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\def\RunningAuthor{Sushanth N K et al.}
\firstPage{2128}
\articleType{Original Article}
\receivedDate{15 Sep 2020}
\acceptedDate{27 Oct 2020}
\revisedDate{19 Oct 2020}
\journalVolume{2020, 11 (SPL4)}
\journalDoi{ijrps.v11iSPL4.4431}
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\title{\textbf{Protective Effect of Hydroalcoholic Extract of \textit{Aloe Vera} Supplemented by Zinc on the Testis of Wistar Albino Rats Exposed to Cadmium} \textbf{\space }}
\author{Sushanth~N~K\textsuperscript{*}\textsuperscript{1},  
            Vijayalakshmi~S\textsuperscript{2},  
            Dayanand~Reddy~G\textsuperscript{3},  
            Sunil~Kumar~K~N\textsuperscript{4}, 
            Shakila~R\textsuperscript{5}~\\[5pt]{\textsuperscript{1}Department of Research and Development, Saveetha University, Thandalam, Chennai - 600077, Tamil Nadu, India}~\\{\textsuperscript{2}Department of Anatomy, Saveetha University, Thandalam, Chennai - 600077, Tamil Nadu, India}~\\{\textsuperscript{3}Department of Phramacology, Siddha Central Research Institute, Arumbakam, Chennai - 600106, Tamil Nadu, India}~\\{\textsuperscript{4}Department of Phramacognosy, Siddha Central Research Institute, Arumbakam, Chennai - 600106, Tamil Nadu, India}~\\{\textsuperscript{5}Department of Chemistry, Siddha Central Research Institute, Arumbakam, Chennai - 600106, Tamil Nadu, India}}

\begin{abstract}
Infertility is a major health concern that may be caused due to inadequate fertility hormones or poor quality of reproductive parameters. The environmental pollutants such as cadmium is one of the causative factors for inducing infertility in men. Cadmium accumulates in various tissues and affects reproductive organs, especially testis which is highly sensitive to cadmium poisoning. In this experiment, protective effect of hydroalcoholic extract (HAE) of Aloe vera with and without zinc has been evaluated. Zinc, protects the testes from damages resulting due to cadmium. Aloe vera is a very good source of antioxidants such as vitamin C and E. In this experiment male Wistar albino rats were grouped into 5. The control group received only the vehicle. Experimental groups were administered in Cd group with 10mg/Kg body weight of CdCl\ensuremath{_{2}}, in Cd+Zn group 40mg/Kg body weight of CdCl\ensuremath{_{2}} and 40mg/Kg bodyweight of ZnCl\ensuremath{_{2}}, in Cd+AV group 10mg/Kg body weight of CdCl\ensuremath{_{2}} and 200mg/Kg body weight of HAE of \textit{Aloe vera} and in Cd+AV+Zn 10mg/Kg body weight of CdCl\ensuremath{_{2}}, 200mg/Kg body weight of HAE of \textit{Aloe vera} and 40mg/Kg bodyweight of ZnCl\ensuremath{_{2.}} The results showed that cadmium caused extensive testicular damage which was protected by HAE of \textit{Aloe vera} and zinc. Hence, protective effect of HAE of \textit{Aloe vera } was exhibited clearly in cadmium treated rats, however, supplementing \textit{Aloe vera} with zinc provided better protection.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Aloe vera,\newline Cadmium,\newline Heavy metal,\newline Infertility,\newline Oxidative stress,\newline Testosterone,\newline Zinc
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Sushanth~N~K~\\ Phone:\ +91-8606287063~\\  Email:\ sushanthanatomy.ss@outlook.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
Infertility due to environmental toxicants has alarmingly increased in the past decade and has raised concerns among couples. In about 20\% infertility cases, male factor is the primary cause \unskip~\citep{910808:20568277}. A variety of environmental, genetic, behavioral and genotoxic factors result in male infertility by impairment of spermatogenesis at various stages \unskip~\citep{910808:20568266}. One of the well-defined causes of impairment of spermatogenesis is exposure to environmental chemicals \unskip~\citep{910808:20568270}. Chemical element cadmium (Cd), a heavy metal and an environmental pollutant, is released into the environment by various human activities \unskip~\citep{910808:20568288}. Industrial and environmental exposure to Cd, results in various health problems. It causes various biochemical and physiological alterations in man and lab animals \unskip~\citep{910808:20568287}. Severe degeneration of testicular tissue and associated seminiferous tubule damage and ischemic necrosis results in rats following exposure to Cd \unskip~\citep{910808:20568296}. Cd induces generation of reactive oxidation species (ROS) in testis which is a by-product of O\ensuremath{_{2}} metabolism and are free radicals having short life, but accumulation is harmful to the cell. 

