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\begin{document}


\def\RunningAuthor{Pankaj Nainwal and Amita B Sati}
\firstPage{1139}
\articleType{Original Article}
\receivedDate{05 Aug 2020}
\acceptedDate{19 Sep 2020}
\revisedDate{15 Sep 2020}
\journalVolume{2020, 11 (SPL)}
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\journalDoi{ijrps.v11iSPL1.3561}
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\title{Assessment of ascorbic acid content and anti-oxidant activity in juices of selected fruits help in the prophylaxis of primary symptoms of COVID-19}
\author{Pankaj~Nainwal\textsuperscript{*}\textsuperscript{1}, 
            Amita~B~Sati\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Pharmacognosy, School of Pharmacy, Graphic Era Hill University, Dehradun, Uttarakhand, India}~\\{\textsuperscript{2}Department of Chemistry, Uttaranchal University, Dehradun, Uttarakhand, India}}

\begin{abstract}
Ascorbic Acid, a regimen claimed to use in the prevention of primary symptoms of COVID-19, is a hydrophilic anti-oxidant found plentiful in fruit juices, which also known to possess total anti-oxidant activity. Fruits play an essential part in our healthy diet. As many fruits contain anti-oxidant properties which means the presence of anti-oxidant constituents. In present study samples of the various fruit juices obtained from the local market were seek out for the ascorbic acid content and estimated for their anti-oxidant activity. For determining ascorbic acid content, the dichlorophenol-indophenol method was employed, while anti-oxidant activity was studied by employing Ferric Reducing Antioxidant Power assay and reducing power assay method. As per the result obtained, the high amount of ascorbic acid was found in amla juice while bael juice was found for good anti-oxidant activity. The conclusion results out from the study that fruits rich in an appreciable amount of ascorbic acid possess good anti-oxidant activity. Those juices results in best anti-oxidant activity indicate the presence of phenolics, i.e. phenolic compounds.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}COVID,\newline Ascorbic acid,\newline anti-oxidant activity,\newline Reducing power
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Pankaj~Nainwal~\\ Phone:\ +917579029583~\\  Email:\ drpankajnainwal@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
In one of the study, patients were given the dose in a combination of hydroxy-chloroquine, Vitamin C, Vitamin D and Zinc tablets to test if this alone or in mixed doses can prevent primary symptoms arises in the patient suffering from COVID-19. The whole treatment will proceed for 11-12 weeks, The fact that Vitamin C can be used in preventing corona disease, hence an objective was made to investigate which fruit in India contain a high amount of ascorbic acid, as it helps in slight speedy up in recovery from corona disease \unskip~\citep{907130:20534628,907130:20534620}. 

Ascorbic acid (vitamin C) mainly found in various foodstuffs like fresh vegetables and fruits especially citrus fruits like orange, lemon etc. \unskip~\citep{907130:20534624}. Vegetable and fruits play a significant function in lower down the risk of long term diseases like cardiovascular diseases, cancer, ageing and obesity. These health consequences can be accredited to the occurrence of various vitamins like as A, E and C (Ascorbic acid), plant fibres in diet and non-essential phytochemicals, for example, phenolic compounds etc. Ascorbic acid helps in biologically synthesising of collagen-a protein, absorption of Fe\ensuremath{^{2+}} (Iron), and activation of immune system responses and also indulged in pharmacological/ therapeutic activity like wound healing property and osteo-genesis \unskip~\citep{907130:20534618}.

Vitamin C (Ascorbic acid) scavenge free radicals which are responsible for the occurrence of disease hence possess a good anti-oxidant activity. 

A study states that if the use of ascorbic acid is done in a high amount, it can lead to various effects like gastritis. The metabolic product of ascorbic acid, i.e. oxalic acid, could be a source of Nephritis \unskip~\citep{907130:20534623}. The phenolics which are present in fruits and vegetables have free radical scavenging property which restrains the anti-oxidants principles to maintain the ranges of phenolics in a minimum amount, which was employed for normal cell functioning. 

