735fd2a}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.50\linewidth-2\tabcolsep}p{\dimexpr.50\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}EXTRACT & TOTAL FLAVONOID CONTENTS\\
\tblmidrule 
CZO &
  71.42\\
Curcuma  longa &
  51.86\\
Zingiber  officinale &
  62.81\\
Ocimum  tenuiflorum &
  76.89\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {TLC} }
\label{tw-9319aafd35e9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1267\linewidth-2\tabcolsep}p{\dimexpr.1395\linewidth-2\tabcolsep}p{\dimexpr.14809999999999999\linewidth-2\tabcolsep}p{\dimexpr.1457\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.NO. & Sample & Mobile  Phase & Reagents  For \mbox{}\protect\newline Detection & Colour  Of  Spot & No. Of  Spots & Rf Value\\
\tblmidrule 
1 &
  CZO &
  Hexane: Ethyl  acetate &
  Iodine  fumes &
  Green  and  yellow &
  3 &
  0.87 \mbox{}\protect\newline 0.6 \mbox{}\protect\newline 0.52\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {The ethanol extracts' capacity to scavenge hydroxyl radicals of CZO} }
\label{tw-3c4a7d8d2f08}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.18669999999999998\linewidth-2\tabcolsep}p{\dimexpr.18680000000000003\linewidth-2\tabcolsep}p{\dimexpr.2079\linewidth-2\tabcolsep}p{\dimexpr.2186\linewidth-2\tabcolsep}p{\dimexpr.20\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Sample & Concentration & Absorbance  (Mean \ensuremath{\pm} SEM) & Percentage inhibition \mbox{}\protect\newline (Mean \ensuremath{\pm} SEM) & IC50\\
\tblmidrule 
 &
  20 \ensuremath{\mu}g/ml &
  0.187\ensuremath{\pm}0.001 &
  39.29\ensuremath{\pm}0.294 &
  \\
 &
  40 \ensuremath{\mu}g/ml &
  0.176\ensuremath{\pm}0.003 &
  45.10\ensuremath{\pm}0.779 &
  \\
Ethanol  extract &
  60 \ensuremath{\mu}g/ml &
  0.162\ensuremath{\pm}0.001 &
  58.58\ensuremath{\pm}0.372 &
  52.20 \ensuremath{\mu}g/ml\\
 &
  80 \ensuremath{\mu}g/ml &
  0.122\ensuremath{\pm}0.006 &
  68.95\ensuremath{\pm}1.532 &
  \\
 &
  100 \ensuremath{\mu}g/ml &
  0.092\ensuremath{\pm}0.005 &
  78.73\ensuremath{\pm}3.351 &
  \\
Ascorbic  acid &
  20 \ensuremath{\mu}g/ml &
  0.142\ensuremath{\pm}0.002 &
  44.93\ensuremath{\pm}0.450 &
  51.41 \ensuremath{\mu}g/ml\\
 &
  40 \ensuremath{\mu}g/ml &
  0.117\ensuremath{\pm}0.001 &
  50.23\ensuremath{\pm}0.226 &
  \\
 &
  60 \ensuremath{\mu}g/ml &
  0.106\ensuremath{\pm}0.003 &
  62.86\ensuremath{\pm}0.756 &
  \\
 &
  80 \ensuremath{\mu}g/ml &
  0.090\ensuremath{\pm}0.004 &
  76.95\ensuremath{\pm}1.001 &
  \\
 &
  100 \ensuremath{\mu}g/ml &
  0.081\ensuremath{\pm}0.002 &
  89.33\ensuremath{\pm}0.388 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Percent inhibition of Protein denaturation} }
\label{tw-48b5a5343cb7}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1476\linewidth-2\tabcolsep}p{\dimexpr.1457\linewidth-2\tabcolsep}p{\dimexpr.1362\linewidth-2\tabcolsep}p{\dimexpr.1305\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} & 10 & 20 & 40 & 60 & 80 & 100\\
\tblmidrule 
ZINGER &
  24.83\ensuremath{\pm}1.91 &
