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\def\RunningAuthor{Syeda Sanobar et al.}
\firstPage{1086}
\articleType{Original Article}
\receivedDate{07 Feb 2021}
\acceptedDate{17 Mar 2021}
\revisedDate{12 Mar 2021}
\journalVolume{2021, 12}
\journalIssue{2}
\journalDoi{ijrps.v12i2.4637}
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\title{\textit{\textbf{In- vitro}}\textbf{ Pharmacological investigation and Molecular Docking validation of } \textbf{\textit{Rhodiola rosea root against Bacterial and Helminthiasis infections}} \textbf{\space }}
\author{Syeda~Sanobar\textsuperscript{*}, 
            Dharmendra~Ahuja~\\[5pt]{Faculity of Pharmaceutical Science, Jayoti Vidyapeeth women's university, Jaipur, Rajasthan, India}}

\begin{abstract}
The present study's objective was to identify the phytoconstituents present in methanolic extract of \textit{Rhodiola rosea (R. rosea)} with a help of Gas chromatography-Mass Spectrometry (GC-MS). The extract was subjected for the antibacterial (\textit{Escherichia coli} ) and anthelmintic (adult Indian earthworms-\textit{Pheretima posthuma)} \textit{in-vitro }activities followed by the validation of the same by subjecting the bioactive compounds obtained in GCMS analysis to molecular docking studies for anthelmintic and antibacterial protein targets\textit{.} Six compounds with molecular formula  C\ensuremath{_{12}}H\ensuremath{_{14}}O\ensuremath{_{4}}NF\ensuremath{_{3,\ }} C\ensuremath{_{9}}H\ensuremath{_{14}}O\ensuremath{_{2}}Si, C\ensuremath{_{29}}H\ensuremath{_{44}}O\ensuremath{_{12,\ }} C\ensuremath{_{18}}H\ensuremath{_{30}}O, C\ensuremath{_{12}}H\ensuremath{_{23}}O\ensuremath{_{2}}N, and C\ensuremath{_{17}}H\ensuremath{_{32}}O  were identified in GCMS analysis. Different extract concentrations viz 50, 75, 100, 150, and 200 mg/ml and 50, 75, 100, 150, and 200 \ensuremath{\mu}g/ml for anthelmintic activity and anti-bacterial activity respectively. For anthelmintic protein target, results of molecular docking (presented by 3D \& 2D model) revealed that the identified compounds (binding energy -6.05 to -8.40 Kcal/mol) exhibited a comparable binding affinity as that of albendazole(binding energy -9.54 Kcal/mol) except C3 (binding energy +33.42 Kcal/mol) compound.  antibacterial protein target, compounds' binding affinities were found to be in the range -4.49 to -6.68 Kcal/mol, while chlorobiocin was found to be -4.75 Kcal/mol. The extract exhibited dose-dependent \textit{in-vitro}anthelmintic (150 and 200 mg/ml) and antibacterial activity (150 and 200 \ensuremath{\mu}g/ml. The minimum lethal amount of extract concentration for anthelmintic activity was found to be 150 \ensuremath{\mu }g/ml. At this concentration, \textit{Pheretimaposthuma died in }33.56 \ensuremath{\pm} 2.15 min while with a standard dose of albendazole, the lethal time was found to be 22.75 \ensuremath{\pm} 1.63 min. Finally concluded that the identified bioactive compounds from \textit{R. rosea have the potential }anthelmintic and antibacterial activity as justified by \textit{in-vitro} biological evaluations and molecular docking studies.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Rhodiola rosea,\newline GC-MS,\newline Earthworm,\newline Escherichia coli,\newline Molecular Docking
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Syeda~Sanobar~\\ Phone:\ +91-8497907995~\\  Email:\ sanoafnan@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
The genus \textit{Rhodiola of} family Crassulaceae nearly around 200 species present, in which almost 20, comprising \textit{Rhodiola rosea}, \textit{Rhodiola alterna, Rhodiola brevipetiolata, Rhodiola crenulata, Rhodiola kirilowi, Rhodiola quadrifida, Rhodiola sachalinensis}, and \textit{Rhodiola sacra}, are used as ethno-medicines in Asia \unskip~\citep{1034685:21664417}\textit{Rhodiola rosea} plants mainly available in the region of the Himalayan belt, Tibet, China, and Mongolia, were as also cultivated in Europe and North America,  \textit{Rhodiolarosea }are available dietary supplements and juice on the market \unskip~\citep{1034685:21664423,1034685:21664414} widely known as "golden root" or "roseroot." The pharmacological effects of \textit{Rhodiola rosea }are adaptogenic or anti-stress, hepatoprotective effects and immunomodulatory, antiviral, anti-inflammatory, and antibacterial activities \unskip~\citep{1034685:21664417,1034685:21664412}\textit{Rhodiola}\textit{rosea }contain secondary metabolites such as polyphenols, flavonoids, proanthocyanidines, tyrosol, and cinnamyl alcohol, glycosides, organic acids, essential oils, sugars, fats, alcohols, and proteins \unskip~\citep{1034685:21664412}. The polyphenol includes Rosavin, cinnamyl alcohol, salidroside and tyrosol, major components of \textit{Rhodiola} \textit{rosea}\unskip~\citep{1034685:21664416,1034685:21664422}.