Current research signifies that phenolics in the fruits and vegetables, employed as anti-oxidant can reduce the risk of Alzheimer's disease \unskip~\citep{907130:20534626,907130:20534625}. India is high cultivator in fruit production, so it is important to evaluate the best ascorbic acid (Vitamin C) content various fruit juices, especially that there requires more attention in such field. 

However, it is necessary to seek out the connection between the Vitamin C rich crude drugs to the presence of Phenolics, as a source of anti-oxidant potential in them.

Therefore the plan of this study was made to analyse ascorbic acid levels in juices from fruits and vegetables and also to seek out the relationship of Vitamin C, i.e. Ascorbic acid to the anti-oxidant activity.
    
\section{Materials and Methods}
\textbf{Chemical \& Instruments required}

2,6-Dichloro-phenol-indophenol sodium Dihydrate (DCP salt) and 2,4,6-Tris(2-pyridyl)-s-triazine procured from CDH, India. Sodium bicarbonate from Qualikems, India. Di sodium phosphate from Rankem, India. Ferric Chloride (anhydrous) from Qualikems, India. Ferrous sulfate was purchased from CDH, India. Potassium ferricyanide from Rankem, India. Sodium acetate from CDH, India. Sodium Phosphate was purchased from Rankem, India. Trichloroacetic acid was purchased from Rankem, India. Analytical balance, Micropipette was purchased from Lab kit Chemicals, India. Spectrophotometer (CAMAG), water bath and Oven purchased from Ambala, Punjab, India.

\textbf{Samples}

The following fruits were purchased from three different places from Uttarakhand. The three places were Uttarkashi, Dehradun and Haldwani, i.e. Orange, Lime, Green Grapes, Plums, Mulberry, Pomegranate, Bael and Amla. These places were chosen because Uttarkashi climate is favourable for fruits production and fresh fruits can be used, Dehradun was chosen as it covers collection of fruits from Garhwal region and Haldwani was chosen as it covers Kumaon region of Uttarakhand. Each fruit was taken for washing than peeling and lastly squeezing then the squeezed material was filtered and kept in the freezer till the commencement of study done. The help of mechanical juicer prepared the juice, and four samples of each of were taken for study. The dilute solution of all juices was made by the addition of distilled water before the study was conducted.

\textbf{Ascorbic Acid Content}

The amount of Ascorbic acid in the test sample of juices by adding DCP sodium salt solution into them. The few amounts of the diluted test sample were then analysed by titration method using titrant as DCP sodium salt solution (0.001 N). The endpoint was estimated when a pale pink colour is observed \unskip~\citep{907130:20534622}.

\textbf{Ferric Reducing Antioxidant Power Assay Method}

The assay mentioned above methods for anti-oxidant activity was estimated by using Ultra-Violet spectrophotometer \unskip~\citep{907130:20534627}. The reduced ferric solution was prepared by making a combination of 300 millimolar acetate buffer solution with pH of 3.5 and 10 millimolar solution of 2,4,6 tripyridyltriazine mixed in 40 millimolar hydrochloric acid and 20 millimolar solution of FeCl\ensuremath{_{3}} in the proportion of 10:1:1 respectively. Now, 1-2 ml of reducing ferric solution was incorporated into 0.1 ml samples of juices. 

The testing samples were then kept for incubation for 5-10 minutes at 25\ensuremath{^{0}}C temperature. The absorbance parameter via UV spectrophotometer was measured at 593 nanometers. All the results were then expressed as per obtained calibration curve of Ferric sulphate in varies concentrations ranging from 150 \ensuremath{\mu }M to 500 \ensuremath{\mu }M, which was then expressed as milliMolarFe\ensuremath{^{2+}} per litre of juice sample. 

