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\def\RunningAuthor{Abdul Khayum K et al.}
\firstPage{2520}
\articleType{Original Article}
\receivedDate{14 Oct 2020}
\acceptedDate{17 Nov 2020}
\revisedDate{15 Nov 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4507}
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\title{Experimental evaluation of anxiolytic activity in mice using Vacha Churanam}
\author{Anamika~P~K\textsuperscript{1},  
            Sulaiman~Mohammed~Alnasser\textsuperscript{2}, 
            Abdul~Khayum~K\textsuperscript{*}\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Department of Pharmacology, CL Baid Metha College of Pharmacy, Chennai, Tamil Nadu,  India}~\\{\textsuperscript{2}Department of Pharmacology and Toxicology, Unaizah College of Pharmacy, Qassim University, Saudi Arabia}~\\{\textsuperscript{3}Department of Pharmacology, PSG College of Pharmacy, Coimbatore, Tamil Nadu, India}}

\begin{abstract}
Vacha Churanam is an Ayurvedic product. It is commonly given traditionally for memory enhancement, antidepressants, anxiolytic and anti-stress. However, there is no scientific evidence for its anxiolytic activity. Therefore the current study is aimed to validate its anxiolytic activity. Kept mice persuaded the anxiety in the room maintained with a temperature of 4\ensuremath{^{0}}C for two h per day. The Vacha Churanam has administrated doses of 200 and 400 mg/kg, p.o for 21 days. The behavioural parameters such as elevated zero maze, light-dark test, Morris water maze, actophtometer, hole board were produced significant activity with Vacha Churanam. The neurotransmitters measures such as dopamine, serotonin and acetylcholine esterase measures were also increased in Vacha Churanam treated mice when matched with negative control group mice. The antioxidant enzyme superoxide dismutase was increased, and lipid peroxidase level was diminished in Vacha Churanam treated mice. It supports the anxiolytic action of Vacha Churanam through an antioxidant mechanism. The Anxiolytic result of Vacha Churanam is associated with essential constituents such as flavonoids, phenolic and steroids. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Hole board,\newline Morris water maze,\newline Dopamine,\newline Serotonin,\newline Superoxide dismutase,\newline Anxiety
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Abdul~Khayum~K~\\ Phone:\ +91-9600331252 ~\\  Email:\ abdulkhayumk7@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Anxiety is a feeling of unease and fear, typically general and unclear overreaction. It is related to restlessness, muscular tension, fatigue and problems in concentration \unskip~\citep{935917:20789993}. As per the World Health Organization (WHO), one in 13 people affected globally with anxiety. The anxiety disorders stand worldwide caused mental disorders with phobia and chief depressive illness \unskip~\citep{935917:20790006}. There are many medications including Escitalopram, Fluvoxamine, Sertraline, Duloxetine, Alprazolam, Chlordiazepoxide, Diazepam, Oxazepam, Lorazepam, Clomipramine, Imipramine, Phenelzine, Hydroxyzine, Buspirone can be utilised currently to the treatment of anxiety \unskip~\citep{935917:20790004}. The difficulty in the synthetic drugs are imperfectly tolerated or assemble substantial undesirable side effects (WHO reports). The widely used medicine to treat anxiety is benzodiazepines as they deliver fast and significant relief however the problem with the benzodiazepines is harm cognitive function while using them, and long-standing practice could cause permanent memory deficiency \unskip~\citep{935917:20790003}. Most people wishing substitute treatment including for anxiety including meditation practice, Ayurveda, Siddha and homoeopathy, play a part in mental, behavioural therapy, decreasing stress and workout \unskip~\citep{935917:20789999}. Vacha Churanam is an ayurvedic product. It is commonly used traditional product for the memory enhancement, antidepressants, anxiety and anti-stress etc., \unskip~\citep{935917:20789992}. However, there is no scientific confirmation for its anxiety and anti-stress activity. Therefore the current study is aimed to validate its anxiety and anti-stress activity. 
    
\section{Materials and Methods}
\textbf{Materials}

The marketed ayurvedic product Vacha Churanam was purchased from online shopping website Ayu svastha. Diazepam was purchased from Sigma Aldrich, USA. All other reagents and chemicals were used under analytical grade.

\textbf{Preliminary phytochemical analysis}

Vacha Churanam was mixed with distilled water and subjected to preliminary phytochemical screening for the confirmation of phytoconstituents such as alkaloids, carbohydrates, protein, steroids, phenols, tannins and flavonoids \unskip~\citep{935917:20789989,935917:20789991}. 