The aim of the present study is to the identification of bio active compounds in methanolic extract\textit{ R. rosea }on the application of Gas chromatography-mass spectrometry (GC-MS) there molecular docking, \textit{in-vitro} antibacterial and anthelmintic activity of \textit{R. rosea}. 
    
\section{Materials and Methods}
\textbf{Collection of plant material}

Dried roots of \textit{Rhdiola rosea} (Golden root) family Crassulaceae were procured from a local vendor, Seremban, Negeri Sembilan, Malaysia. Roots are authenticated by Dr. Long chiau Ming, Associated Professor, Deputy Dean faculty of Pharmacy, Quest international university Perak. (QUIP-RR/02/2019).


\bgroup
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\centering \makeatletter\IfFileExists{images/80375651-cfc8-431e-8124-735d283d4ee3-upicture1.png}{\includegraphics{images/80375651-cfc8-431e-8124-735d283d4ee3-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Docked complex of all the Test Compounds (C1to C6) and Standard Ligand (albendazole sulphoxide) in the binding pocket of 1OJ0.}}
\label{f-51970c7fef42}
\end{figure}
\egroup
\textbf{Preparation of extraction}

The roots material was powdered using a mixer grinder and passed through sieve no 85. About The dried 150gm powder was subjected to soxhlets apparatus extraction using methanol solvent for 72 hrs. The extract were concentrated in rotary flash evaporators and stored in refrigerator.

\textbf{Culture media bacteria }

\textit{E.coli }micro organism provided by Owaisi Hospital and Research Center, department of microbiology, Hyderabad, Mueller--Hinton agar (Himedia, Lot 0000333943, Code M173) NaCl, Beef Extract procured by Himedia Pvt. Ltd., India.

\textbf{GC-MS analysis}

Chromatographic data were obtained using Clarus 680 GC. A fused silica column packed with Elite-5MS (containing 5\% biphenyl 95\% dimethylpolysiloxane, 30 m \ensuremath{\times} 0.25 mm ID \ensuremath{\times} 250\ensuremath{\mu }m df), separation of components was carried out using Helium gas with a constant flow rate of 1ml/minute. Whereas, the injector temperature was maintained at 260\ensuremath{^\circ}C throughout the chromatographic. The volume of the extract injected into the instrument was 1 \ensuremath{\mu }L. The oven temperature was initially maintained at 60 \ensuremath{^\circ}C for two minutes, followed by 300 \ensuremath{^\circ}C at the rate of 10 \ensuremath{^\circ}C min\ensuremath{-}1 for 6 minutes.

Meanwhile, for the mass detector, transfer line temperature and ion source temperature was maintained at 240\ensuremath{^\circ} C. Ionization mode was set with an electron impact of 70 eV with 0.2 seconds, scan time and 0.1-sec scan interval. Fragmentation was observed between 40 to 600 Da. The resultant spectra were compared with the reference spectrum using GC-MS NIST (2008) library.