  29.12\ensuremath{\pm}0.38 &
  33.15\ensuremath{\pm}0.38 &
  48.13\ensuremath{\pm}0.87 &
  57.71\ensuremath{\pm}0.21 &
  65.13\ensuremath{\pm}1.28\\
TURMERIC &
  30.24\ensuremath{\pm}0.30 &
  41.31\ensuremath{\pm}0.19 &
  43.53\ensuremath{\pm}0.17 &
  49.7\ensuremath{\pm}0.17 &
  57.26\ensuremath{\pm}0.21 &
  62.54\ensuremath{\pm}0.25\\
TULSI &
  24.8\ensuremath{\pm}0.36 &
  30.39\ensuremath{\pm}0.30 &
  48.49\ensuremath{\pm}0.24 &
  52.86\ensuremath{\pm}0.25 &
  55.23\ensuremath{\pm}0.182 &
  60.12\ensuremath{\pm}1.78\\
PHF-1 &
  21.19\ensuremath{\pm}0.33 &
  27.12\ensuremath{\pm}0.97 &
  38.14\ensuremath{\pm}0.78 &
  43.74\ensuremath{\pm}0.38 &
  69.26\ensuremath{\pm}0.26 &
  78.72\ensuremath{\pm}0.29\\
PHF-2 &
  37.12\ensuremath{\pm}0.28 &
  39.16\ensuremath{\pm}0.86 &
  47.14\ensuremath{\pm}0.96 &
  58.62\ensuremath{\pm}0.46 &
  64.20\ensuremath{\pm}0.178 &
  77.16\ensuremath{\pm}0.76\\
PHF-3 &
  62.519\ensuremath{\pm}0.50 &
  69.89\ensuremath{\pm}8.87 &
  78.98\ensuremath{\pm}0.65 &
  83.12\ensuremath{\pm}0.54 &
  89.16\ensuremath{\pm}0.65 &
  98.12\ensuremath{\pm}0.78\\
PHF-4 &
  64.47\ensuremath{\pm}0.86 &
  69.26\ensuremath{\pm}0.91 &
  71.90\ensuremath{\pm}0.31 &
  79.90\ensuremath{\pm}0.32 &
  69.88\ensuremath{\pm}0.76 &
  79.898\ensuremath{\pm}0.145\\
DICLO &
  57.14\ensuremath{\pm}0.64 &
  62.12\ensuremath{\pm}0.721 &
  64.15\ensuremath{\pm}1.32 &
  71.287\ensuremath{\pm}0.33 &
  72.16\ensuremath{\pm}0.24 &
  98.16\ensuremath{\pm}1.78\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {HRBC Membrane Stabilization} }
\label{tw-715b75a18bc1}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.16413333333333334\linewidth-2\tabcolsep}p{\dimexpr.16413333333333334\linewidth-2\tabcolsep}p{\dimexpr.16413333333333334\linewidth-2\tabcolsep}p{\dimexpr.16413333333333334\linewidth-2\tabcolsep}p{\dimexpr.16413333333333334\linewidth-2\tabcolsep}p{\dimexpr.17933333333333337\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} & 20 & 40 & 60 & 80 & 100\\
\tblmidrule 
PHF-1 &
  42.15\ensuremath{\pm}0.17 &
  51.27\ensuremath{\pm}0.18 &
  61.11\ensuremath{\pm}0.176 &
  68.12\ensuremath{\pm}0.74 &
  68.54\ensuremath{\pm}0.89\\
PHF-2 &
  34.18\ensuremath{\pm}0.72 &
  39.76\ensuremath{\pm}0.14 &
  40.10\ensuremath{\pm}0.72 &
  51.82\ensuremath{\pm}0.14 &
  66.74\ensuremath{\pm}0.14\\
PHF-3 &
  48.25\ensuremath{\pm}0.19 &
  58.16\ensuremath{\pm}0.82 &
  61.11\ensuremath{\pm}0.14 &
  66.52\ensuremath{\pm}0.79 &
  82.74\ensuremath{\pm}0.95\\
PHF-4 &
  41.16\ensuremath{\pm}0.19 &
  47.82\ensuremath{\pm}0.17 &
  54.18\ensuremath{\pm}0.26 &
  65.19\ensuremath{\pm}0.99 &
  79.16\ensuremath{\pm}1.24\\
DICLO &
  51.25\ensuremath{\pm}2.15 &
  62.25\ensuremath{\pm}0.19 &
  74.12\ensuremath{\pm}0.98 &
  78.52\ensuremath{\pm}0.89 &
  84.12\ensuremath{\pm}1.02\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Characteristics of gel formulation made with Carbapol} }
