Acute toxicity studies of an novel natural polymer vigna mungo in swiss albino rats

Krishnaveni Manubolu*1, Sreenivasulu Munna2, Chandrasekhar Kothapalli Bonnoth3 1Research Scholar, Department of Pharmaceutical Sciences, Jawaharlal Nehru technological university anantapur, Ananthapuramu-515002, Andhra Pradesh, India 2Department Of Pharmaceutical Chemistry, Santhiram College Of Pharmacy, Nandyal, Kurnool518501, Andhra Pradesh, India 3Department of chemistry, Krishna University, Machilipatnam, Krishna521001, Andhra Pradesh, India


INTRODUCTION
Polymers are the backbone of drug delivery systems. These are the carriers of inert nature. Polymers alter the pharmacokinetics, pharmacodynamics proper-ties of the dosage forms. Polymers are natural, synthetic and semisynthetic based on the source of origin. Many natural source origin polymers, especially proteins, polysaccharides used as carriers in the tissue engineering, targeting, and in bio response stimuli drug delivery systems (GIL and HUDSON, 2004). These are used as binders, ilm formers, arti icial organs linings, immunological testing, and as substrates for cell growth. Smart polymers are materials of choice in the dosage forms which undergo physical or chemical change in response to external stimuli. Hydrogels are the hydrophilic polymeric networks capable of taking a large amount of water or biological luids (Qiu and Park, 2001).
Natural polymers are gaining popularity in the modern era due to biocompatibility, biodegradability, no-toxicity, economic, safety, easy availability. Nat-ural polymers are produced from plants, animals sources (Shanmugam et al., 2005). Herbal polymers are largely contributing in many industries. These polymers are obtained from gummy exudates, and plant ibres are used in these areas (Sailaja et al., 2011). Like Acacia, carrageenan, agar, Ispagul a, tragacanth many polymers are used in various formulations (Gandhi et al., 2019). Even though a lot of research work was done in the exploration of natural polymer associated dosage forms, yet to ind and con irm the safety pro ile of the Vigna mungo polymer.
Novel Vigna mungo polymer is obtained from Vigna mungo (black gram) seeds pulverised portion using non-solvent acetone.
Although research work regarding the Vigna mungo polymer was done still, there is a need to establish toxicity pro iling to carryout invivo administration of dosage form. To carry out this exploration, acute toxicity pro iling was performed using OECD guideline 420 (Sravani et al., 2011;Yadav et al., 2009).
Toxicity studies are used in hazard identi ication and risk management in the context of production, handling, and use of chemicals (Aneela et al., 2011). Acute oral toxicity study is imperatively required not exclusively to distinguish the scope of test substance that could be utilized, yet in addition to reveal the clinical signs evoked by the substances under scrutiny. It is likewise a valuable boundary to researching the helpful record of medications and xenobiotics (Sailaja et al., 2011). For a traditional LD50 study, research facility mice and rodents are the species normally chose. Frequently both genders should be utilized for administrative purposes. In this study, female rats were used as per OECD 420 guidelines.
Acute oral toxicity refers to those adverse effects occurring following oral administration of a single dose of a substance or multiple doses given within 24 hours. Toxicological studies are used to assess the acceptance for clinical use. Toxic doses and therapeutic indices of drugs can be determined from toxicity study. Chronic toxicity tests determines the affected organs after completion of toxicity study (Robinson et al., 2009). Safety must be explored in animals to predict safe human doses (da Silva et al., 2015).

Plant Material
The Vigna mungo seeds were collected from the local market Nellore, Andhra Pradesh. It was authenticated by Dr A.Sreenivasulu, Department of Botany, Visvodaya govt. Degree College, Venkatagiri, Nellore, Andhra Pradesh.

Preparation of Vigna mungo polymer
100g of powdered Vigna mungo seeds were soaked in 300ml of distilled water for 24 hours. Soaked material was iltered and to iltrate, add a double portion of acetone as non-solvent to separate the polymer. The polymer was dried at 40 ºC. After drying, powder the material and sieve through sieve no.20.

Experimental animals
Healthy adult Swiss Albino female rats weighing 130-150 g acclimatized for 14 days. The animals were housed under standard conditions, and room temperature (25±2 o C) and the relative humidity was at 50-60%. Arti icial lighting and 12 hours light and 12 hours dark was maintained (E, 1998). The animals were fed with a balanced pellet and water adlibitum (Sholikhah et al., 2020). The experimental protocol of acute toxicity studies was approved by the Institutional Animal Ethical Committee of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Acute Toxicity Study
The acute oral toxicity study was carried out according to OECD guideline 420, and a ixed-dose procedure was used (WHO, 1993). Considering the unavailability of in vitro and in vivo toxicological data of the Vigna mungo polymer extract, 300mg/kg of initial dose was chosen in the sighting study. At 300 mg/ kg of dose did not produce any toxic symptoms on one rat. In the preliminary tests, 2000mg/kg of dose did not produce toxic symptoms. In the main test, 2000mg/kg of the dose was continued on a test group containing 3 rats and a control group treated with distilled water. Animals were observed for a period of 24hrs and 14days to record the toxicity symptoms like body weight, treatment-related changes like respiration rate and heart rate and behavioural signs like apathy, reduced locomotor behaviour, licking activity (Pradeep et al., 2020).
After the treatment period of 14 days, the vital organs (heart, liver, brain and kidneys) of animals under chloroform anaesthesia were preserved for histopathological examination (Saleem et al., 2017). Then the blood samples were collected by cardiac puncture, subjected to haematological biochemical analysis (Chunlaratthanaphorn et al., 2007).