\textbf{Docking (Molecular) Evaluation}

Molecular docking with anthelmintic and antibacterial target proteins was carried out on the bioactive molecules/moieties obtained from the GCMS analysis results. The biological targets are Model of \textit{Haemonchuscontortus } \ensuremath{\beta }-tubulin bound to albendazole sulphoxide (anthelmintic protein target - PDB ID: 1OJ0, retrieved from \BreakURLText{www.modelarchive.org),} and DNA gyrase from \textit{E-coli } (antibacterial protein Target - PDB ID: 1KZN, retrieved \BreakURLText{fromwww.rcsb.org).} The existing ligands and water molecules were deleted, and protons were included. The sketching of the compounds and standard ligand (internal ligand) was sketched with ChemSketch software. The ligand moieties were bound to the target pro


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\centering \makeatletter\IfFileExists{images/2428f6d4-bc25-41eb-abed-eed7cdd4abd0-upicture2.png}{\includegraphics{images/2428f6d4-bc25-41eb-abed-eed7cdd4abd0-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Molecular interactions between 1OJ0 amino acid residues and Test Compounds (C1 to C6) and Standard Ligand (STD) (albendazole sulphoxide) }}
\label{f-5ecbaa258af0}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {GC{\textendash}MS analysis of extract \textit{R.rosea}} }
\label{tw-29cb07f7cff1}
\def\arraystretch{1.1}
\ignorespaces 
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\begin{tabulary}{\linewidth}{p{\dimexpr.0935\linewidth-2\tabcolsep}p{\dimexpr.3664\linewidth-2\tabcolsep}p{\dimexpr.1489\linewidth-2\tabcolsep}p{\dimexpr.1337\linewidth-2\tabcolsep}p{\dimexpr.1323\linewidth-2\tabcolsep}p{\dimexpr.1252\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}S. no  & Peak name & Molecular formula & Molecular weight & Retention time & \% Area\\
\tblmidrule 
1 &
  N-(2-(3,4-dimethoxyphenyl)-2-hydroxyethyl)-2,2,2-trifluoroacetamide  &
  C12H14O4NF3 &
  293 &
  19.595 &
  42.844\\
2 &
  Phenol, 3-[(trimethylsilyl)oxy-1 &
  C9H14O2Si &
  182 &
  21.321 &
  35.954\\
3 &
  4-((1R,11R,17R)-1,5,11,14-tetrahydroxy-10-(hydroxymethyl)-13-methyl-3-((3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one &
  C29H44O12 &
  584 &
  24.457 &
  3.639\\
4 &
  (5E,9E)-6,10,14-trimethylpentadeca-5,9,13-trien-2-one &
  C18H30O &
  262 &
  25.122 &
  2.017\\
5 &
  1-Hexyl-1-nitrocyclohexane &
  C12H23O2N &
  213 &
  25.448 &
  3.543\\
6 &
  14-heptadecenal &
  C17H32O &
  252 &
  26.468 &
  12.002\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Docking scores and estimated inhibition constant (Ki) of Test Ligands (C1 to C6) and Standard Ligand (albendazole sulphoxide) against 1OJ0} }
\label{tw-8c2b217c7e13}
\def\arraystretch{1.1}
\ignorespaces 
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\begin{tabulary}{\linewidth}{p{\dimexpr.434\linewidth-2\tabcolsep}p{\dimexpr.25720000000000002\linewidth-2\tabcolsep}p{\dimexpr.3088\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Compound & Docking Score (Kcal/mol) & Estimated Inhibition Constant Ki (\ensuremath{\mu}M)\\
\tblmidrule 
C1 &
  -7.47 &
  3.37\\
C2 &
  -7.25 &
  4.83\\
C3 &
  +33.42 &
  Not Applicable\\
C4 &
  -8.40 &
  0.69\\
C5 &
  -6.05 &
  36.92\\
C6 &
  -6.72 &
  11.85\\
StandardLigand (STD) (albendazolesulphoxide) &
  -9.54 &
  0.10\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Docking scores and estimated inhibition constant (Ki) of Test Ligands and Standard Ligand (Clorobiocin) against 1KZN} }
\label{tw-6d1f83fca8b4}
\def\arraystretch{1.1}
\ignorespaces 
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\begin{tabulary}{\linewidth}{p{\dimexpr.3633\linewidth-2\tabcolsep}p{\dimexpr.29669999999999998\linewidth-2\tabcolsep}p{\dimexpr.34\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Compound & Docking Score (Kcal/mol) & Estimated Inhibition Constant Ki (\ensuremath{\mu}M)\\
\tblmidrule 
C1 &
  -5.21 &
  151.07\\
C2 &
  -5.97 &
  41.92\\
C3 &
  -4.49 &
  509.37\\
C4 &
  -6.68 &
  12.59\\
C5 &
  -5.38 &
  114.19\\
C6 &
  -4.65 &
  390.64\\
Standard Ligand (STD) (Clorobiocin) &
  -4.75 &
  322.71\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
tein, and the binding affinities were determined using AutoDock 4 tools.\unskip~\citep{1034685:21664419} Molec