\textbf{Reducing Power Assay Method}

The test sample of juices was estimated under reducing power assay method by using UV-Spectrophotometer \unskip~\citep{907130:20534617}. 1 ml of diluted test juice sample was taken. It was added to 0.6 ml of 0.2 Molar phosphate buffer with a pH of 6.5 and 1ml of 1\% solution of Potassium Ferricyanide. The solution was mixed, and the solution samples were kept for heating at 50\text{\textordmasculine}C for 20 min in a water bath. After this, the solution was added to 1 ml of trichloroacetic acid's 10\% w/v solution. Now, 1ml 


\begin{table*}[!htbp]
\caption{\boldmath {Ascorbic acid content in fruit juices} }
\label{tw-2fd7858daefb}
\centering 
\begin{threeparttable}

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\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Juice type  & \cAlignHack Sample  & \cAlignHack Ascorbic acid concentration \\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack (mg/100ml) \\
\tblmidrule 
\cAlignHack Orange  &
  \cAlignHack D &
  \cAlignHack 68.21 \ensuremath{\pm} 1.354\\
\cAlignHack Orange  &
  \cAlignHack U~&
  \cAlignHack 97.34 \ensuremath{\pm} 2.433\\
\cAlignHack Orange &
  \cAlignHack H &
  \cAlignHack 89.35 \ensuremath{\pm} 1.54 \\
\cAlignHack Amla &
  \cAlignHack D &
  \cAlignHack 78.522 \ensuremath{\pm} 4.255 \\
\cAlignHack Amla  &
  \cAlignHack U~&
  \cAlignHack 112.485 \ensuremath{\pm} 2.54 \\
\cAlignHack Amla  &
  \cAlignHack H &
  \cAlignHack 101.03 \ensuremath{\pm}1.86 \\
\cAlignHack Lime &
  \cAlignHack D &
  \cAlignHack 72.15 \ensuremath{\pm} 5.432\\
\cAlignHack Lime &
  \cAlignHack U~&
  \cAlignHack 101.85 \ensuremath{\pm} 1.098\\
\cAlignHack Lime &
  \cAlignHack H &
  \cAlignHack 94.64 \ensuremath{\pm} 2.084 \\
\cAlignHack Grapefruit  &
  \cAlignHack D &
  \cAlignHack 89.93 \ensuremath{\pm} 3.15\\
\cAlignHack Grapefruit  &
  \cAlignHack U~&
  \cAlignHack 64.16 \ensuremath{\pm} 4.11\\
\cAlignHack Grapefruit &
  \cAlignHack H &
  \cAlignHack 76.98 \ensuremath{\pm} 0.412 \\
\cAlignHack Mulberry  &
  \cAlignHack D &
  \cAlignHack 45.21 \ensuremath{\pm} 0.543\\
\cAlignHack Mulberry &
  \cAlignHack U~&
  \cAlignHack 51.87 \ensuremath{\pm} 1.78\\
\cAlignHack Mulberry  &
  \cAlignHack H &
  \cAlignHack 59.54 \ensuremath{\pm} 2.936\\
\cAlignHack Bael &
  \cAlignHack D &
  \cAlignHack 42.97\ensuremath{\pm}4.231\\
\cAlignHack Bael &
  \cAlignHack U~&
  \cAlignHack 50.97\ensuremath{\pm}2.14\\
\cAlignHack Bael &
  \cAlignHack H &
  \cAlignHack 54.97 \ensuremath{\pm}1.54\\
\cAlignHack Pomegranate  &
