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\def\citeauthor{~Badugu~Kranthi~~Kumar,
              ~Marella~priyachandana,
              ~Allu~Sree~Reddy,
              ~Bhura~akhila,
              ~Gaddam~sushma,
              ~Naragoni~shireesha,
              ~Ade~Srinivash}
\def\citetitle{Phytochemical screening and pharmacological actions of Ocimumgratissimum}
\def\copyrightstmt{This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial- ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.}
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\begin{document}


\def\RunningAuthor{Badugu Kranthi Kumar et al.}
\firstPage{41}
\articleType{Original Article}
\receivedDate{02 Feb 2023}
\acceptedDate{14 Mar 2023}
\revisedDate{13 Mar 2023}
\journalVolume{2}
\journalIssue{2}
\journalDoi{ijebr.v2i2.457}
\copyrightYear{2023}
\eISSN{2583-5254}
\journalDoi{ijebr.v2i2.457}
\def\authorCount{7}
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\def\journalTitle{International Journal of Experimental and Biomedical Research}

\title{Phytochemical screening and pharmacological actions of Ocimumgratissimum}
\author{Badugu~Kranthi~~Kumar\textsuperscript{*}\textsuperscript{1}\textsuperscript{\href{https://orcid.org/0000-0003-0320-9603}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}},  
            Marella~priyachandana\textsuperscript{2}\textsuperscript{\href{https://orcid.org/0000-0003-0259-0588}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}},  
            Allu~Sree~Reddy\textsuperscript{2}\textsuperscript{\href{https://orcid.org/0000-0002-6696-5310}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}},  
            Bhura~akhila\textsuperscript{2}\textsuperscript{\href{https://orcid.org/0000-0002-5909-5388}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}},  
            Gaddam~sushma\textsuperscript{2}\textsuperscript{\href{https://orcid.org/0000-0003- 4003-9746}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}},  
            Naragoni~shireesha\textsuperscript{2}\textsuperscript{\href{https://orcid.org/0000-0002-5208-351X}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}}, 
            Ade~Srinivash\textsuperscript{2}\textsuperscript{\href{https://orcid.org/0009-0005-9378-1874}{\XeTeXLinkBox{\includegraphics[height=7pt]{orcid-logo-pharmascope.pdf}}}}~\\[5pt]{\textsuperscript{1}Department of Pharmacognosy, St.Mary's group of institution, Deshmukhi (Village), Pochampally (Mandal), Yadadri Bhuvanagiri (Dist), Telangana-508284, India}~\\{\textsuperscript{2}St. Mary's group of institution, Deshmukhi (Village), Pochampally (Mandal), Yadadri Bhuvanagiri(Dist), Telangana-508284, India}}

\begin{abstract}
The vast majority of these organic substances are secondary plant metabolites. With such in mind, there is a desire to synthesise certain natural goods as well as investigate their bioactivity. Additionally, plans have been made to isolate several bioactive natural substances. the species of plant Ocimum gratissimum, which is naturally occurring throughout India. The goal of the work is to identify several phytochemicals, including Alkaloids, Flavonoids, Carbohydrates, Tannins and Steroids from the leaves of Ocimumgratisimum The selected phytochemical compounds obtained from the plant by extraction are evaluated for antibacterial activity against the chosen species of microorganisms by utilising several types of solvents in the extraction process.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Phytochemical,\newline pharmacological,\newline Ocimumgratisimum
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright{\Large$^\ast$}Corresponding Author\par Name:\ Badugu~Kranthi~~Kumar~\\ Phone:\ +91 9160394919~\\  Email:\ badugu.kranthi19@gmail.com
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        \makeatother
        
    
\section{Introduction}
Organic chemistry is a science that is also an art. One of the most fascinating and best branches of modern chemistry is the art of synthesis and isolation of natural products and bioactive chemicals \unskip~\cite{1956344:28180481}. The crucial role that plant-based systems play in the healthcare of many cultures has been well-documented. According to the World Health Organisation, 65{\textendash}80\% of the world's population primarily uses traditional medicines made from plants for their main health care needs.  The world's population is now turning towards alternative traditional systems of medicine like Ayurveda, Unani, Homeopathy, Siddha and Yoga the herbal drugs show evidence of remarkable efficiency in the treatment of persistent chronic ailments \unskip~\cite{1956344:28180482}. The world health organization has suggested that nowadays people realize herbal medicine form of drugs there are more than 21,000 plants medicinal use around the world. Traditional medicinal systems followed by different tribal peoples of many countries in last few decades are very popularly known as tribal or folk medicine. In these systems that have the awareness of curing illnesses, preserves this information as a well kept secret and passes it down orally to the following generation. Still no written text evidence On these platforms, information is available, and various tribes use various time-tested practises \unskip~\cite{1956344:28180476}. In addition to the recommendation of lengthy as well as mystic ceremonies, the treatment of 2 days. In Indian system of traditional medicine after thousands of years of routine use a variety of plants have been utilized worldwide in the form of food preservation process, pharmaceutical products and natural medicine \unskip~\cite{1956344:28180485}. 
    