\bgroup
\fixFloatSize{images/93e7792a-b518-4664-9cb7-125c4d59cc81-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/93e7792a-b518-4664-9cb7-125c4d59cc81-upicture3.png}{\includegraphics{images/93e7792a-b518-4664-9cb7-125c4d59cc81-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Docked complex of all the Test Compounds (C1to C6) and Standard Ligand (Clorobiocin)in the binding pocket of 1KZN.}}
\label{f-0a82fa71929f}
\end{figure}
\egroup
ular interaction between protein and ligands were viewed with Molegrow Molecular Viewer 2.5 and Discovery Studio 2017.

\textbf{Preparation of extracts}

The \textit{Rhdiolarosea }methanolic extract different concentration were prepared 50, 75,100,150 and 200 mg/ml for anthelmintic activity and 50, 75,100,150 and 200 \ensuremath{\mu}g/ml for anti-bacterial activity. Albendazole 20mg/kg bw and Chlorobiocin apply as standard. In each Group contain 3-4 \textit{Pheretima Posthuma }earthworms of approximately equal size are individually placed in each Petri dish fill with10-15 ml normal saline solution and desired concentration of drug and extracts.

\textbf{Antibacterial testing by agar well diffusion method}

Assessment of antibacterial activity \textit{R.rosea} extracts was determined by agar well diffusion method described \unskip~\citep{1034685:21664411}. Inoculums of \textit{Escherichia coli} bacterial strains were plated using sterile swabs into Petri dishes containing approximately 25 ml of Nutrient agar media, where 6 mm wells were made and filled with different concentration of 50,100,150 and 200\ensuremath{\mu }l/ml of extracts. The Petri dishes were pre-incubated for 3 h at room temperature, allowing the complete diffusion of the samples and then incubated at 37\ensuremath{\pm}1\ensuremath{^\circ}C for 24 h. Further antibacterial activity of the above samples was determined by measuring the zone of inhibition millimeters (mm) according to the standard clinical parameters.\textbf{\space }

\textbf{Anthelmintic activity}

The \textit{in-vitro }anthelmintic study was done as per the protocol described by \unskip~\citep{1034685:21664410,1034685:21664409}.

\textbf{Group 1: }Normal Control Group using normal saline 0.5\% w/v \textbf{\space }

\textbf{Group 2: }Standard GroupAlbendazole 20 mg/ml kg bw

\textbf{Group 3: } \textit{R.rosea} methanolicextract50 mg/ml

\textbf{Group 4: } \textit{R.rosea} methanolic extract 75 mg/ml 

\textbf{Group 5: } \textit{R.rosea} methanolic extract 100 mg/ml 

\textbf{Group 6: } \textit{R.rosea} methanolic extract 150 mg/ml

\textbf{Group 7: } \textit{R.rosea} methanolic extract 200 mg/ml 

\textit{Pheretima Posthuma }adult Indian earthworms (n=3, or 4) were placed in each Petri dish. The worms were observed for the time of loss of movement (paralysis) and death. Average time for the loss of movement (paralysis) and complete loss of movement even upon aggressive shaking (death) was recorded. Albendazole was used as the reference standard to compare the activity of the extract. \textbf{\space }
    
\section{Results}
GC{\textendash}MS chromatogram of extract \textit{R.rosea }clearly shows six peaks representing the presence of 6 bio active compounds. Bioactive compounds from the extracts were identified from the data obtained in terms of the peak area, retention time and the molecular formula. (Table~\ref{tw-29cb07f7cff1}).