\textbf{Experimental animals}

Albino mice (22-30g body weight) were attained from the animal household of C.L. Baid Metha College of Pharmacy, Thoraipakkam, Chennai-97. All experimentations were supported to the guidelines for attention and usage of experimental animals and approved by the Committee for Control and Supervision of Experiments on Animals (CPCSEA). The experimental ethical was permitted by the Institutional Animal Ethical Committee (IAEC). IAEC reference no: IAEC/LI/01/CLBMCP/2017. 

\textbf{Acute toxicity studies}

The OECD guideline 423 was followed for the acute toxicity to the fixing of experimental dose. In acute toxicity study, 2000 mg/kg, p.o different dose of Vacha Churanam was given for three animal containing groups. Then mice have withheld the feed for 3-4 h and monitored carefully for the marks of toxicity for 15 successive days (OECD-423). \unskip~\citep{935917:20789994}

\textbf{Grouping of animals }

Totally 30 mice were separated into five groups and each for six animals. Group I: Control mice were only received normal saline. Group II: Negative Control mice were received normal saline. Group III: Standard mice served with Diazepam 5mg/kg, i.p. Group IV: Vacha Churanam low dose mice served with Vacha Churanam 200mg/kg, p.o. Group V: Vacha Churanam high dose mice served with Vacha Churanam 400mg/kg, p.o. 

\textbf{Induction of anxiety}

The negative control, standard treatment and Vacha Churanam treatment groups, were preserved in a room maintained with the temperature of 4\ensuremath{^{0}}C for two h per day for the generation of stress followed by anxiety. The normal control group was only received normal saline for the entire study. The treatments for the respective groups were for 21 days. The final dose was given 60 min earlier to behavioural testing \unskip~\citep{935917:20789995}. 

\textbf{Estimation of behavioural parameters}

The behavioural examinations were conducted on the 21\ensuremath{^{st}} day of the experiment. The behavioural examinations including locomotors test (Actophotometer) \unskip~\citep{935917:20790005}, Elevated zero maze \unskip~\citep{935917:20790002}, Dark-light compartment \unskip~\citep{935917:20789998} and Marble burying tests \unskip~\citep{935917:20790008} were conducted by standard procedure. 

\textbf{Estimation of neurotransmitter level}

After the experimental period, all group mice were humanly killed by cervical decapitation and Brain were isolated. The isolated brain was homogenate with HCl {\textendash} Butanol solution and pH 7.4 solutions and allowed 10 min for centrifugation at 10000 rpm \unskip~\citep{935917:20790000}. The obtained supernatant undergoes the valuation of neurotransmitter including Dopamine, Serotonin and Acetylcholinesterase enzyme using standard procedure followed by literature \unskip~\citep{935917:20789997}.

\textbf{Estimation of antioxidants levels}

On the completion of the investigational period, all group mice were humanly killed by cervical decapitation for isolation of Brain. The isolated brain was homogenate with ice-cold TCA pH 7.4 solutions and allowed 10 min for centrifugation at 10000 rpm \unskip~\citep{935917:20789996}. The obtained supernatant undergoes the valuation of \textit{in vivo} antioxidants levels including Lipid peroxidation (LPO) and Superoxide dismutase (SOD) as per standard process followed by literature \unskip~\citep{935917:20790001,935917:20790066}. 

\textbf{Histopathological study}

On the final day experiment, the brains of all group animals were carefully detached without damage. Then the isolated brains were rinsed using ice-cold saline solution and stable in 10\% neutral buffered formalin. The five \ensuremath{\mu} sections of tissues were stained using Mayer's Haematoxylin Eosin dye. 

\textbf{Statistical analysis}

The statistical analysis was carried by one-way analysis of variance (ANOVA) monitored by Dunnet's test. P values {\textless}0.05 (95\% confidence limit) was measured statistically significant, by Software Graph pad Prism 6.0. 
    
\section{Results}
\textbf{Preliminary phytochemical Qualitative analysis}

The preliminary phytochemical examination endorses the numerous phytoconstituents including alkaloids, carbohydrates, Glycosides, Saponins, Anthraquinones, protein, steroids, phenols, tannins and flavonoids existence in Vacha Churanam. 


