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\def\RunningAuthor{Goli Penchala Prasad et al.}
\firstPage{160}
\articleType{Original Article}
\receivedDate{11.08.2019}
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\title{\textbf{Pharmacognostical standardization of balamula churna} \textbf{ (\textit{Sida acuta} Burm.f. root powder)}}
\author{Jyostna~Kumari~T\textsuperscript{1},  
            Penchala~Pratap~G\textsuperscript{2},  
            Murthy~P~H~C\textsuperscript{1}, 
            Goli~Penchala~Prasad\textsuperscript{*}\textsuperscript{3}~\\[5pt]{\textsuperscript{1}NRS Medical College, Vijayawada, Andhra Pradesh,India}~\\{\textsuperscript{2}Central Research Institute of Unani Medicine, Hyderabad,Andra Pradesh,India}~\\{\textsuperscript{3}Regional Ayurveda Institute for Fundamental Research, Kothrud, Pune, Maharashtra,India}}

\begin{abstract}
 In this study, an effort was performed to produce the data on, physicochemical parameters, preliminary phytochemical screening, histochemical studies, and HPTLC data required for the standardization of Balamula Churna concerning root powder of \textit{Sida acuta}. The physicochemical parameters such as water-soluble extractive, alcohol soluble extractive, and loss on drying at 105 \ensuremath{^\circ}C, total ash, and insoluble acid ash were determined according to standard methods. HPTLC studies were carried, and Rf values were documented. Physicochemical parameter values were recorded as pH (6.73\%) LOD (7.2\%) water-soluble extractive value (4.48\%), alcohol soluble extractive value (6.88\%) total Ash (11.00\%) and Acid Insoluble Ash (2.72\%). Preliminary phytochemical screening reveals the presence of glycoside, alkaloid, carbohydrate, phenol, flavonoid, and saponin. HPTLC screening showed the occurrence of significant phytoconstituents with Rf values 0.07, 0.21, 0.54, and 0.86. The obtained results from this study can be effective in the evaluation of quality, detection of adulteration and substitution, and highlighting the importance of standardization.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Balamula,\newline Bala,\newline Churna,\newline Sida acuta,\newline Standardization
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Goli~Penchala~Prasad~\\ Phone:\ 09441654500~\\  Email:\ drprasadgoli@yahoo.com
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\section{Introduction}
 In Ayurveda, medicinal plants were practiced to cure human ailments in every possible condition of the time immemorial. In the recent era, we have the advantage of practice them over the synthetic molecules because of minor side effects \unskip~\citep{644554:15144944}. Ayurveda system of medicine is flourishing due to antiquated knowledge, experiences, and practices based on the theories and expertise of many practitioners in India. Standardization of herbal drug is significant because, in some cases, desirable pharmacological actions are not achieved due to the utilization of adulterants. In such is a case Standardization is the only inherent step for ensuring the quality control of herbal drugs. To achieve quality-oriented herbal products, attention should be taken a right from the proper identification of plants \unskip~\citep{644554:15144949}.

In Ayurveda system, Balamula churna is an important major ingredient in medicated oil Ksheerabala Thailam. Ksheerabala Thailam is practiced for the therapy of diseases associated with the musculoskeletal system. It is also desirable for internal (oral) intake for soothing Vata Dosha in the body. It relieves pain, stiffness, inflammation, swelling, and other symptoms of complicated and increased Vata Dosha. It is a rejuvenating agent, which assists to rebuild the damaged tissue. It also furnishes nourishment to the nerves, brain, and spinal structures. 

In India, three species of\textit{ Sida, } concerning\textit{ S. acuta Figure~\ref{f-4b03bbed1aa1},S. cordifolia }and\textit{ S. retusa }are used as Bala in different parts of our country. To make the efficacy of the drug present study is aimed to standardize the Balamula churna concerning the root powder of \textit{S. acuta}. The root Figure~\ref{f-ed2a8ff79e9f} is said to possess aphrodisiac, antirheumatic, stomachic, diaphoretic, diuretic, antipyretic, and wound healing properties \unskip~\citep{644554:15144945}. 