\label{tw-a23ddbd58ab4}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.1167\linewidth-2\tabcolsep}p{\dimexpr.1184\linewidth-2\tabcolsep}p{\dimexpr.1349\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Formulation code & Conc. (\%) & pH* & Viscosity* (poise) & Spread  ability*gcm/sec & Drug  content*  (\%) & Extrudability* & Nature of  gel\\
\tblmidrule 
F1 &
  0.5 &
  7.56 &
  0.381 &
  31.13 &
  99.3 &
  Good &
  Pale green,  smooth, homogenous,  translucent\\
F2 &
  1 &
  7.48 &
  0.382 &
  44.03 &
  99.6 &
  Good &
  Pale green,  smooth, homogenous, \mbox{}\protect\newline translucent\\
F3 &
  1.5 &
  7.67 &
  0.388 &
  55.29 &
  98.3 &
  Excellent &
  green,  smooth, \mbox{}\protect\newline homogenous, translucent\\
F4 &
  2 &
  6.88 &
  0.367 &
  66.20 &
  104 &
  Good &
  Dark Green,  smooth, homogenous, \mbox{}\protect\newline translucent\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/58dd37be-4922-4436-bbeb-3a9664de0c4c-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/58dd37be-4922-4436-bbeb-3a9664de0c4c-upicture2.png}{\includegraphics{images/58dd37be-4922-4436-bbeb-3a9664de0c4c-upicture2.png}}{\includegraphics{58dd37be-4922-4436-bbeb-3a9664de0c4c-upicture2.png}}
\makeatother 
\caption{\boldmath {Soxhlet extraction of Polyherbal compound}}
\label{f-d9916140e1d5}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/c74b8769-3843-4ba0-963e-1f7871f387e7-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c74b8769-3843-4ba0-963e-1f7871f387e7-upicture3.png}{\includegraphics{images/c74b8769-3843-4ba0-963e-1f7871f387e7-upicture3.png}}{\includegraphics{c74b8769-3843-4ba0-963e-1f7871f387e7-upicture3.png}}
\makeatother 
\caption{\boldmath {Recovery of solvent}}
\label{f-f9150df00ced}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/e48e8970-3c3c-4011-b187-bee77399c974-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/e48e8970-3c3c-4011-b187-bee77399c974-upicture4.png}{\includegraphics{images/e48e8970-3c3c-4011-b187-bee77399c974-upicture4.png}}{\includegraphics{e48e8970-3c3c-4011-b187-bee77399c974-upicture4.png}}
\makeatother 
\caption{\boldmath {CZO ethanolic extracts' hydroxyl radical scavenging properties }}
\label{f-126fb1e8aabc}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/3e58cb8d-9cc9-47be-983b-390ac56887f1-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/3e58cb8d-9cc9-47be-983b-390ac56887f1-upicture5.png}{\includegraphics{images/3e58cb8d-9cc9-47be-983b-390ac56887f1-upicture5.png}}{\includegraphics{3e58cb8d-9cc9-47be-983b-390ac56887f1-upicture5.png}}
\makeatother 
\caption{\boldmath {Inhibition of Protein Denaturation}}
\label{f-1e97f90f6dfb}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/8624bf4e-3d5b-4821-bf73-79dd5a0d34e6-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/8624bf4e-3d5b-4821-bf73-79dd5a0d34e6-upicture6.png}{\includegraphics{images/8624bf4e-3d5b-4821-bf73-79dd5a0d34e6-upicture6.png}}{\includegraphics{8624bf4e-3d5b-4821-bf73-79dd5a0d34e6-upicture6.png}}
\makeatother 
\caption{\boldmath {Method for HRBC Membrane Stabilisation}}
\label{f-244989a4bc25}
\end{figure}
\egroup
\textbf{Physicochemical Parameters}