\bgroup
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\centering \makeatletter\IfFileExists{images/c49d0b1a-952e-4794-909a-681cc84225e1-upicture1.png}{\includegraphics{images/c49d0b1a-952e-4794-909a-681cc84225e1-upicture1.png}}{}
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\caption{\boldmath {Effect of Vacha churnaon Behavioral parameters}}
\label{f-d931fac31e2b}
\end{figure*}
\egroup

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\caption{\boldmath {Histopathological study of Brain}}
\label{f-b092ebd52719}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Effect of Vacha Churanam on neurotransmitters} }
\label{tw-7e183f25555e}
\centering 
\begin{threeparttable}

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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No & \cAlignHack Group & \cAlignHack Acetylcholine Esterase (ng/mg wet tissue) & \cAlignHack Dopamine  \mbox{}\protect\newline  & \cAlignHack Serotonin  \mbox{}\protect\newline (ng/mg wet tissue)\\
\tblmidrule 
\cAlignHack 1 &
  Normal control &
  \cAlignHack 25.63\ensuremath{\pm}0.57 &
  \cAlignHack 430.23\ensuremath{\pm}20.34 &
  \cAlignHack 243.34\ensuremath{\pm}5.6\\
\cAlignHack 2 &
  Negative Control &
  \cAlignHack 12.34\ensuremath{\pm}0.67 &
  \cAlignHack 308.45\ensuremath{\pm}2.45 &
  \cAlignHack 181.33\ensuremath{\pm}5.34\\
\cAlignHack 3 &
  Standard &
  \cAlignHack 23.34\ensuremath{\pm}0.12** &
  \cAlignHack 400.23\ensuremath{\pm}3.2**  &
  \cAlignHack 235.55\ensuremath{\pm}5.32**\\
\cAlignHack 4 &
  Vacha Churanam 200mg/kg &
  \cAlignHack 15.34\ensuremath{\pm}0.2* &
  \cAlignHack 239.47\ensuremath{\pm}2.55   &
  \cAlignHack 203\ensuremath{\pm}2.72ns\\
\cAlignHack 5 &
  Vacha Churanam 400mg/kg &
  \cAlignHack 20.98\ensuremath{\pm}0.11** &
  \cAlignHack 381.45\ensuremath{\pm}4.34*  &
  \cAlignHack 225.34\ensuremath{\pm}2.89* \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{No. of Animals 6, Data were analysed as Mean \ensuremath{\pm} SEM, One way ANOVA followed by Dunnett's test, All groups were matched with negative control, *p{\textless}0.05, **p{\textless}0.01,***p\textless\ 0.001}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Effect of Vacha Churanam on antioxidants} }
\label{tw-ca8f96f59098}
\centering 
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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No & \cAlignHack Group & \cAlignHack SOD (Units/mg wet tissue) & \cAlignHack LPO (Units/mg wet tissue)\\
\tblmidrule 
\cAlignHack 1 &
  Normal control &
  \cAlignHack 9.32\ensuremath{\pm}0.4 &
  \cAlignHack 70.1\ensuremath{\pm}0.3\\
\cAlignHack 2 &
  Negative Control &
  \cAlignHack 4.1\ensuremath{\pm}0.25 &
  \cAlignHack 109.3\ensuremath{\pm}0.23\\
\cAlignHack 3 &
  Standard &
  \cAlignHack 9.02\ensuremath{\pm}0.41**  &
  \cAlignHack 72.34\ensuremath{\pm}1.3** \\
\cAlignHack 4 &
  Vacha Churanam 200mg/kg &
  \cAlignHack 5.99\ensuremath{\pm}0.3\ensuremath{^{ns}} &
  \cAlignHack 95.45\ensuremath{\pm}1.23\ensuremath{^{ns}}\\
\cAlignHack 5 &
  Vacha Churanam 400mg/kg &
  \cAlignHack 7.187\ensuremath{\pm}0.21*  &
  \cAlignHack 80.23\ensuremath{\pm}1.32* \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{No. of Animals 6, Data were analysed as Mean \ensuremath{\pm} SEM, One way ANOVA followed by Dunnett's test, All groups were matched with negative control, *p{\textless}0.05, **p{\textless}0.01,***p\textless\ 0.001}
\end{tablenotes}
\end{threeparttable}

\end{table*}
\textbf{Acute oral toxicity studies}

A single dose (2000 mg/kg p.o) of the Vacha Churanam did not persuade any sign of toxicity. It confirms the safe dose of Vacha Churanam at 2000 mg/kg. Therefore we fixed the 1/10\ensuremath{^{th}} (200mg/kg) and 1/5\ensuremath{^{th}} (400 mg/kg) dose for further experimentation. 