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\caption{\boldmath { Overlay Densitogram of \textit{S. acuta} root Powder at UV 366nm}}
\label{f-1d551fb2b155}
\end{figure}
\egroup

    
\section{Materials and Methods}
\textbf{Plant material}

Roots of \textit{S. acuta} were collected from hilly areas of Tirupathi, Andhra Pradesh. 

\textbf{Identification and authentication of plant material}

Plant material was identified and authenticated with the help of the Flora \unskip~\citep{644554:15144943} Plant material was also compared with the herbarium specimen available in Sri Venkateswara University, Tirupati. The herbarium accession number SVUBS1257, respectively.

\textbf{Powder preparation}

Shade dried root pieces were made into powder using a grinding mill, passed through \#60 sieve, and kept in an airtight sealed container for further study \unskip~\citep{644554:15144944}. 

\textbf{Organoleptic evaluation }

 Root powder was evaluated for organoleptic properties. 

\textbf{Determination of pH}

10\% solution of Root powder extract in water was prepared, and the pH of the liquid was calculated with the help of pH meter. 

\textbf{Determination of physicochemical parameters}

Physicochemical parameters, namely loss on drying, ash value, insoluble acid ash, water-soluble extractive, alcohol soluble extractive, were performed as per the standard procedures \unskip~\citep{644554:15144947,644554:15144939}. 


\begin{table*}[!htbp]
\caption{\boldmath {pH and physicochemical parameter (s) } }
\label{tw-94f31b16c745}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10330000000000002\linewidth-2\tabcolsep}p{\dimexpr.6207000000000001\linewidth-2\tabcolsep}p{\dimexpr.276\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S. No & Parameter Value & (\%)\\
\tblmidrule 
1 &
  pH &
  6.73\\
2 &
  Loss on Drying &
  7.2\\
3 &
  Water-soluble extractive value &
  4.48\\
4 &
  Alcohol soluble extractive value &
  6.88\\
5 &
  Total Ash &
  11\\
6 &
  Acid Insoluble Ash &
  2.72\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Preliminary phytochemical studies} }
\label{tw-722c7f5748bf}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1016\linewidth-2\tabcolsep}p{\dimexpr.6363\linewidth-2\tabcolsep}p{\dimexpr.2621\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Secondary metabolites  & Result\\
\tblmidrule 
1 &
  Glycoside &
  +\\
2 &
  Alkaloid  &
  +\\
3 &
  Carbohydrate &
  +\\
4 &
  Phenol  &
  +\\
5 &
  Flavonoid &
  +\\
6 &
  Saponin &
  +\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Histochemistry \unskip~\citep{644554:15144948}.}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Histochemical studies} }
\label{tw-f9e647dbb9c9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10819999999999999\linewidth-2\tabcolsep}p{\dimexpr.21009999999999998\linewidth-2\tabcolsep}p{\dimexpr.33309999999999995\linewidth-2\tabcolsep}p{\dimexpr.3486\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S.No & Test & Reagent used & Nature of change\\
\tblmidrule 
1 &
  Lignins &
  1\% Safranine &
  Red Figure~\ref{f-5a627f467516}\\
2 &
  Mucilage &
  Ruthenium Red &
  Pink Figure~\ref{f-c5590f9e7b0c}\\
3 &
  Starch &
  Iodine &
  Blue Figure~\ref{f-a4ecfa6cdb43}\\
4 &
  Alkaloids &
  Wagner's Reagent &
  Orange Figure~\ref{f-955437e9600a}\\
5 &
  Tannins &
  Dil.FeCl\ensuremath{_{3}} solution &
  Bluish Black Figure~\ref{f-d23ee780f262}\\
6 &
  Saponins &
  Conc.H\ensuremath{_{2}}So\ensuremath{_{4}} &
  Light Yellow Figure~\ref{f-602a7e7d5eec}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Peak(s) list of \textit{S. acuta} root Powder at UV 366nm} }
\label{tw-2916050ce4b9}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.092\linewidth-2\tabcolsep}p{\dimexpr.15309999999999995\linewidth-2\tabcolsep}p{\dimexpr.1726\linewidth-2\tabcolsep}p{\dimexpr.17849999999999998\linewidth-2\tabcolsep}p{\dimexpr.19000000000000004\linewidth-2\tabcolsep}p{\dimexpr.2138\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Peak no & Y-Pos & Area & Area (\%) & Height & Rf value\\
\tblmidrule 
1 &
  10.2 &
  546.07 &
  51.99 &
  231.06 &
  0.02\\
2 &
  23.8 &
  17.55 &
  1.67 &
  8.46 &
  0.21\\
3 &
  46.2 &
  63.45 &
  6.04 &
  26.60 &
  0.51\\
4 &
  53.5 &
  19.12 &
  1.82 &
  10.26 &
  0.62\\
5 &
  78.2 &
  404.18 &
  38.48 &
  96.60 &
  0.96\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Preliminary phytochemical screening}

