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\def\RunningAuthor{Delvin Thozhuthumparambil Robin et al.}
\firstPage{2184}
\articleType{Original Article}
\receivedDate{30 May 2021}
\acceptedDate{05 Jul 2021}
\revisedDate{03 Jul 2021}
\journalVolume{2021, 12}
\journalIssue{3}
\journalDoi{ijrps.v12i3.4832}
\copyrightYear{2021}
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\title{\textbf{Preliminary Physico-Chemical Profile of Yashtimadhu Ghrita}}
\author{Swathy~Vijayakumar\textsuperscript{1},  
            Mahadevan~Subramaniam\textsuperscript{2},  
            Vandana~Rani~Madhavan\textsuperscript{3},  
            Anusree~Dileep\textsuperscript{3},  
            Ramesh~Narve~Venkatesha\textsuperscript{4}, 
            Delvin~Thozhuthumparambil~Robin\textsuperscript{*}\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Research Scholar, Department of Swasthavritta (Social and Preventive Medicine), Amrita School of Ayurveda, Amritapuri, Amrita Vishwa Vidyapeetham, Kerala, India}~\\{\textsuperscript{2}Acara Laboratory, Amrita School of Ayurveda, Amritapuri, Amrita Vishwa Vidyapeetham, Kerala, India}~\\{\textsuperscript{3}Department of Swasthavritta (Social and Preventive Medicine), Amrita School of Ayurveda, Amritapuri, Amrita Vishwa Vidyapeetham, Kerala, India}~\\{\textsuperscript{4}Department of Rasashastra and Bhaishajya Kalpana (Medicinal Chemistry and Pharmaceutics), Amrita School of Ayurveda, Amritapuri, Amrita Vishwa Vidyapeetham, Kerala, India}}

\begin{abstract}
\textit{Yashtimadhu ghrita} is a single-herbal ghee formulation mentioned in the classical texts of Ayurveda and is indicated in \textit{sadya kshatavrana} (an acute inflammatory condition or an acute wound associated with trauma). It also minimizes the pain related to the wound. The present study is intended to create a finger print profile of \textit{Yastimadhu ghrita} through its pharmacognostical identification and the physico-chemical analysis.  The genuineness of the dry specimen of \textit{Yashtimadhu} (Glycyrrhiza glabra Linn.) was confirmed through organoleptic evaluation and cross-sectional microscopy. \textit{Yashtimadhu } ghrita was prepared as per the \textit{Sneha paka (}preparation of unctuous formulations using ghee, oil etc.\textit{)} procedure mentioned in \textit{Sarngadhara Samhita} \textit{Madhyama khanda} and the standard operative procedures specified in Ayurvedic pharmacopoeia of India. The test result of the finished product shows Acid value 6.7, saponification value 213.4, and Refractive index value 1.455 at room temperature, Iodine value 38.7, LOD 0.64. TLC and HPTLC were carried out for the characterization of the plant material in an appropriate solvent system. A total of nine spots were distinguished in 254 nm and five spots in 366 nm during the HPTLC scanning. The Rf value pertaining to the spots were identical in the methanolic extract which indicates the presence of definite constituents in Yashtimadhu ghrita.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Yashtimadhu ghrita,\newline TLC,\newline HPTLC,\newline Pharmacognosy,\newline Physico-Chemical Analysis,\newline Acute Injuries
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Delvin~Thozhuthumparambil~Robin~\\ Phone:\ +91-9400853232~\\  Email:\ delvintroby@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Yashtimadhu ghrita is a potent single herb ghee formulation mentioned in the classical Ayurveda texts like \textit{Ashtanga hrudaya,}\textit{Sushruta Samhita,} \textit{Bhaishajyaratnavali}, \textit{Chakradutta,}\unskip~\citep{1159646:22724741,1159646:22724752,1159646:22724842}  in the context of management of \textit{sadya kshatavrana (}wound of an acute origin as a result of injury, burns, contact with poisonous substances, strong and penetrating medications etc.) \unskip~\citep{1159646:22724910}. It was also found to be effective in controlling the radiation and chemotherapy induced side effects in the head and neck malignancies \unskip~\citep{1159646:22724996}. Addition of liquorice not only improves therapeutic efficacy of ghee but also increases its shelf life by exerting a resistance towards the oxidative deterioration of the ghee \unskip~\citep{1159646:22725006}. The objective of this study is to create a finger print profile of \textit{yashtimadhu ghrita} as a reference standard for future studies through its physico-chemical analysis. This was carried out systematically by procuring the herbal medicine, authentication of the specimen through organoleptic evaluation and cross-sectional microscopy, preparation of the formulation as per the classical texts of \textit{Ayurveda, }physico-chemical analysis through TLC and HPTLC.
    