Statistical analysis
The obtained results were presented as mean ±SEM, and the statistical signi icance between the

DISCUSSION
Gums and mucilaginous substances are used in the medicinal system from the olden days (Anroop et al., 2006). Polymers are macro molecular structures with a wide variety of physicochemical characteristics. Polymers are a signi icant part of drug delivery systems to alter the release pro ile characteristics. Polymers are used in the extension of the release pattern of both hydrophilic and hydrophobic drug molecules. With the exploration of polymers in the drug delivery systems well de ined and predicted therapeutic bene it and release kinetics (Anepu et al., 2016). Pharmaceutical active ingredients are associated with the carrier systems to allow transport and target the particular site. Polymer physicochemical characteristics allow understanding of the underlying mechanisms and behaviour in the biological environment (Liechty et al., 2010). Suf icient concentration of the drug has to reach the target site to exert the desired therapeutic bene it. This is achieved by using a drug delivery system of suitable characteristics by employing polymeric structures (Santoshnaidu et al., 2014;Durga et al., 2020). Table 1 clearly shows the bodyweight of each rat before and after treatment with Vigna mungo polymer. The observations exhibited normal after a week and 14 days of a period. The weight gain observations explored normal values during experimentation of 7 days and 14 days treatment with Vigna mungo polymer. The observations of body weight recorded at different time periods, i.e. before treatment, after 7 days of treatment and after 14 days of treatment and the same is illustrated in Figure 1. The body weight changes are the markers for identifying the toxicity in the animals. The experimental data is statistically analysed by 2-way ANOVA. It is clearly shown that there is no interaction overall, and the treatment periods effect was considered extremely signi icant (p=0.001). Comparing the bodyweight results in the control and test group were found to be not signi icant.
The vital organ weights (heart, liver, brain and kidney) of control and test groups after 14 days period presented in Table 2. Mean vital organ weight of control and test groups rats after treatment with Vigna mungo polymer after 14 days period illustrated in Figure 2. Many researchers studied liver and kidney in the acute toxicity study to evaluate the safety and toxicity of the Phyto chemicals. Pradeep D P.et.al studied the effect of plant extract on vital organ weight. The study indings are in similarity with the present research results (Shende and Marathe, 2016). The results of test group animal and control group animals for gross pathological changes in view of size, shape and colour did not show noticeable variations. 2-ANOVA tests performed on experimental data. It was found that there is no signi icant difference observed in the test group compared to the control group.
Microscopic exploration of vital organs, heart, liver, brain and kidney were prepared for histo pathological studies. The prepared transverse sections of vital organs observed under a microscope. Histo pathological pictures represented in Figure  Blood parameters are sensitive indicators of any alterations in the physiological environment (Pradeep et al., 2020). Haematological examination results for haemoglobin, total RBC, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), total white blood cells (WBC), Eosinophils, Basophils, Neutrophils are presented in Table 3. By observing the haematological parameters of the control and test group were assessed (Jain et al., 2009). There is no signi icant difference, and the p-value is <0.0001. A signi icant difference compared to the control group using the two-way ANOVA was performed. There is no signi icant difference value of these groups to the control group was within the normal range (Robinson et al., 2009).
Information obtained from toxicity studies helps in the determination of organ toxicity, the relation between dose and response (Jothy et al., 2011). Toxicity levels of the medicinal substance makes it unsuitable for treatment (Ayodeji et al., 2019). Many plant-based products are safe, but it was proven false in many research indings (Pradeep et al., 2020). Mulchand A.Shende et al. documented acute and chronic oral toxicity indings of Hibiscus esculentus mucilage on Swiss albino rats (Shende and Marathe, 2016). Divvela HN et al. explored acute oral toxicity studies of Araucaria heterophylla novel natural polysaccharide gum in albino mice (Divvela et al., 2016). Ez Zoubi Yassine et al. studied the acute toxicity using mucilage of aerial part (branches, lowers and leaves) of Lavandula stoechas L from morocco (Yassine et al., 2016). All the above Plantbased mucilage works justify the research indings of Vigna mungo polymer. Hence, it is found that Vigna mungo polymer obtained from the seeds of Vigna mungo is safe to use in animals up to the dose of 2000mg/kg and did not produce any sort of abnormal and toxic changes.

CONCLUSION
In the present research acute oral toxicity study, extracted Vigna mungo polymer was used up to 2000mg/kg. And this dose did not produce any toxicity and abnormal symptoms in the test group, i.e. in the preliminary study with one rat and in the main test with three rats. According to OECD guideline 420, 2000mg/kg dose produced no toxicity symptoms, and it is categorised as unclassi ied material. It is concluded that Vigna mungo polymer extracted using acetone is nontoxic material.

Con lict of Interest
The authors declare that they have no con lict of interest for this study.

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