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\begin{document}


\def\RunningAuthor{Ravi Dabas et al.}
\firstPage{1074}
\articleType{Original Article}
\receivedDate{10 Feb 2021}
\acceptedDate{16 Mar 2021}
\revisedDate{13 Mar 2021}
\journalVolume{2021, 12}
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\journalDoi{ijrps.v12i2.4635}
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\title{\textbf{Physiochemical Analysis of \textit{Nilotapaladi} \textit{Yoga}}}
\author{Ravi~Dabas\textsuperscript{*}\textsuperscript{1},  
            Sweety\textsuperscript{2},  
            Dixit~VK\textsuperscript{3}, 
            Kar~AC\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Vikriti Vigyan, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India}~\\{\textsuperscript{2}Department of Agada Tantra evam Vidhivaidyaka, Uttarakhand Ayurved University, Haridwar, India}~\\{\textsuperscript{3}Department of Gastroenterology, Faculty of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India}}

\begin{abstract}
In \textit{Ayurveda} many \textit{Ayurvedic} formulations are explained, which used to treat a variety of illness since ancient time and to protect patient faith in \textit{Ayurveda} and \textit{Ayurvedic} treatment, scientifically proven safety, standard and quality of herbs is the need of the hour. \textit{Nilotpaladi yoga }(NY)\textit{} is mentioned in\textit{ Ayurvedic }texts\textit{}for the Treatment of\textit{ Raktatisara } ({\texttildeapprox}haemorrhagic diarrohea). \textit{Nilotpaladi yoga} consists of four ingredients such as\textit{\space }\textit{Nilakamala }(\textit{Nymphaea nouchal}i Burm. f.), \textit{Mocharasa} (\textit{Salmalia malabarica } Schott \& Endl.), \textit{Lajavanti } (\textit{Mimosa pudica } Linn.) and \textit{Kamalakesara} (\textit{Nelumbo nucifera} Gaertn.). \textit{Nilotpaladi yoga} ingredient like \textit{Mochrasa} (\textit{Salmalia malabarica } Schott \& Endl.) is known to be effective as \textit{Shonita sthapaka gana } (a group of drugs that act as haemostatics). Physiochemical analysis of \textit{Nilotpaladi} yoga is not explored in detail. So, this article is an attempt to present a physiochemical analysis of ingredients of \textit{Nilotpaladi} \textit{yoga} by using Modern analytical Techniques. Physio-chemical analysis, HPTLC etc., were carried out as per standard methods. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}HPTLC,\newline Mochrasa,\newline Nilotpaladi yoga,\newline Raktatisara
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Ravi~Dabas~\\ Phone:\ +917565087467~\\  Email:\ ravidabas1988@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
In \textit{Charaka Samhita}, \textit{Nilotpaladi} \textit{Yoga} is mentioned for the treatment of \textit{Raktatisara}\unskip~\citep{1033380:21660098}. 

Ayurveda described \textit{Raktatisara} ({\texttildeapprox}haemorrhagic diarrhoea), which has symptoms similar to ulcerative colitis, i.e. \textit{Shula} ({\texttildeapprox}pain in abdomen), \textit{Gudapaaka} ({\texttildeapprox}burning sensation in rectum) and \textit{Trishna} ({\texttildeapprox}excessive thirst) \unskip~\citep{1033380:21660112}. 

Flowers of \textit{Nymphea} \textit{nouchali} were found antibacterial functions against bacteria like Bacillus cereus and Staphylococcus aureus \unskip~\citep{1033380:21660106}. 

Some Studies also indicated that\textit{ Mochrasa} (\textit{Salmalia malabarica } Schott \& Endl.) has haemostatic, antidiarrhoeal, antidysenteric and antipyretic properties \unskip~\citep{1033380:21660107,1033380:21660105}. 

The \textit{Mocharasa} (\textit{Salmalia malabarica } Schott \& Endl.) is known to contain tannic and gallic acids, which acting as astringents and precipitate proteins which is helpful in the healing of the damaged mucosal lining of ulcerated mucosa \unskip~\citep{1033380:21660102,1033380:21660096}. 