\section{Methodology:}
\textbf{Collection of Plant Material}

The Plant was collected from Venkatachalam, Nellore. Authentification of the plant was carried out by the Botanist Dr.Joy (HOD of BOTANY), D.K.W Government Degree College {\textendash} Nellore.

\textbf{Preparation of Powder}

The soil and other adherent materials were removed from the leaves after they had been harvested by washing them in fresh water. A sufficient quantity of leaves were allowed to dry at room temperature in the shade before being ground into a coarse powder.

\textbf{Preparation of Extract}

\textbf{Solvent Extraction by continuous hot Soxhlet Extractor}

A Soxhlet is a piece of lab equipment created by Franh von Soxhlet in 1879. Only when the desired component has a restricted solubility in a solvent and the impurity is insoluble in the solvent is a soxhlet extraction necessary. 

\textbf{Procedure}

Above 30 gm of powder of Ocimumgratissimum  leaves were loaded into the main chamber of the Soxhlet extractor, which is kept at a temperature, and contained in a thimble manufactured from thick paper of starting at 450 C. On top of a flask containing the extraction solvent, the Soxhlet extractor was put. \unskip~\cite{1956344:28180474} A condensor was included with the Soxhlet. The solvent was reflux-heated. Warm solvent that contains some of the desired compounds is slowly poured into the chamber where the solid substance is located. \unskip~\cite{1956344:28180491} When the Soxhlet chamber is filled, the solvent flows via a syphon tube and into the distillation flask.

The extraction process was subsequently completed by soaking the marc in ethanol for roughly 6 hours, or until it was colourless. To create a semi-solid mass, the extract was concentrated under reduced pressure as well as dried under vacuum \unskip~\cite{1956344:28180475}.

\textbf{Phytochemical investigations }

\textbf{Qualitative phytochemical studies }

Chemical analysis of a courtallam plant sample's methanol extracts was done qualitatively using established techniques to determine the main phytochemical components \unskip~\cite{1956344:28180490}. 

\textbf{Test for alkaloids: }

\textbf{Hager's test: }

Extracts were dissolved individually with Hager's reagent. Formation of reddish brown colored precipitate indicated the presence of alkaloids \unskip~\cite{1956344:28180484}. 

\textbf{Reagent preparation: }

Dissolve 100 ml of water and 1 g of picric acid.

\textbf{Test for flavonoids: }

\textbf{Alkaline reagent test: }

A few drops of sodium hydroxide (NaOH) solution were added to the extract. The presence of flavonoids was revealed by the formation of a bright yellow hue that became colourless when a few drops of diluted acetic acid were added \unskip~\cite{1956344:28180488}. 

\textbf{Test for phenolic compounds: }

5 ml of distilled water were used to dissolve 1 ml of the extract. A few drops of 5\% neutral ferric chloride solution were then added to this. There were phenolic chemicals present because of the dark green colour \unskip~\cite{1956344:28180487}.

\textbf{Test for tannins: }

Two drops of 5\% FeCl3 were added to in a test tube containing plant extract. A dirty green precipitate indicated the presence of tannins \unskip~\cite{1956344:28180479}. 