Oxidative stress affects fertility potential due to depletion of antioxidants by free radicals affecting the normal balance between the two resulting in accelerated tissue damage. Testicular antioxidant defense system plays a major role in protecting the testis from damages caused by oxidative stress due to ROS. Severe impairment of testicular function including germ cell death and inhibition of steroidogenesis results by acute Cd administration in male rats \unskip~\citep{910808:20568267,910808:20568271,910808:20568263}. Antioxidants are free radical scavengers and they suppress ROS generation and their actions \unskip~\citep{910808:20568295,910808:20568268}.

Zinc (Zn) is a member of the same family as Cd, but is essential in cellular response to DNA repair, oxidative stress and apoptosis \unskip~\citep{910808:20568280}. It is an important antioxidant since superoxide dismutase, which it is a part of, converts superoxide anion into H\ensuremath{_{2}}O\ensuremath{_{2}}+O\ensuremath{_{2}}\unskip~\citep{910808:20568294}. It is important for the physiology of spermatozoa, sperm production and viability \unskip~\citep{910808:20568275}. In rat and mice testes, Zn provides protection against acute Cd toxicity \unskip~\citep{910808:20568286}. 

Even though variety of chemical drugs are available for infertility treatment, traditional medicine is gaining popularity due to its less toxicity and adverse effects. Medicinal plant contains active compounds that can be used for therapeutic purposes \unskip~\citep{910808:20568272}. \textit{Aloe vera}, a cactus like perennial herb, showcases its antioxidant role by strengthening Vitamin C and E \unskip~\citep{910808:20568273}. 

The present study is to evaluate the protective effects of hydroalcoholic extract of \textit{Aloe vera} in combination with Zn and without it on the effects of cadmium induced toxicity in the testis of Wistar rats. \textit{Aloe vera}, is expected exhibit its antioxidant properties and Zn, to act as antioxidant as well as chelating agent thereby prevent testicular damage caused by Cd. 
    
\section{Materials and Methods}
The study was conducted on Wistar albino rats. The rats procured from Biogen (Bengaluru, India) were kept under controlled condition throughout the experiment in the animal house of Siddha Central Research Institute (Chennai, India). After 4 days of acclimatisation, the animals were divided into following groups (Table~\ref{tw-fdcd9e602e74}). The experiment was conducted for 56 days. After the completion of the experiment, the rats were sacrificed, blood, testes with epididymis were collected and properly preserved for semen and hormonal analysis. 

\textbf{Preparation of hydroalcoholic extract of \textit{Aloe vera}}

Fresh \textit{Aloe vera} plants were obtained from Amudham Nandavanam Garden in Chennai, India. The plants were certified by a Pharmacognosist. They were washed, cut, shade dried and hydro alcoholic extract (HAE) was prepared by using ethyl alcohol and water. 10 kilos of fresh plants were used and the yield of extract was 292 g.

\textbf{Drug administration}

Control: 1 ml of 0.5\% carboxymethyl cellulose (CMC).

Cd10: 10mg/Kg body weight of CdCl\ensuremath{_{2}} (NICE Chemicals) in 0.5\% CMC

Cd10+Zn: 10mg/Kg body weight of CdCl\ensuremath{_{2}} and 40mg/Kg bw of ZnCl\ensuremath{_{2}} in 0.5\% CMC

Cd+AV: 10mg/Kg body weight of CdCl2 and 200mg/Kg bw of HAE of \textit{Aloe vera} in 0.5\% CMC

Group 5: received 10mg/Kg body weight of CdCl2, 40mg/Kg bw of ZnCl2 and 200mg/Kg bw of HAE of \textit{Aloe vera} in 0.5\% CMC.

CdCl2, ZnCl2 and CMC: NICE Chemicals, Sudhagar Biological and Chemicals, Chennai, India.
    
\section{Results and Discussion}
\textbf{Sperm concentration}

The concentration of sperm in Cd (24.2\ensuremath{\pm} 3.1X10\ensuremath{^{6}}/mL) was significantly 3 times lower than control; in Cd+Zn (47.8\ensuremath{\pm} 3.4X10\ensuremath{^{6}}/mL) was significantly 1.5 times lower than control and significantly 2 times more than Cd; in Cd+AV (62\ensuremath{\pm} 3.2X10\ensuremath{^{6}}/mL) was significantly 2.5 times higher than Cd but there was no significant difference from control group; in Cd+AV 200+Zn (55.4\ensuremath{\pm}3.1X10\ensuremath{^{6}}/mL) itwas significantly 1.3 times lower than control and significantly 2.3 times more than Cd.