The Nelumbo Nucifera is used to treat bleeding, gastritis and has anti-inflammatory effects \unskip~\citep{1033380:21660097,1033380:21660100}. \textit{Nelumbo} leaf extract (NLE) was also shown antibacterial activity \unskip~\citep{1033380:21660117}. 
    
\section{Materials and Methods}
\textit{Nilotpaladi yoga} (NY) ingredients were purchased from the Gola dinanath market of Varanasi. The identity was confirmed with the help of Sri Dharmasthala Manjunatheshwara centre for research in Ayurveda and Allied Sciences (AYUSH Centre for Excellence and Recognized SIROs by DSIR) Laxminarayana Nagar, P.O. Kuthpady{\textendash}UDUPI Karnataka. \textit{Nilotpaladi yoga }ingredients means gum of \textit{Salmalia malabarica } Schott \& Endl., Seeds of\textit{ Lajavanti } (\textit{Mimosa pudica } Linn.), Stamen of \textit{Kamalakesara} (\textit{Nelumbo nucifera } Gaertn.) and petals of \textit{Nilakamala } (\textit{Nymphaea nouchal}\textit{i } Burm. f.) were converted into \textit{Churna} ({\texttildeapprox}powder form). Samples were sent to \textit{Sri }\textit{Dharmasthala } \textit{Manjunatheshwara} Centre for Research in \textit{Ayurveda} and allied Sciences (AYUSH Centre for Excellence and Recognized SIROs by DSIR). Kuthpady, UDUPI  Karnataka, where Physicochemical parameters as depicted in (Table~\ref{tw-3579e8c7ec5b}) were estimated as per Standard methods described in Ayurvedic Pharmacopeia of India and world health organization standard and HPTLC values were evaluated by using Linomat 5 TLC applicator. 

\textbf{Methodology of Various Parameters and Results}

\textit{\textbf{Loss on drying at 105\ensuremath{^{o}}C}}

10 g of each ingredients sample of \textit{Nilotpaladi} \textit{yoga} were taken separately in a separate tared evaporating dish. It was then dried at temperature 105{\textdegree}C for 5 hours in the oven and then weighed. The drying continued till the difference between two continuous weights didn't more than 0.01 after cooling by using a desiccator. Moisture percentage calculated in relation to the weight of the sample. 

\textbf{\textit{Total Ash}}

2 g of sample was incarnated in tared platinum crucible at a temperature not exceeding 450{\textdegree}C until carbon-free ash is obtained. The percentage of ash was calculated in relation to the weight of the sample. 

\textit{\textbf{Acid insoluble Ash}} \textit{\space }

To the crucible containing total ash, add 25ml of dilute HCl and boil. Collect the insoluble matter on ashless filter paper (Whatmann 41) and wash with hot water until the filtrate is neutral. Transfer the filter paper containing the insoluble matter to the original crucible, dry on a hot plate and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 30 mins and weigh without delay. Calculate the content of acid-insoluble ash with reference to the air-dried drug.

\textit{\textbf{Water-soluble ash}} \textit{\space }

Boil the ash for 5 min with 25 ml of water; collect insoluble matter on an ashless filter paper, wash with hot water, and ignite for 15 min at a temperature not exceeding 450{\textdegree}C. Subtract the weight of the insoluble matter from the weight of the ash; the difference in weight represents the water-soluble ash with reference to the air-dried sample.

\textit{\textbf{Alcohol soluble extractive}} \textit{\space }

Weigh accurately 4 g of the sample in a glass stoppered flask. Add 100 ml of distilled Alcohol (approximately 95\%). Shake occasionally for 6 hours. Allow to stand for 18 hours. Filter rapidly, taking care not to lose any solvent. Pipette out 25ml of the filtrate in a pre-weighed 100 ml beaker. Evaporate to dryness on a water bath. Keep it in an air oven at 105\ensuremath{^\circ}C for 6 hours, cool in a desiccator for 30 minutes and weigh. Calculate the percentage of Alcohol extractable matter of the sample. Repeat the experiment twice, and take the average value. 