\section{Materials and Methods}
\textit{Yashtimadhu} in its dried form was procured from cultivated resources at Hamirpur, Himachal Pradesh and Organic ghee from Ambadi hi-tech dairy farm, Tiruvamandoor, Kerala. The genuineness of the dry specimen of \textit{Yashtimadhu} (Glycyrrhiza glabra Linn.) was confirmed through organoleptic evaluation and cross-sectional microscopy. The purity of the ghee was tested in the QC lab of the Department of Medicinal Chemistry and Pharmaceutics, Amrita School of Ayurveda. Furfural and Baudouin tests were performed to rule out the presence of vanaspati hydrogenated fat and refined vegetable oil. The purity of the \textit{ghee} is ascertained and \textit{Yashtimadhu} \textit{ghrita} is prepared in the Department of \textit{Rasashastra } and \textit{Bhaishajya Kalpana}. The composition and ratio of ingredients of \textit{Yashtimadhu ghrita } is mentioned in Table~\ref{tw-7573279b5fdd}.

Method of Preparation of \textit{Yashtimadhu Ghrita}

The medicine was prepared as per the \textit{sneha paka (}preparation of unctuous  formulations using ghee, oil etc.\textit{)} procedure mentioned in \textit{Sarngadhara Samhita } \textit{Madhyama khanda} and the standard operative procedures specified in Ayurvedic pharmacopoeia of India \unskip~\citep{1159646:22725272,1159646:22725477}. \textit{Yashtimadhu} decoction is made using eight kilogram of coarse powdered specimen in 64 Liters of water [Figure~\ref{f-bf0b9638b3a9}]. It is reduced to 32 Liters [Figure~\ref{f-cc96bd778717}]. Into it eight liters of cow's ghee and two kilograms of fine powdered \textit{yashtimadhu kalka } are added and cooked in low flame until the entire moisture disappears and proper \textit{ghrita paka} (consistency) is obtained. A final quantity of 6.5 liters of ghee is obtained [Figure~\ref{f-bb932ea9dd9c}]. Flame test confirmed the absence of moisture and the residue was perfectly rolled into a wick between fingers. The entire procedure is done using a traditional bronze vessel in \textit{manda agni} (low flame). The finished product \textit{Yashtimadhu ghrita} was prepared in four days and the finished product is semi solid\textit{\space }in consistency, resembling colour of honey with a peculiar aroma and a mild sweet taste. Later the physico-chemical analysis of the finished product was carried out in the QC Laboratory of Arya Vaidyasala Kottakkal, Kerala.