\textbf{Molecular docking analysis}

The \textit{R.rosea }methanolic extract compounds were found to possess a range of binding affinities for the studied macro molecular targets as observed by docking scores shown in Tables~\ref{tw-8c2b217c7e13} and~\ref{tw-6d1f83fca8b4} for anthelmintic and antibacterial activity. The binding affinities of the compounds (being in range -6.05 to -8.40 Kcal/mol for anthelmintic protein target\textbf{) }found to be in good agreement with standard albendazole (-9.54 Kcal/mol) except C3 (+33.42 Kcal/mol).In the antibacterial protein target, the compounds' binding affinities are found to be in the range -4.49 to -6.68 Kcal/mol, while the standard Chlorobiocin was found to be-4.75 Kcal/mol.  Compounds C1, C2, C4, and C5 showed a higher binding affinity for the antibacterial protein target than the standard Chlorobiocin. The docked complex of all the compounds with 1OJ0 and their binding interaction maps were shown in Figures~\ref{f-51970c7fef42} and~\ref{f-5ecbaa258af0}.

Figure~\ref{f-5ecbaa258af0} Shows, C1- N-(2-(3,4-dimethoxyphenyl)-2-hydroxyethyl)-2,2,2-trifluoroacetamide,C2- Phenol, 3-[(trimethylsilyl)oxy-1,C3-4-((1R,11R,17R)-1,5,11,14-tetrahydroxy-10-(hydroxymethyl)-13-methyl-3-((3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one,C4- (5E,9E)-6,10,14-trimethylpentadeca-


\bgroup
\fixFloatSize{images/790c652d-a022-4f56-ad9d-0dfdd55b9484-upicture4.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/790c652d-a022-4f56-ad9d-0dfdd55b9484-upicture4.png}{\includegraphics{images/790c652d-a022-4f56-ad9d-0dfdd55b9484-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Molecular interactions between 1KZN amino acid residues and Test Compounds (C1 to C6) and Standard Ligand (STD) (Clorobiocin). }}
\label{f-290b546dc27b}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/a5084d2b-d226-4489-a14e-aaaef4b830c4-upicture5.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/a5084d2b-d226-4489-a14e-aaaef4b830c4-upicture5.png}{\includegraphics{images/a5084d2b-d226-4489-a14e-aaaef4b830c4-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Showing the anti bacterial effect of \textit{R. rosea }different concentrations against \textit{E. Coli}}}
\label{f-59ac64bb25d8}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/3e3c1009-b15f-4a59-ad47-e433ecca830a-upicture6.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/3e3c1009-b15f-4a59-ad47-e433ecca830a-upicture6.png}{\includegraphics{images/3e3c1009-b15f-4a59-ad47-e433ecca830a-upicture6.png}}{}
\makeatother 
\caption{\boldmath {showing anthelmintic activity \textit{R.rosea} (a) Normal (b)Albendazole and (c) \textit{R.rosea} extract.}}
\label{f-24456fce1482}
\end{figure*}
\egroup
5,9,13-trien-2-one, C5-1-Hexyl-1-nitrocyclohexane and C6-14-heptadecenal

Also, the docked complex of all the compounds with 1KZN and their binding interaction maps were shown in Figures~\ref{f-0a82fa71929f} and~\ref{f-290b546dc27b}.

Figure~\ref{f-290b546dc27b} shows, C1- N-(2-(3,4-dimethoxyphenyl)-2-hydroxyethyl)-2,2,2-trifluoroacetamide,C2- Phenol, 3-[(trimethylsilyl)oxy-1,C3-4-((1R,11R,17R)-1,5,11,14-tetrahydroxy-10-(hydroxymethyl)-13-methyl-3-((3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one,C4- (5E,9E)-6,10,14-trimethylpentadeca-5,9,13-trien-2-one, C5-1-Hexyl-1-nitrocyclohexane and C6-14-heptadecenal

The antimicrobial activity of extracts \textit{R.rosea} were studied in different concentrations (50, 100,150 and 200 \ensuremath{\mu }g/ml) against Gram-negative \textit{Escherichia coli} pathogenic bacterial strains. The extract shows that  varying degree of antimicrobial activity 150 and 200 \ensuremath{\mu }g/ml as shown in (Figure~\ref{f-59ac64bb25d8})