\begin{table*}[!htbp]
\caption{\boldmath {The compound showed good to moderate antimicrobial activity} }
\label{tw-2334a8f3496c}
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\tbltoprule \rowcolor{kwdboxcolor}S.No & Phytochemicals & Tests\\
\tblmidrule 
1 &
  Alkaloids &
  Wagner dragender offs test\\
2 &
  Tannins &
  Ferric chloride test\\
3 &
  Flavonoids &
  Ammonium and Sodium hydroxide acid test\\
4 &
  Steroids &
  Liberman Burchard and Salkowkis test\\
5 &
  Carbohydrates &
  Molisch's test\\
\tblbottomrule 
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\begin{table*}[!htbp]
\caption{\boldmath {Preliminary phytochemical screening of tests} }
\label{tw-50d4da649938}
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\tbltoprule \rowcolor{kwdboxcolor}Observation & Inference\\
\tblmidrule 
Brown precipitate that picric acid transforms into a bright yellow colour &
  Alkaloids  present\\
Greenish-Black precipitate &
  Tannins  present\\
Yellow color which turns colorless on addition of acid &
  Flavonoids  present\\
Brownish color Red color at interference &
  Steroids  present\\
Bluish violet zone formed &
  Carbohydrates present\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Table of Observation} }
\label{tw-1ca2336e43a1}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.5664000000000001\linewidth-2\tabcolsep}p{\dimexpr.4335999999999999\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Chemical Test & Extract\\
\rowcolor{kwdboxcolor} & Ethanol\\
\tblmidrule 
Alkaloids &
  +\\
Carbohydrates &
  +\\
Sugar &
  -\\
Steroids &
  +\\
Tannins &
  +\\
Proteins &
  \_\\
terpenoids &
  +\\
Flavenoids &
  +\\
Anthocyanin &
  \_\\
Quinines &
  \_\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Antibacterial zone of inhibition (mm) of extracted compound using disc-diffusion method (100\ensuremath{\mu}g/ml)} }
\label{tw-30a84c843e16}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.36650000000000006\linewidth-2\tabcolsep}p{\dimexpr.35989999999999995\linewidth-2\tabcolsep}p{\dimexpr.2736\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Compound & Zone of inhibition(mm) & \\
\rowcolor{kwdboxcolor} & Anti-bacterial activity & \\
\rowcolor{kwdboxcolor} & Staphylococcus aureus & Escherichia coli\\
\tblmidrule 
Ocimumgratissimum &
  23 &
  24\\
Ciprofloxacin \mbox{}\protect\newline  &
  25 \mbox{}\protect\newline  &
  26\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Test for steroids and triterpenoids }

\textbf{Salkowski's test:}

Chloroform was used to treat the extracts before filtering. A few drops of concentrated sulfuric acid were added to the filtrates, and they were then thoroughly shaken and left to stand \unskip~\cite{1956344:28180483}. The lower layer's appearance of red hue suggested the presence of steroids. After carefully adding concentrated sulfuric acid to the side (without shaking), the interface developed a reddish brown hue, signifying the presence of terpenoids.

\textbf{Test for Saponins }

A test tube was filled with 0.5g of the plant sample and 5ml of distilled water. The mixture was aggressively agitated to check for a stable, long-lasting foam. Three drops of extra-virgin olive oil were added to the foam, which was rapidly shook before being examined for the development of an emulsion \unskip~\cite{1956344:28180489}.

\textbf{Test for cardiac glycosides }

\textbf{Keller Killiani test: }

Two millilitres of glacial acetic acid containing a few drops of the FeCl3 solution was added to the test solution. Carefully adding 1 ml of concentrated H2SO4 along the test tube's side revealed the presence of cardenoloides' deoxysugar by the appearance of a brown ring at the interface. In the acetic acid layer, a greenish ring may also slowly emerge across the layer, while a violet ring may also appear beneath the brown ring \unskip~\cite{1956344:28180480}.

\textbf{Preparative Thin Layer Chromatography (TLCP) }

Glass plates were coated (1mm) with silica gel GF254 Merck, Darmstadt, Germany were used for the preparative thin layer chromatography (TLCP) separation process. Thin Layer Chromatography Plate (PTLCP) Preparation In a 500 mL rubber-stopper Erlenmeyer flask, 25g of Merck silica gel was suspended in 50ml of deionized water and violently shaken for 45 seconds. To make identical plates, the thickened slurry was poured into the glass and pulled with a ruler in two sides at 1mm trailing edge. The dishes were then placed in an oven set to 50 degrees Celsius for 30 minutes to air dry (until they were white). After drying, each plate was put into a separate glass container with the mobile phase being a solvent mixture of chloroform: methanol (9:1) and hexane: acetone (9:1 v/v). Each plate was taken out of the glass chamber and given a separate air drying after the movement of the solvent at the top of the plates. When the spots had dried, their fluorescence was used to identify them under long and short wave UV light (254 and 366 nm, respectively) \unskip~\cite{1956344:28180478,1956344:28180493} .