\textbf{Determination of  Alcohol  Soluble  Extractive  value:}

The alcohol-soluble components of the polyherbal formulation (such as alkaloids, proteins, amino acids, and carbohydrates) were determined using pharmacopoeial procedures and in-house standards; the findings are shown in Table (Tables~\ref{tw-2dcd2a146841}, \ref{tw-34f2ffe3e433} and~\ref{tw-5cdb729f973f}).

\textbf{Evaluation of Polyherbal Powder:}

\textbf{Qualitative  phytochemical  test  for CZO  extract}

Several assays were used to conduct qualitative phytochemical screening, and the results demonstrated that ethanol extract of CZO.  powder contains phenols, flavonoids and absence of alkaloids,  carbohydrates  and steroids \& triterpenoids (Tables~\ref{tw-9667f735fd2a}, \ref{tw-9319aafd35e9} and~\ref{tw-3c4a7d8d2f08}). 

\textbf{Estimation of Total  Flavonoids}

Using the regression equation y = 0.0043x + 0.0104, R2 = 0.9968, the amount of total flavonoid content was calculated from the gallic acid calibration curve. The overall flavonoid content of the  ethanolic extract of CZO was 71.42, of curcuma longa was 51.86, of zingiber officinale was  62.81, of Ocimum tenuiflorum was 76.86, gallons of gallic acid equivalents per kilogramme of dry material. Within these portions, Ocimum tenuiflorum contains high flavonoid content as compared to curcuma  longa  and zingiber officinale (Tables~\ref{tw-48b5a5343cb7}, \ref{tw-715b75a18bc1} and~\ref{tw-a23ddbd58ab4}). 

The  thin  layer  chromatography  was  used  to isolate  the  non-volatile  mixtures.  The  PHE  was used in TLC to estimate the RF values for individual components. The PHE consists of three  mixtures  so,  the  TLC  results  in  three  spots.  The  RF values  of the  three  spots  are(0.87, 0.6,0.52).

\textbf{In-vitro antioxidant Activity}

\textbf{Hydroxyl  radical  scavenging  activity  of  extracts:}

The values are expressed as the MeanSEM (n=3). The breakdown of deoxyribose by the Fe+3-ascorbic acid-EDTA-H2O2 system was greatly reduced by CZO and ascorbic acid at concentration ranges of 20-100 g/ml in a dose dependent manner, demonstrating Poly herbal extract's considerable hydroxyl radical scavenging action. 

The antioxidant activity of the ethanol extract was significant (IC50=52.20 g/ml) compared  to  standard  (IC50=51.41  \ensuremath{\mu}g/ml).  

At  100  \ensuremath{\mu}g/ml,  the  percentage  inhibition.  Value  was  78.73\%  for  ethanol  extract,  while  ascorbic  acid  possesses  79.33\%  scavenging  activity  at same concentration.

\textbf{Protein denaturation technique for in vitro anti-inflammatory action}

The table as well as image demonstrate how zinger, turmeric, tulsi, and PHF 1{\textendash}4 ethanolic extracts affect the prevention of protein denaturation. All samples were at various concentrations (between 20 and 100 g). offered significant defence against protein denaturation. Comparing PHF-3 to different formulations and individual ethanolic extracts, the highest \% inhibition was found at 100g/ml. It has sizable activity comparable to that of diclofenac sodium standard. The majority of researchers have noted that one underlying cause of rheumatoid arthritis is protein denaturation. In some rheumatic illnesses, the production of autoantibodies may be triggered by the denaturation of proteins. 

The work is motivated by the need for effective natural anti-inflammatory agents with fewer side effects to replace chemical therapies. Table and image depict the impact of PHFs 1 through 4 on the stabilisation of the HRBC membrane. Highest level of stabilisation was  observed in PHF -3 (82.74\% at 100\ensuremath{\mu}g/ml) as compared to other formulations. It possesses significant activity equivalent to that of diclofenac sodium. It is possible that the substantial anti-inflammatory efficacy is related to  the presents of secondary  metabolites such as  flavanoids,  terpenoids, phenols.

\textbf{Polyherbal evaluations}

Carbopol 934, various amounts of ethanolic extract of PHE, methylparaben, distilled water, and triethanolamine were used to make the polyherbal gel. The appearance, viscosity, spreadability, pH, and homogeneity of the prepared gels were tested, and the findings are reported in Table. All gel compositions are pale green in colour, transparent, and have a smooth application feel. All of these formulas demonstrated optimal viscosity. All developed formulations had pH values ranging from 6 to 7, which is regarded as appropriate to avoid skin irritation when applied to the skin. When made and after one month, all formulations are homogenous and free of gritty particles.



\subsection{Conclusion}Using the study as a foundation, the findings revealed that the polyherbal gels made from the Polyherbal extracts (Ginger, Turmeric and Tulasi) When compared to normal Diclofenac gel, it demonstrated strong anti-inflammatory effect. Because phytochemical studies revealed the presence of glycosides, carbohydrates, flavonoids, steroids, and resin in the ethanolic extracts, these compounds may restrict the synthesis of prostaglandins and bradykinins, antagonise their action, as well as exert their function. When compared to individual extracts, the polyherbal gels demonstrated a synergistic effect that may be effective for the treatment of local inflammation.
    
\section{Acknowledgement}
We would like to thank principal \& staff Narayana Pharmacy College for their immense support. 

\textbf{Conflict of interest}

The authors declare no conflict of interest, financial or otherwise.

\textbf{Funding Support}

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

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