Preliminary qualitative tests were performed by standard methods \unskip~\citep{644554:15144947,644554:15144939,644554:15144940}. 

\textbf{Heavy metal analysis}

Determination Heavy metals tests were performed by standard methods \unskip~\citep{644554:15144944}.

\textbf{Histochemistry\ensuremath{^{}}}

\unskip~\citep{644554:15144942,644554:15144941}

Hand sections of fresh root were stained with a series of histochemical reagents. \unskip~\citep{644554:15144942,644554:15144941}.

 Safranine (1\% safranine in 50\% alcohol); lignins (appears in red color ); Ruthenium red; mucilage (appears in pink color); Iodine; starch (appears in blue color); Wagner's reagent; alkaloids (appears in pink orange color);  Dilute FeCl\ensuremath{_{3}} solution; tannins (appears in bluish black color);  conc. H\ensuremath{_{2}}SO\ensuremath{_{4}}; saponins (appears in light yellow color).  

\textbf{High-performance thin-layer chromatography}

Thin-layer chromatography of Alcoholic extract of the powder drug was made using solvent system toluene: ethyl acetate (6:4), which was saturated for 45 minutes in CAMAG twin trough chamber. \unskip~\citep{644554:15144946} The extract was applied manually on TLC Silica gel 60 F254 Aluminum coated plate and run up to 8 cm. Plates were observed under daylight, ultraviolet light at 366 nm, and subsequently derivatized with Iodine vapor and anisaldehyde-sulphuric acid. Developed band colours and retention factor (Rf) were recorded \unskip~\citep{644554:15144948,644554:15144944}. 


    
\section{Results and Discussion}
 The root powder is cream in color, coarse fibrous, the odor is pleasant, and an image of powder is depicted in Figure~\ref{f-653bf5326ac5}. The value of pH and physicochemical parameters such as loss on drying, total ash, insoluble acid ash, water-soluble ash, water, and alcohol soluble extracts are depicted in Table~\ref{tw-94f31b16c745}. In the heavy metal analysis, arsenic and lead content were found to be less than 2 and 20 PPM, respectively. Preliminary phytochemical screening revealed the occurrence of phytoconstituents, as depicted in Table~\ref{tw-722c7f5748bf}. Histochemical studies revealed the presence of Lignins, Mucilage, Starch, Alkaloids, Tannins, and Saponins described in Table~\ref{tw-f9e647dbb9c9}. 

 TLC chromatograms of root powder with solvent system toluene: ethyl acetate (6: 4) at UV 366nm Figure~\ref{f-0d109835a93a}, UV 254nm Figure~\ref{f-edd6b65ed4fc}, under expose to Iodine vapour Figure~\ref{f-a93bdb29d366} and derivatized with Anisaldehyde sulphuric acid Figure~\ref{f-62be645a4b47}.

TLC Plate shows five major spots under UV 366nm at Rf values 0.07 (blue), 0.21 (blue), 0.51 (blue), 0.54 (blue) and 0.86 (blue); and under Iodine vapours shows one spot at Rf value 0.71 (brown); and under visible region after derivatizing with anisaldehyde sulphuric acid shows one spot at Rf value 0.71 (purple) Figures~\ref{f-0d109835a93a} and~\ref{f-1d551fb2b155}, Table~\ref{tw-2916050ce4b9}.
    
\section{\textbf{\space }Conclusions}
The results of these study would be beneficial for identification, authentication and standardization of Balamula churna concerning \textit{S. acuta root} powder and also helpful in distinguishing spurious adulteration of the genuine drug. 
    

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