\textbf{Antimicrobial Activity}

\textbf{Collection of test microorganisms}

Gramme positive (Staphylococcus aureus) as well as Gramme negative (Escherichia coli) test organisms culture were collected from Veterinary College, Namakkal in Tamilnadu. The collected test pathogenic culture was sub-cultured in MH (Mueller- Hinton) Broth media the growth was observed to have increasing OD value under spectrophotometer. \unskip~\cite{1956344:28180492} Maintenance of Culture Maintain the test pathogenic culture which were inoculated into MHA slant by streaking and were incubated at 37oC for 24 hours and then stored at 4\ensuremath{^{0 }} C for further studies.

\textbf{Preparation of Mueller -Hinton media: }

Mueller-Hinton (MHB) Broth Composition 

Beef Infusion : 0.2g 

Casein hydrolysate : 1.75g 

Starch : 0.15g D. 

H2O :100 ml 

pH :7.4 \ensuremath{\pm} 0.2

\textbf{Composition of Mueller-Hinton Agar Media }

Beef extract : 150.0 gm 

Peptone : 8.75 gm 

Starch : 0.75 gm 

Agar : 8.5 gm 

D. H2O : 500 ml 

pH : 7.4 \ensuremath{\pm} 0.2 

\textbf{Antibacterial Activity }

For the Antimicrobial study, the various solvent concentrated extracts of all five samples of Ocimumgratisimum were diluted with 5mg, 10mg, 20mg and 40mg/ ml of same solvents respectively \unskip~\cite{1956344:28180486}. Antimicrobial activity was evaluated by well diffusion method on MHA medium. The sterile medium (20ml) was poured into Petri plates. The medium was allowed to cool in a 46 sterile condition and plates were then inoculated with 1x105 cfu (Colony-Forming Unit) of both cultures of test bacteria. 

The concentration of bacterial cells in the suspension was adjusted to minimum of 1x105 cfu/ ml in Muller Hinton broth solution \unskip~\cite{1956344:28180477}. Agar well of 6mm in diameter were made in the plates using sterile cork borer. The desired (5, 10, 20 and 40 mg/ml) different concentrations of the leaf extracts of 100 \ensuremath{\mu}l were added to separate well into MHA plates already seeded with the standardized inoculums (5 \ensuremath{\times} 105) of the test bacterial culture. The control antibiotic is used as ciprofloxacin. The antibiotic control was prepared by taking 10mg/ml of DMSO (Dimethyl Sulfoxide). All test plates were incubated at 37\ensuremath{^\circ}C for 24 hours.

Preliminary Phytochemical analysis was performed to identify [Table~\ref{tw-50d4da649938}] the presence of chemical constituents on support of literature review.

The chemical test revealed that the ethonolic extract was found to have more phytoconstituents [Table~\ref{tw-2334a8f3496c}].

Ascending thin layer chromatography (TLC) on silica gel G plates was used to measure and visualise the homogeneity of the extracted chemical. Chloroform: methanol (4:1) were the evaporating solvents for the compound. 

The extracted compound were evaluated for in vitro antimicrobial activity against Staphylococcus aureus (gram+ve); Escherichia coli (gram-ve), by disc diffusion method.

\textbf{Table of observation }

The chemical test revealed that the ethonolic extract was found to be the constituents was present in the passed chemical test of alkaloids, carbohydrates, steroids, tannins, terpenoids, flavonoids [Table~\ref{tw-1ca2336e43a1}]. 

\textbf{Evaluation of Anti-Microbial Activity}

Antimicrobial screening was performed by using disk diffusion method. The antimicrobial properties of the extraction were investigated against bacterial strains. The zone of inhibition was measured [Table~\ref{tw-30a84c843e16}]. 

This thesis deals with the ``Phytochemical screening and pharmacological actions of Ocimum gratissimum''. Isolated different types of phytochemicals like Alkaloids, Flavonoids, Carbohydrates, Tannins and Steroids Ocimum gratisimum leaves are extracted using a variety of solvents, and then some of the phytochemical components that were extracted from the plant are evaluated. Analyses of the compounds' antibacterial activity against the chosen species of microorganisms were conducted.
    
\section{ACKNOWLEDGEMENT}
I would like to thank Principal sir (Dr.V. Goutham) St. Mary's Group of Institutions, Deshmukhi (Village), Pochampally (Mandal), Yadadri Bhuvanagiri (Dist), Telangana-508284, India.

\textbf{Conflict of Interest}

The authors attest that they have no conflict of interest in this study.

\textbf{Funding}

No Funding.
    
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