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\caption{\boldmath {Concentration, motility and viability of sperm. Values expressed as mean + SD.1-Control, 2-Cd, 3-Cd+Zn, 4-Cd+AV, 5-Cd+AV+Zn.}}
\label{f-13c54b49f963}
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\caption{\boldmath {Serum TST and LH levels. Values expressed as mean + SD. 1-Control, 2-Cd, 3-Cd+Zn, 4-Cd+AV, 5-Cd+AV+Zn.}}
\label{f-bf44a873b29c}
\end{figure}
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\begin{table}[!htbp]
\caption{\boldmath {Control and various experimental groups. Abbreviations used: Cd- Cadmium, Zn-Zinc, AV- \textit{Aloe vera}.} }
\label{tw-fdcd9e602e74}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.43489999999999995\linewidth-2\tabcolsep}p{\dimexpr.56510000000000005\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Sl No  & Groups \\
\tblmidrule 
1  &
  Control \\
2  &
  Cd \\
3  &
  Cd+Zn \\
4  &
  Cd+AV \\
5  &
  Cd+AV+Zn \\
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\textbf{Sperm motility}

In Cd, sperm motility (3.4\ensuremath{\pm} 0.5\%) significantly decreased 17.4 times in comparison to control; in Cd+Zn (39.3\ensuremath{\pm} 2.2\%) it significantly decreased 1.5 times compared to control and significantly increased 11.2 times compared to Cd; in Cd+ AV (49.0\ensuremath{\pm} 1.7\%) it significantly increased 14.4 times than Cd, however any difference that was significant from control could be found. In Cd+AV+ Zn (46.5\ensuremath{\pm}3\%) motility significantly increased 13.7 times compared to Cd but without any significant difference from control group.

\textbf{Sperm viability}

In Cd (13.9 \ensuremath{\pm} 1.8 \%), sperm viability significantly decreased 5.2 times compared to control; in Cd+ Zn (51.5\ensuremath{\pm} 2\%) it decreased significantly 1.5 times compared to control but significantly 3.7 times more than Cd. In groups Cd+ AV (53.8\ensuremath{\pm} 2.8\%) and Cd+ AV 200+Zn (62.4\ensuremath{\pm}2.6\%) the viability increased significantly 3.9 and 13.7 times respectively in comparison to Cd, but any significant difference could not be observed compared to control in this group (Figure~\ref{f-13c54b49f963}).

\textbf{Testosterone hormone analysis (TST)}

In Cd the TST level (1.9 ng/mL) significantly decreased 2.6 times compared to control; in Cd+Zn it (TST level of 3.4ng/mL) significantly increased 1.8 times in comparison to Cd, without any significant difference compared to control. In Cd+ AV, TST level (3.9 ng/mL) significantly increased 2 times compared to Cd, here too no significant difference in comparison to control group; in Cd+ AV +Zn, in compa~ rison to Cd, TST level (4.2 ng/mL) significantly increased 2.2 times. In this group there was no significant difference of values compared to control. 

\textbf{Luteinizing hormone analysis (LH)}

The level of LH in groups Cd (22.6 ng/mL) significantly decreased 2.6 times compared to control, in Cd +Zn (53.8 ng/mL) it increased significantly 2.4 times compared to Cd, without any significant difference compared to control group. In Cd+ AV the level of LH (63.6 ng/mL) significantly increased 2.8 times in comparison to Cd, however any significant difference from control could not be found. In Cd+ AV+Zn with LH level 62.7 ng/mL, it was observed that it significantly increased 2.8 times compared to Cd, without any significant difference compared to control (Figure~\ref{f-bf44a873b29c}). 