\textit{\textbf{HPTLC}}

1g of each of \textit{Nilakamala}, \textit{Mocharasa}, \textit{Lajavanti}, \textit{Kamalakesara} and Churna formulation (1part of each of the ingredient) was extracted with 10 ml of alcohol. 5\ensuremath{\mu}l of each of the above extract was applied on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator. The plate was developed in Toluene: Ethyl Acetate (9.0: 1.0). The developed plates were visualized in under short UV, long UV and then derivatised with vanillin sulphuric acid and scanned under UV 254nm, 366nm and 620nm (the following derivatisation). Rf, colour of the spots and densitometric scan were recorded (Tables~\ref{tw-c48266fd9319}, \ref{tw-c9da0d1624e5} and~\ref{tw-7c9b137954fc}) and Figures~\ref{f-774a727e1a52} and~\ref{f-05a7c12348a4} a-d,  Figure~\ref{f-81913f1c7b3f} a-d \& Figure~\ref{f-fa50e7164332} a-d). 


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\caption{\boldmath {HPTLC photo documentation of Alcoholic fraction of Nilakamala, Mocharasa, Lajavanti, Kamalakesara Churna}}
\label{f-774a727e1a52}
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\caption{\boldmath {Densitometric scan at 254nm}}
\label{f-05a7c12348a4}
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\caption{\boldmath {Densitometric scan at 366nm}}
\label{f-81913f1c7b3f}
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\caption{\boldmath {Densitometric scan at 620nm}}
\label{f-fa50e7164332}
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\begin{table*}[!htbp]
\caption{\boldmath {Results of \textit{Niltpaladi yoga} ingredients standardization parameters} }
\label{tw-3579e8c7ec5b}
\def\arraystretch{1.1}
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\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.38269999999999996\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}p{\dimexpr.14530000000000001\linewidth-2\tabcolsep}p{\dimexpr.13189999999999996\linewidth-2\tabcolsep}p{\dimexpr.1801\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Parameter Results n = 3 \%w/w (Avg) & Nilakamala  & Mocharasa  & Lajavanti  & Kamalakesara \\
\tblmidrule 
Loss on Drying &
  9.46 &
  10.97 &
  6.77 &
  9.22\\
Total Ash &
  10.83 &
  8.22 &
  5.44 &
  7.18\\
Acid Insoluble Ash &
  0.89 &
  3.47 &
  0.29 &
  2.58\\
Water soluble Ash &
  4.08 &
  3.18 &
  0.29 &
  2.67\\
Alcohol soluble extractive value &
  4.88 &
  12.40 &
  16.59 &
  5.06\\
Water soluble extractive value &
  14.19 &
  20.47 &
  47.80 &
  16.03\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {R\ensuremath{_{f\ }}values At short UV} }
\label{tw-c48266fd9319}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2334\linewidth-2\tabcolsep}p{\dimexpr.2459\linewidth-2\tabcolsep}p{\dimexpr.2479\linewidth-2\tabcolsep}p{\dimexpr.2728\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Nilakamala & Mocharasa & Lajavanti & Kamalakesara\\
\tblmidrule 
- &
  - &
  - &
  -\\
- &
  - &
  - &
  -\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*D{\textendash} dark; L {\textendash} light; F {\textendash} fluorescent}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {R\ensuremath{_{f\ }}values At long UV} }
\label{tw-c9da0d1624e5}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.2293\linewidth-2\tabcolsep}p{\dimexpr.2707\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} Nilakamala &  Mocharasa &  Lajavanti & Kamalakesara\\
\tblmidrule 
 - &
   - &
   - &
   -\\
 - &
   - &
   - &
   -\\
 - &
   - &
   - &
   -\\
 -  &
   -  &
   -  &
   - \\
 -  &
   -  &
   -  &
   - \\
 0.38 (F red) &
   - &
   - &
   -\\
 0.48 (F aqua blue) &
   0.48 (FL blue) &
   - &
   0.48 (F green)\\
 0.55 (F blue) &
   0.55 (FD blue) &
   0.55 (FD blue) &
   0.55 (F blue)\\
 0.69 (F blue) &
   &
   0.69 (FL blue) &
   0.69 (F blue)\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*D{\textendash} dark; L {\textendash} light; F {\textendash} fluorescent}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {R\ensuremath{_{f}} values After derivatisation} }
\label{tw-7c9b137954fc}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.26450000000000003\linewidth-2\tabcolsep}p{\dimexpr.23549999999999997\linewidth-2\tabcolsep}p{\dimexpr.2479\linewidth-2\tabcolsep}p{\dimexpr.2521\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Nilakamala & Mocharasa & Lajavanti & Kamalakesara\\
\tblmidrule 
- &
  - &
  - &
  -\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{*D {\textendash} dark; L {\textendash} light; F {\textendash} fluorescent}
\end{tablenotes}
\end{threeparttable}