\begin{table*}[!htbp]
\caption{\boldmath {Composition of Yashtimadhu Ghrita} }
\label{tw-7573279b5fdd}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.12959999999999999\linewidth-2\tabcolsep}p{\dimexpr.2231\linewidth-2\tabcolsep}p{\dimexpr.4885999999999999\linewidth-2\tabcolsep}p{\dimexpr.15870000000000001\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Sl.No. & Components & Botanical name/Common name & Quantity\\
\tblmidrule 
 1. &
  \textit{Yashtimadhu} &
  Glycyrrhiza glabra Linn. &
  1 Part\\
 2. &
  \textit{Go ghrita} &
  Cow's ghee &
  4 Parts\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Macroscopic Features of Glycyrrhiza glabra Linn } }
\label{tw-cf65e7a3a9e8}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.334\linewidth-2\tabcolsep}p{\dimexpr.666\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Part used & Root\\
\tblmidrule 
Nature of powder &
  Coarse and fine\\
Color &
  Brownish yellow\\
Taste &
  Sweet, Bitter\\
Odour &
  Sweetish (felt in the throat on sniffing)\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Results of physico-chemical analysis of finished product} }
\label{tw-7e204b8745d5}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1359\linewidth-2\tabcolsep}p{\dimexpr.3641\linewidth-2\tabcolsep}p{\dimexpr.16080000000000002\linewidth-2\tabcolsep}p{\dimexpr.3392\linewidth-2\tabcolsep}}
\tbltoprule 
S. No. &
  Test Parameter &
  Unit &
  Result\\
01 &
  Description &
  - &
  Pale yellow ghee\\
02 &
  LOD &
  \% W/W &
  0.64\\
03 &
  Acid Value &
   &
  6.7\\
04 &
  Iodine value &
   &
  38.7\\
05 &
  Saponification value &
   &
  213.4\\
06 &
  Refractive Index &
   &
  1.455\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Rf value and percentage area of Yahtimadhu ghrita sample at 254 nm} }
\label{tw-74eadeb1e190}
\def\arraystretch{1.1}
\ignorespaces 
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\begin{tabulary}{\linewidth}{p{\dimexpr.2438\linewidth-2\tabcolsep}p{\dimexpr.2562\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Peak No. & Rf Value & Area (AU) & \% Area (AU)\\
\tblmidrule 
1 &
  0.22 &
  145.1 &
  1.75\\
2 &
  0.33 &
  444.4 &
  5.35\\
3 &
  0.39 &
  1989.2 &
  23.97\\
4 &
  0.45 &
  971.2 &
  11.70\\
5 &
  0.52 &
  1326.6 &
  15.98\\
6 &
  0.57 &
  1731.2 &
  20.86\\
7 &
  0.67 &
  935.4 &
  11.27\\
8 &
  0.83 &
  269.7 &
  3.25\\
9 &
  0.92 &
  487.6 &
  5.87\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Rf Value and percentage Area of Yashtimadhu Ghrita sample at 366 nm} }
\label{tw-2ae0c5a06718}
\def\arraystretch{1.1}
\ignorespaces 
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\tbltoprule \rowcolor{kwdboxcolor}Peak No. & Rf Value & Area (AU) & \% Area (AU)\\
\tblmidrule 
1 &
  0.31 &
  272.4 &
  1.94\\
2 &
  0.38 &
  1884.3 &
  5.35\\
3 &
  0.46 &
  6953.7 &
  23.97\\
4 &
  0.58 &
  4771.6 &
  11.70\\
5 &
  0.70 &
  159.6 &
  15.98\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