The \textit{R.rosea} extract was studied in different concentrations (50, 75,100,150 and 200 mg/ml) anthelmintic activity on \textit{Pheretima posthuma }worms. The extract shows significant activity in a dose-dependent manner, as shown in (Figure~\ref{f-24456fce1482}). The anthelmintic activity \textit{R.rosea} extract in lower concentration not significant, in higher concentration extract 150,200 mg/ml exhibit significantly 31.15 \ensuremath{\pm} 4.35for paralysis and 29.54 \ensuremath{\pm} 3.21 minute, in death time of the worms 37.28 \ensuremath{\pm} 4.42 minute and 33.56 \ensuremath{\pm} 2.15 minute. Albendazole22.75 \ensuremath{\pm} 1.63 and 31.20 \ensuremath{\pm} 1.25. The effectiveness of the extract \textit{R. rosea} was inversely proportional to the time for paralysis (vermifuge) and death (vermicidal) of the worms\textbf{\space }
    
\section{Discussion}
Resistance to antibiotics is of more significant concern worldwide due to rising incidences of resistance in the pathogens against the antimicrobials and antibiotics. This increase in the resistance is attributed to the random, indiscriminate use of the available antimicrobial drugs. Therefore, scientists are continually looking for newer antimicrobial agents, particularly from natural sources \unskip~\citep{1034685:21664424}.

Natural sources, particularly plants and herbs, have always been the scientist's first choice in the discovery and development of the therapeutic agents due to the presence of the phytoconstituent with various medicinal properties. Besides, medicinal agents obtained from plants are generally considered safe and are recommended for various ailments \unskip~\citep{1034685:21664418,1034685:21664420}. Many plants have been studied and are reported to contain the phytoconstituent effective against various pathogens \unskip~\citep{1034685:21664413}.

Phenylethanoids, phenylproponoids, and their glycosides, monoterpene alcohols and their glycosides, aryl glycosides, cyanogenic glycosides, proanthocyanidins, flavonlignans, flavonoids, and gallic acid derivatives, phenolic compounds tyrosol, salidroside, and rosavin derivatives are reported to be present in higher amount in the roots and rhizomes of Rhodolia rosea \unskip~\citep{1034685:21664416,1034685:21664412}.

\textbf{Molecular docking analysis}

Molecular docking analyses validated the \textit{in-vitro} anthelmintic and antibacterial activities demonstrated by the compounds'. The compounds' demonstrated good binding affinity levels for the protein target \ensuremath{\beta }-tubulin and bacterial DNA gyrase. The main target for inhibiting helminth development is \ensuremath{\beta }-tubulin, as it is the sub unit of microtubules involved in essential cellular functions, including mitosis, motility, and transport. \unskip~\citep{1034685:21664421} Bacterial DNA gyrase from \textit{E-coli} is one of the significant bacterial proteins involved in replication and transcription and catalyzes bacterial circular DNA's negative super coiling. DNA gyrase is perhaps a recognized target for antibacterials because bacterial death is caused by its blocking. \unskip~\citep{1034685:21664415}.\textbf{\space }Hence, the anthelmintic and antibacterial potential of the compounds' could be linked with the ability to interact with and possibly regulate the studied protein targets' activities.
    
\section{Conclusions}
Rhodiola rosea has gained popularity as a medicinal herb and has been used in Chinese medicine for its adaptogenic properties. This research study was aimed at investigating the antibacterial and anthelmintic effect of \textit{R. rosea} extracts. Besides, the extracts were evaluated for potential phytoconstituent effective against the microbes and helminth parasites. The extracts under study demonstrated a significant antibacterial and anthelminthic activity. Antibacterial and anthelminthic effects of the extracts may be ascribed to the presence of various phytoconstituent, including alcohol derivatives, flavonoids, and phenolic compounds, a variety of glycosides, and essential oils. \textit{\space }
\section*{Acknowledgement}Authors would like to acknowledge and thanks to Dr. Long chiau Ming, Associated Professor, Deputy Dean faculty of Pharmacy, Quest international university Perak for authentication of a plant. We also thank and appreciate MEDICULE for scientific solution Hyderabad, providing chemicals. We heartly gratitude to Assoc. Prof Dr Abdullah Khan KPJ Healthcare University College, Nilai, Malaysia and Assoc prof Dr K.M Noorulla, College of Health Sciences, Arsi University, Ethopia for his valuable suggestion in research work.

\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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