\end{table*}

    
\section{Results and Discussion}
Physiochemical analysis is an important tool to find out the quality, standard and efficacy of Ayurvedic drugs and by correct identification, it can be helpful in preventing adulteration \unskip~\citep{1033380:21660114}. Ash values used to be an important tool to assess the purity of herbs. The Ash and extractive values is the fastest means of determining the quality and efficacy of Herbs. Acid insoluble ash is a part of total ash and tells us the amount of siliceous earth \unskip~\citep{1033380:21660114}. Physiological Parameters evaluation of Nilotpaladi yoga is depicted in (Table~\ref{tw-3579e8c7ec5b}). Loss of drying was maximum seen in \textit{Mocharasa} (\textit{Salmalia malabarica } Schott \& Endl.) (10.97\%) and minimum in\textit{ Lajavanti } (\textit{Mimosa pudica } Linn.) (6.77\%). Total ash values maximum \textit{Nilakamala } (\textit{Nymphaea nouchal} \textit{i} Burm. f.) (10.83) and minimum \textit{Lajavanti } (\textit{Mimosa pudica } Linn.) (5.44\%). Each \textit{Nilotpaladi} \textit{yoga} ingredients Standardization Parameters Results depicted in (Table~\ref{tw-3579e8c7ec5b}). \textbf{\space }

With HPTLC, authentication of many types of plant possible, as well as the analysis of stability and consistency of their preparations from various manufactures \unskip~\citep{1033380:21660113}. HPTLC Fingerprinting, by examining R\ensuremath{_{f}} values and color of the spots, is one of the easiest pharmacopeial parameters to evaluate the qualitative compositional characteristics of any crude drug extracts. HPTLC of ethanolic extract of \textit{Nilotpaladi Yoga }TLC photo documentation confirmed the detection of various photoconstituents with separate R\ensuremath{_{f}} values (Figure~\ref{f-774a727e1a52}, Tables~\ref{tw-c48266fd9319}, \ref{tw-c9da0d1624e5} and~\ref{tw-7c9b137954fc}). HPTLC photo documentation of Alcoholic fraction of \textit{Nilakamala}, \textit{Mocharasa}, \textit{Lajavanti}, \textit{Kamalakesara} \textit{Churna} (Figure~\ref{f-774a727e1a52}), under Short UV and After derivatization showed no any spots (Figure~\ref{f-774a727e1a52}). Therefore, R\ensuremath{_{f\ }} Value at short UV and after dervivatization can not be observed (Figure~\ref{f-774a727e1a52}, Tables~\ref{tw-c48266fd9319} and~\ref{tw-7c9b137954fc}). HPTLC photo documentation at Long UV of \textit{Nilkamala} showed 3 spots (in different colors) at R\ensuremath{_{f\ }} Values ranging from 0.38 to 0.69. \textit{Mocharasa} (\textit{Salmalia malabarica } Schott \& Endl.) showed 2 spots (both F.blue) at R\ensuremath{_{f\ }} values ranging from 0.48 to 0.55. \textit{Lajavanti }(\textit{Mimosa pudica } Linn.)\textbf{\space }showed 2 spots (F. Dark \& F. Light) at R\ensuremath{_{f}} values ranging from 0.55 to 0.69. \textit{Kamalakesara} (\textit{Nelumbo nucifera } Gaertn.) showed 5 spots (in different colors) at R\ensuremath{_{f}} values ranging from 0.08 to 0.69 (Figure~\ref{f-774a727e1a52}, Table~\ref{tw-c9da0d1624e5}).