The testicular damage is mainly by disruption of blood-testis barrier (BTB) \unskip~\citep{910808:20568286}. BTB is highly vulnerable to Cd at the microvascular level in rats \unskip~\citep{910808:20568274}. Cd in the form of Cd\ensuremath{^{2+ }} has similarities to Ca \ensuremath{^{2+}} or Zn \ensuremath{^{2+}} ions and is likely to replace these ions in crucial physiological processes resulting in inhibition or activation of a number of signalling pathways. Cd can cause oxidative stress by depleting glutathione \unskip~\cite{910808:20568283} an important antioxidant. Oxidative-stress due to ROS generated by Cd can severely damage basement of the seminiferous epithelium and the spermatogenic cells supported by it and this can be attributed to increased metabolic rate and proliferative nature of tissues of the testes. \unskip~\citep{910808:20568285,910808:20568269}. ROS generated due to Cd disturb the process of steroidogenesis and production of certain hormones \unskip~\citep{910808:20568264}. Cd, because it affects production and regultion of some hormones, it is described as a disroptor of endocrine system \unskip~\citep{910808:20568288,910808:20568292}.  It alters the concentration of certain hormones like testosterone and luteinizing hormones in blood very significantly \unskip~\citep{910808:20568282}.  It decreases the level of (StAR) protein, and luteinizing hormone receptors of testis \unskip~\citep{910808:20568260}. The StAR protein facilitates delivery of cholesterol, the raw material for steroidogenesis, to cytochrome P450scc on the inner mitochondrial membrane. Another way Cd can readily modify level of hormone by influencing hypothalamic pituitary testicular (HPT) axis or Leydig cells \unskip~\citep{910808:20568282}. In this study, the Cd treated rats showed decreased testosterone and LH levels which could be due to decreased level of StAR protein and effect of Cd on H-P-T axis. This effect is reflected on the sperm concentration, which could be due to reduced spermatogenesis because of decreased TST level, since for spermatogenesis to take place one of the requirements is adequate level of TST. 

To counteract the oxidative stress, testis has a number of enzymatic antioxidants such as SOD, catalase and GPx. However, upon exposure to Cd, the levels of these enzymes decrease \unskip~\citep{910808:20568279,910808:20568290}. Hence it is imperative that enzymatic or non-enzymatic antioxidants may prevent or reduce testicular damage induced by Cd. Zn could be used to prevent the testicular damage by Cd \unskip~\citep{910808:20568286}. In the present study, in Cd+Zn and Cd+AV+Zn treatment groups, Zn clearly exhibited its protective effect due to its antioxidant properties \unskip~\citep{910808:20568262,910808:20568284,910808:20568293}. Furthermore, Zn supplementation prevents increased rate of lipid peroxidation \unskip~\citep{910808:20568291}. Zn exhibited its protective nature when Cd was co-administered with it, and the toxicity due to Cd was countered better by Zn than when Cd was administered alone \unskip~\citep{910808:20568286,910808:20568281}. Similar results were found in the present study. Zn is required for the normal physiology of spermatozoa in addition to its contribution towards sperm production and its viability \unskip~\citep{910808:20568275}. The Zn treated rats in the present study, showed increased concentration and viability compared to Cd group due to this reason. 

Substances rich in antioxidant enzymes such as Vitamin C and E, reduce and or prevent oxidative stress and damages due to Cd in the testis of rats \unskip~\citep{910808:20568289}. Treating the rat testes with antioxidants like Vitamin C and Vitamin E prior to Cd exposure has shown to restore normal testicular function due to their ability to counter production of excess ROS \unskip~\citep{910808:20568290}. Aloe vera is one such medicinal plant which has about 75 active compounds that include antioxidant enzymes such as Vitamin C and E \unskip~\citep{910808:20568278}. A study observed that hydroalcoholic extract of Aloe vera increased the level of testosterone and LH hormones, and also rise in sperm concentration and motility \unskip~\citep{910808:20568276}.  In this study, rats administered with Cd+AV and Cd+AV+Zn, similar result was observed. This is due to the antioxidant properties of \textit{Aloe vera}, which by strengthening Vitamin C and E enhances reproductive parameters and improves fertility potential. It stimulates Leydig cells and thereby increases testosterone hormone \unskip~\citep{910808:20568273}. In addition, \textit{Aloe vera} could have increased intracellular levels of reduced glutathione thus providing protection against Cd \unskip~\citep{910808:20568265}. Due to the combined actions of hydroalcoholic extract of \textit{Aloe vera} and Zn, in the treatment group Cd+AV+Zn, the functional integrity was very well maintained. Here \textit{Aloe vera} is believed to exhibit its antioxidant properties thus offering protection to testis from damages due to ROS generated by Cd. Cd, in presence of Zn has to compete for physiological binding sites due to similarity between these ions, and it is understood that Cd is failed by Zn mainly by chelating as well as antioxidant properties of Zn. \ensuremath{^{}}
    
\section{Conclusions}
Cd induces testicular damage in rat testis. Zn acts as an antioxidant and chelating agent. Thus, co-treatment of Cd with Zn protects testis from damages due to Cd effectively. Combined actions of HAE extract of \textit{Aloe vera} and Zn protects the rat testis from Cd induced alterations very effectively.

\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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