  \cAlignHack D &
  \cAlignHack 65.97\ensuremath{\pm}1.654 \\
\cAlignHack Pomegranate  &
  \cAlignHack U~&
  \cAlignHack 89.234\ensuremath{\pm}0.825\\
\cAlignHack Pomegranate  &
  \cAlignHack H &
  \cAlignHack 76.654\ensuremath{\pm}2.467\\
\cAlignHack Plum &
  \cAlignHack D &
  \cAlignHack 59.45\ensuremath{\pm}1.543\\
\cAlignHack Plum &
  \cAlignHack U~&
  \cAlignHack 63.743\ensuremath{\pm}2.562\\
\cAlignHack Plum &
  \cAlignHack H &
  \cAlignHack 67.35\ensuremath{\pm}1.986\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{n=4, (n= no. of observation),   D= Dehradun, U= Uttarkashi, H= Haldwani}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Antioxidant study on juices of fruits} }
\label{tw-6ead1e23f95e}
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\begin{tabulary}{\linewidth}{p{\dimexpr.13790000000000001\linewidth-2\tabcolsep}p{\dimexpr.25220000000000002\linewidth-2\tabcolsep}p{\dimexpr.31850000000000005\linewidth-2\tabcolsep}p{\dimexpr.29139999999999997\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Juice type  & \cAlignHack FRAP  & \cAlignHack Reducing Power \\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack  & \cAlignHack (mM Fe\ensuremath{^{+2}}/L\ensuremath{_{juice}})  & \cAlignHack (mM Fe\ensuremath{^{+2}}/L\ensuremath{_{juice}}) \\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack Orange &
  \cAlignHack 20.45 \ensuremath{\pm} 0.4 &
  \cAlignHack 32.93 \ensuremath{\pm} 2.01\\
\cAlignHack 2 &
  \cAlignHack Amla  &
  \cAlignHack 38.67 \ensuremath{\pm} 1.1 &
  \cAlignHack 40.54 \ensuremath{\pm} 1.53\\
\cAlignHack 3 &
  \cAlignHack Lime &
  \cAlignHack 39.87 \ensuremath{\pm} 1.2 &
  \cAlignHack 37.67 \ensuremath{\pm} 1.48\\
\cAlignHack 4 &
  \cAlignHack Grapefruit &
  \cAlignHack 55.23 \ensuremath{\pm} 1.5 &
  \cAlignHack 51.43 \ensuremath{\pm} 2.14\\
\cAlignHack 5 &
  \cAlignHack Mulberry  &
  \cAlignHack 76.23 \ensuremath{\pm} 2.1  &
  \cAlignHack 75.33 \ensuremath{\pm} 1.19 \\
\cAlignHack 6 &
  \cAlignHack Bael &
  \cAlignHack 89.67 \ensuremath{\pm} 0.4  &
  \cAlignHack 92.45 \ensuremath{\pm} 0.12 \\
\cAlignHack 7 &
  \cAlignHack Pomegranate  &
  \cAlignHack 57.77 \ensuremath{\pm} 0.5  &
  \cAlignHack 51.63 \ensuremath{\pm} 0.12 \\
\cAlignHack 8 &
  \cAlignHack Plum &
  \cAlignHack 61.34 \ensuremath{\pm} 1.2  &
  \cAlignHack 72.22 \ensuremath{\pm} 0.03 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
from the mixed sample was taken and to it distilled water and 1ml of 0.1 \% ferric chloride solution added to it. 