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\caption{\boldmath {Preparation of \textit{Yashtimadhu }decoction}}
\label{f-bf0b9638b3a9}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/0fd34d9a-cbb9-467f-b4a9-4bbaaaaee4ca-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/0fd34d9a-cbb9-467f-b4a9-4bbaaaaee4ca-upicture2.png}{\includegraphics{images/0fd34d9a-cbb9-467f-b4a9-4bbaaaaee4ca-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Decoction reduced to 32 Liter's}}
\label{f-cc96bd778717}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/d3e978fc-21e9-460f-bd38-66c56446d218-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/d3e978fc-21e9-460f-bd38-66c56446d218-upicture3.png}{\includegraphics{images/d3e978fc-21e9-460f-bd38-66c56446d218-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Finished stage of \textit{Yashtimadhu ghrita}}}
\label{f-bb932ea9dd9c}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/5727e65d-3142-4130-96f1-2fea1af8560c-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/5727e65d-3142-4130-96f1-2fea1af8560c-upicture4.png}{\includegraphics{images/5727e65d-3142-4130-96f1-2fea1af8560c-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Overview graph of \textit{Yashtimadhu ghrita} sample at 254nm}}
\label{f-1deb77196d2a}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/973718a2-6710-41ce-92e5-b4f4e4cd3feb-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/973718a2-6710-41ce-92e5-b4f4e4cd3feb-upicture5.png}{\includegraphics{images/973718a2-6710-41ce-92e5-b4f4e4cd3feb-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Overview graph of Yashtimadhu ghrita sample at 366nm}}
\label{f-8be88519f621}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/5fc2be3b-dcae-4ff6-b923-c9db724ccf6c-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/5fc2be3b-dcae-4ff6-b923-c9db724ccf6c-upicture6.png}{\includegraphics[width=.25\linewidth]{images/5fc2be3b-dcae-4ff6-b923-c9db724ccf6c-upicture6.png}}{}
\makeatother 
\caption{\boldmath {TLC Plate Views of Yashtimadhu Ghrita Sample (At 254nm)}}
\label{f-d45d5b045578}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/53cd47d3-db29-49b2-bd6b-1848150da878-upicture7.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/53cd47d3-db29-49b2-bd6b-1848150da878-upicture7.png}{\includegraphics[width=.25\linewidth]{images/53cd47d3-db29-49b2-bd6b-1848150da878-upicture7.png}}{}
\makeatother 
\caption{\boldmath {TLC Plate Views of Yashtimadhu Ghrita Sample (At 366nm)}}
\label{f-ff28a90322c8}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/0c913412-474c-4d6f-93f4-9e3b69a1f70d-upicture8.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/0c913412-474c-4d6f-93f4-9e3b69a1f70d-upicture8.png}{\includegraphics[width=.25\linewidth]{images/0c913412-474c-4d6f-93f4-9e3b69a1f70d-upicture8.png}}{}
\makeatother 
\caption{\boldmath {TLC Plate Views of Yashtimadhu Ghrita Sample (At White Light)}}
\label{f-0eb72b347780}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/cdfc483f-2dc4-4e14-adb3-883915a84123-upicture9.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/cdfc483f-2dc4-4e14-adb3-883915a84123-upicture9.png}{\includegraphics[width=.26\linewidth]{images/cdfc483f-2dc4-4e14-adb3-883915a84123-upicture9.png}}{}
\makeatother 
\caption{\boldmath {Derivatized TLC Plate Views of Yashtimadhu Ghrita Sample (At 254nm)}}
\label{f-e8ef1ebac466}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/f5cae487-645f-4c9d-96e0-6a34f5842056-upicture10.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/f5cae487-645f-4c9d-96e0-6a34f5842056-upicture10.png}{\includegraphics[width=.24\linewidth]{images/f5cae487-645f-4c9d-96e0-6a34f5842056-upicture10.png}}{}
\makeatother 
\caption{\boldmath {Derivatized TLC Plate Views of Yashtimadhu Ghrita Sample (At 366nm)}}
\label{f-950671bd04f3}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/8f50d470-8147-47bf-8950-7f389c618e9e-upicture11.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/8f50d470-8147-47bf-8950-7f389c618e9e-upicture11.png}{\includegraphics[width=.25\linewidth]{images/8f50d470-8147-47bf-8950-7f389c618e9e-upicture11.png}}{}
\makeatother 
\caption{\boldmath {Derivatized TLC Plate Views of Yashtimadhu Ghrita Sample (At White Light)}}
\label{f-9b21f70dd680}
\end{figure}
\egroup
\textbf{Pharmacognostical evaluation of ingredients of \textit{Yashtimadhu ghrita}}

\textbf{Organoleptic evaluation}

The dry specimen powder is subjected to various sensory perceptions like color, taste, smell, and touch and recorded. Cross-sectional microscopy: A thin section of the specimen is observed under a compound microscope of 10X-15X resolution and studied. Physico-chemical evaluation: \textit{Yashtimadhu ghrita} was analyzed using different definitive physico-chemical parameters like Acid value, saponification value, refractive index and Iodine value. HPTLC was carried out for the characterization of the plant material after making appropriate solvent system having  methanolic extract of \textit{Yashtimadhu ghrita}.

\textbf{High Performance Thin Layer Chromatography (HPTLC)}

Both TLC and HPTLC were performed for obtaining the characterization of plant material. HPTLC is an instrumental version of TLC in order to get a reproducible result and is done using instruments where all parameters are fixed. 10 gm of the \textit{ghrita} sample is taken and is extracted with 10 ml Methanol and spotted as 10 micro liters.  Post chromatographic derivatization was done with Iodine vapor.