\textbf{Effect of Vacha Churanam on Behavioral parameters}

Our finding in elevated zero maze test shown that negative control mice were decreased in percentage time consumed within open arm and percentage passes within an open arm. In the light-dark test, negative control mice were decreased the time consumed in the light compartment. Similarly, in the whole, board test, negative control mice have decreased the sum of line crossing, head dipping and amount of rearing. These decreased levels were enhanced significantly improved in after 21 days Vacha Churanam (200 mg/kg and 400 mg/kg) and standard diazepam administration. However, negative control mice were buried more amount of marbles in the marble-burying experiment, higher escape latency in the Morris water maze experiment. They augmented the locomotors activity in Actophotometer test. In Vacha Churanam (200 mg/kg and 400 mg/kg) and diazepam treated mice groups were reported significantly lowered (Figure~\ref{f-d931fac31e2b}).

From Figure~\ref{f-d931fac31e2b}, A: Elevated zero maze test, B: Light-dark test, C: Marbleburying test, D: Morris water maze test, E: Locomotors test (Actophoto meter)and F: Hole board test. (Data is expressed as Mean \ensuremath{\pm} SEM; n=6, One way ANOVA followed by Dunnett's test; ns = non-significant; Compared with disease control;* p{\textless}0.05, ** p{\textless}0.01, *** p{\textless}0.001).

\textbf{Effect of Vacha Churanam on neurotransmitters}

Three neurotransmitter endogenous chemical messengers including Acetylcholine esterase, Serotonin and Dopamine measures were significantly diminished in negative control mice when compared with normal control mice. Interestingly, both Vacha Churanam (200 mg/kg and 400 mg/kg) and standard diazepam treated mice were significantly increased (Table~\ref{tw-7e183f25555e}).

\textbf{Effect of Vacha Churanam on antioxidants level}

The antioxidant levels, including SOD and LPO, were estimated. The brain SOD measures in negative control mice were significantly declined, while LPO measures were significantly augmented when both of them were matched with normal control. In contrast, Vacha Churanam (200 mg/kg and 400 mg/kg) and standard diazepam treated mice showed that antioxidant measures were significantly normalised (Table~\ref{tw-ca8f96f59098}).

\textbf{Histopathological study of Brain}

In the negative control mice, the density of neuronal cells and disturbance of neuronal cells were decreased. In the other hand, groups treated with Vacha Churanam (200 mg/kg and 400 mg/kg) and standard diazepam exhibited improved neuronal configurations (Figure~\ref{f-b092ebd52719}).

From Figure~\ref{f-b092ebd52719}, A: Normal Control, B:Negative Control, C: Standard, D: Vacha churna 200mg/kg and E: Vacha churna400mg/kg. Mayer's Haematoxylin Eosin stain was used for staining. 
    
\section{DISCUSSION }
Benzodiazepines are expansively used anxiolytic synthetic drugs. But commonly Benzodiazepines associated with unwanted side effects so that alternative treatments are needed. Medicinal herbals and herbal therapy like Siddha, Ayurveda and Unani are an excellent foundation to find new remedies to treat these disorders. In the study for a substitute, more specific and perhaps cost-free therapy, researchers are focusing on investigating natural anxiolytic drugs. The Vacha Churanam is an Ayurvedic product marketed for controlling the anxiety. In the current research, the anxiolytic action of 200 mg/kg and 400 mg/kg of Vacha Churanam on the Elevated zero maze, light-dark test, actophtometer, morris water maze, hole board were almost same comparable with 4 mg/kg of diazepam. The neurotransmitters, including dopamine, serotonin and acetylcholine esterase measures were also significantly elevated because of anxiolytic activity. The elevated measures of SOD, deteriorated measures of LPO in Vacha Churanam confirm the antioxidant action of anxiolytic activity. These observations indicate that Vacha Churanam exerts an anxiolytic activity. The anxiolytic effect of Vacha Churanam is might be associated with essential constituents, including flavonoids, steroids and phenolic composites \unskip~\citep{935917:20789990}. The literature review has suggested that many scientific trials have been stated that Vacha Churanam exhibited many properties in the central nervous system (CNS) disorders. Vacha Churanam is also intended for the managing of CNS disorders. Vacha Churanam might be appreciated in the controlling of neurodegenerative diseases including Alzheimer's disease, memory-improving property and anticholinesterase activity. 
    
\section{Conclusion}
In conclusion, the current research confirms the anxiolytic activity of Vacha Churanam against cold-induced stress on mice. The effect of Vacha Churanam might be the manifestation of phytochemicals such as flavonoids, steroids and phenolic. Further molecular pharmacological and chemical researches are required to expose the exact mechanisms of anxiolytic action and to separate the active phytochemicals. 

\textbf{Funding Support}

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

\textbf{Conflict of Interest }

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

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