Then the solution was estimated for absorbance under UV spectroscopy at 700 nanometers. If there is an increase in absorbance, it means the reducing power of the sample is increased. However directly the absorbance was not estimated as due to the differences in dilution of every juice sample hence to reduce this confusion a calibration curve of FeSO\ensuremath{_{4}} was plotted prior within the concentration ranging from 250 \ensuremath{\mu }M to 750 \ensuremath{\mu }M and the result was then expressed as milliMolar Fe\ensuremath{^{2+}} per litre of juice sample.

\textbf{Statistical Analysis}

The statistical analysis of all data was obtained by mean \ensuremath{\pm} standard deviation of duplicate determination. The contrast between both anti-oxidant methods was determined ANOVA.
    
\section{Results and Discussion}
Fruits collected from different locations were analysed firstly for ascorbic acid content in juices of all fruits. Sample from each location was tested for ascorbic acid content. This acid content from the juice of orange fruit collected from Uttarkashi region was found high content 97.34mg/100ml while in orange juice collected from Dehradun local market low content was found, i.e. 68.21mg/100 ml. 

The ascorbic acid content in Amla juice collected from Uttarkashi region was found high content 112.49 mg/100ml while low content was found 78.53mg/100ml of amla juice collected from Dehradun local market. Lime juice of Uttarkashi region was found high content 101.85mg/100ml while low content was found 72.15mg/100ml of Lime juice collected from Dehradun local market. Green grapefruit juice from Dehradun region was found high content 89.93 mg/100ml while low content was found 64.16 mg/100ml of grapefruit juice collected from Uttarkashi. Mulberry juice from Haldwani region was found high content 59.54 mg/100ml while low content was found 45.21 mg/100ml of Mulberry juice collected from Dehradun local market. Bael juice from Haldwani region was found high content 54.97 mg/100ml while low content was found 42.97 mg/100ml of bael juice collected from Haldwani. Pomegranate juice of Uttarkashi region was found high content 89.23 mg/100ml while low content was found 65.97 mg/100ml of pomegranate juice collected from Dehradun local market. Plum juice from Haldwani region was found high content 67.35 mg/100ml while low content was found 59.45 mg/100ml of plum juice collected from Dehradun local market. Result from Table~\ref{tw-6ead1e23f95e} the Ferric Reducing Anti-oxidant method Bael and Mulberry were found high, i.e. 89.67 mM Fe\ensuremath{^{+2}}/L\ensuremath{_{juice\ }}and 76.23 mM Fe\ensuremath{^{+2}}/L\ensuremath{_{juice}} respectively. Reducing power assay of Bael and Mulberry was found 92.45 mM Fe\ensuremath{^{+2}}/L\ensuremath{_{juice}} and 75.33 mM Fe\ensuremath{^{+2}}/L\ensuremath{_{juice}}.

Differences in the ascorbic acid content in the same fruits of different location occurs may be due to climatic conditions and factors which affect cultivation. From Table~\ref{tw-2fd7858daefb}, it can be concluded that some of the fruits like amla, lemon, orange show good ascorbic acid concentration require subtropical region for their better growth and constituents in them. This result also shows that which location best suits for the cultivation of fruits. Results in Table~\ref{tw-6ead1e23f95e} shows that Bael and Mulberry show good anti-oxidant activity as many studies reveal that it is due to the presence of a high amount of phenolics. 

Bael juice shows a good anti-oxidant activity than those of orange and grapefruit, in contrast, that its content of ascorbic acid was lower. A study reveals that ascorbic acid is responsible for showing only 10-15\% of the total anti-oxidant activity. At the same time, certain flavonoids and tannins are significant contributors. \unskip~\citep{907130:20534621} Studies also reported that there's a direct relationship between the anti-oxidant activity and the total phenolic content. \unskip~\citep{907130:20534620,907130:20534619}
    
\section{Conclusion}
Coronavirus disease 2019 (COVID-19), previously called `2019 novel coronavirus (2019-nCoV), is an acute respiratory disease. It is caused by the coronavirus called SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona virus-2).\ensuremath{^{}} There is a widespread belief that vitamin C strengthens the immune system and therefore, could work for treating or preventing respiratory infections, such as the common cold. Besides, there is some evidence that suggests that vitamin C might help to manage the immuno-pathological responses contributing to the pathogenesis of severe respiratory viral infections. Hence based on these objective fruits bear good ascorbic acid content as well as good anti-oxidant potential fruits were selected and was investigated for their potential and contents so that it might be a holistic approach to treat the primary symptoms of COVID infection. However, further research is still needed to prove its effectiveness in corona patients. 
\section*{Acknowledgement}My sincere thanks to Chancellor Prof. Kamal Ghansala, Vice-Chancellor Prof. Sanjay Jasola to facilitate me with all amenities and funds required for conducting this project. I~also thank Director Pharmacy Prof. Nardev Singh for his support and guidance during the whole study. 

\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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