\textbf{Chromatographic Conditions}


  
  \begin{enumerate}
  \item \relax Stationary Phase: Merk, 1.05554.0007, TLC Silica gel 60 F\ensuremath{_{254,\ }}10x10 cm Aluminium sheet. 
  \item \relax  Mobile Phase: Toluene: Ethyl acetate: Formic acid: Methanol (7:5:1:0.5).
  \end{enumerate}
  \textbf{HPTLC Instrumentation}


  
  \begin{enumerate}
  \item \relax Camag HPTLC instrument. 
  \item \relax Application mode: CAMAG Linomat 5
  \item \relax Scanner: CAMAG TLC Scanner 3, CAMAG Reprostar 3.
  \item \relax Development chamber: CAMAG 10 x 10 cm Twin trough chamber.
  \item \relax Plates: Precoated silica gel 60 F\ensuremath{_{254,\ }}plates
  \item \relax Chamber saturation: 30 min
  \item \relax Development time: 30 min
  \item \relax Development distance: 7 cm
  \item \relax Detection: Deuterium lamp, Tungsten lamp
  \end{enumerate}
  \textbf{Thin Layer Chromatography (TLC)}

TLC is exclusively used for herbal drug analysis in herbal chemistry. TLC depends on the principle of separation through adsorption, where it relies on the affinity of the compound towards a mobile phase and a stationary phase. The sample is run through a solvent system in a chamber. Phytochemicals will move (ascending technique as per the retention time of the chemical component). Spot is the representation of a chemical compound. These spots obtained is observed by exposing the developed plate in UV light of wave length 254nm, 366 nm, and white light.
    
\section{Results }
Macroscopic features of \textit{Yashtimadhu } can be seen in Table~\ref{tw-cf65e7a3a9e8}. Various stages of preparation of Yashtimadhu ghrita shown in [Figures~\ref{f-bf0b9638b3a9}, \ref{f-cc96bd778717} and~\ref{f-bb932ea9dd9c}]. The results obtained by evaluating the physico-chemical parameters of the finished product is given in the [Table~\ref{tw-7e204b8745d5}].

The HPTLC Results of \textit{Yashtimadhu ghrita} is shown in the [Figure~\ref{f-1deb77196d2a}] and [Table~\ref{tw-74eadeb1e190}] at 254 nm and in [Figure~\ref{f-8be88519f621}] and [Table~\ref{tw-2ae0c5a06718}] at 366nm respectively. The TLC documentation picture profiles of \textit{yashtimadhu ghrita } sample at 254nm, 366 nm and in white light is shown in [Figure~\ref{f-d45d5b045578}], [Figure~\ref{f-ff28a90322c8}] and [Figure~\ref{f-0eb72b347780}] respectively. The derivatized TLC plate view of the \textit{ghrita} sample is shown in [Figure~\ref{f-e8ef1ebac466}], [Figure~\ref{f-950671bd04f3}] and [Figure~\ref{f-9b21f70dd680}] respectively. The total number of peaks obtained is nine in a total area of 8300.4 (AU) at 254 nm and total number of peaks is five in a total area of 14041.6 (AU) at 366 nm.
    
\section{Discussion}
\textit{Yashtimadhu ghrita} is a single herb ghee-based formulation having cold potency and wound healing property. On organoleptic evaluation and cross-sectional microscopy, the authenticity of the collected dry specimen of \textit{Yashtimadhu} was confirmed.

The physicochemical parameters when applied for assessing the finished product revealed an acid value of 6.7 which indicates the quantity of fatty acid present in the \textit{ghrita} formulation, a saponification value 213.4 is close to that of genuine cow's ghee. The refractive index value 1.455 at room temperature is also close to that of genuine ghee. An Iodine value of 38.7 within the range of genuine ghee, implies the iodine molecules consumed by the free fatty acids. Loss on drying (LOD), 0.64 is in the admissible range.

During the HPTLC scanning a total of nine spots were distinguished at 254nm and five spots at 366 nm frequencies. Among the spots, the Rf values 0.33, 0.39, 0.45 and 0.57 are common in both 254 nm and 366nm.
    
\section{Conclusion}
The preliminary pharmacognostical study confirms the genuineness of the dry specimen of \textit{Yashtimadhu.} The test result of the finished product shows Acid value 6.7, saponification value 213.4, and Refractive index value 1.455 at room temperature, Iodine value 38.7, LOD 0.64. The HPTLC scanning, distinguished nine peaks under 254nm and 5 peaks under 366nm. The test results of the present study can be considered as a reference standard and can be used for quality assessment of similar research undertakings in future.

\textbf{Conflict of Interest}

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

\textbf{Source of Funding}

\textbf{\space }The authors declare that they have no funding support for this study.
    

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