\textbf{High-performance thin-layer chromatography} \textbf{\space }

\textit{Nilakamala (\textit{Nymphaea nouchali} Burm. f.). }On the densitometric scan of the plates, 3, 9 and 10 bands were detected under 254nm, 366nm and 620nm, respectively (Figure~\ref{f-05a7c12348a4} a, Figure~\ref{f-81913f1c7b3f} a \& Figure~\ref{f-fa50e7164332} a). Out of 3 peaks seen on densitometric scan at 254nm, compounds with R\ensuremath{_{f}}0.03 (78.31\%) was the major peaks (Figure~\ref{f-05a7c12348a4} a); at 366, out of 9 peaks, peak with R\ensuremath{_{f}} 0.67 (41.26\%) was the major peak detected (Figure~\ref{f-81913f1c7b3f} a); at 620nm, out of 10 peaks, peak with R\ensuremath{_{f}} 0.08 (34.09\%) was the major peak seen (Figure~\ref{f-fa50e7164332} a). \textit{Mocharasa} (\textit{Salmalia malabarica } Schott \& Endl.) on a densitometric scan of the plates, 3, 8 and 5 bands were detected under 254nm, 366nm and 620nm, respectively (Figure~\ref{f-05a7c12348a4} b, Figure~\ref{f-81913f1c7b3f} b \& Figure~\ref{f-fa50e7164332} b). Out of 3 peaks seen on densitometric scan at 254nm, compounds with R\ensuremath{_{f}}0.03 (92.92\%) was the major peaks (Figure~\ref{f-05a7c12348a4} b); at 366, out of 8 peaks, peak with R\ensuremath{_{f}} 0.66 (37.26\%) was the major peak detected (Figure~\ref{f-81913f1c7b3f} b); at 620nm, out of 5 peaks, peak with R\ensuremath{_{f}} 0.05 (89.80\%) was the major peak seen (Figure~\ref{f-fa50e7164332} b). \textit{Lajavanti } (\textit{Mimosa pudica } Linn.) on densitometric scan of the plates, 9, 3 and 5 bands were detected under 254nm, 366nm and 620nm, respectively (Figure~\ref{f-05a7c12348a4} c, Figure~\ref{f-81913f1c7b3f} c \& Figure~\ref{f-fa50e7164332} c). Out of 3 peaks seen on densitometric scan at 254nm, compounds with R\ensuremath{_{f}}0.04 (52.85\%) was the major peaks (Figure~\ref{f-05a7c12348a4} c); at 366, out of 3 peaks, peak with R\ensuremath{_{f}} 0.66 (57.64\%) was the major peak detected (Figure~\ref{f-81913f1c7b3f} c); at 620nm, out of 5 peaks, peak with R\ensuremath{_{f}} 0.07 (34.05\%) was the major peak seen (Figure~\ref{f-fa50e7164332} c). \textit{Kamalakesara} (\textit{Nelumbo nucifera } Gaertn.) on a densitometric scan of the plates, 4, 5 and 7 bands were detected under 254nm, 366nm and 620nm, respectively (Figure~\ref{f-05a7c12348a4} d, Figure~\ref{f-81913f1c7b3f} d \& Figure~\ref{f-fa50e7164332} d). Out of 4 peaks seen on densitometric scan at 254nm, compounds with R\ensuremath{_{f\ }}0.04 (64.48\%) was the major peaks (Figure~\ref{f-05a7c12348a4} d); at 366, out of 5 peaks, peak with R\ensuremath{_{f}} 0.65 (60.34\%) was the major peak detected (Figure~\ref{f-81913f1c7b3f} d); at 620nm, out of 7 peaks, peak with R\ensuremath{_{f}} 0.04 (39.10\%) was the major peak seen Figure~\ref{f-fa50e7164332} d). These fingerprints will be helpful to serve the purpose of evaluation of the identity of chemical composition qualitatively.  
    
\section{Conclusion}
The results and the standards used in Physiochemical analysis and HPTLC findings of \textit{Nilotpaladi} \textit{Yoga} ingredients surely be useful as an important part for validation and quality checking of crude drugs used in NY. This study may be useful for Researches etc. As this article is only dealt with Physio-chemical analysis, the implementation and outcome of this Nilotpaladi Yoga will be published separately.\textbf{\space }

\textbf{Conflict of Interest}

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

\textbf{Funding Support}

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

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