IMPACT OF Pleurotus tuber-regium (Rumph. ex Fr.) EXTRACT-LOADED NANOPARTICLES ON THYROID PROFILE OF RATS

Aim. The aim of this study was to investigate the impact of Pleurotus tuber-regium extract loaded silver nanoparticles (SNPs) on thyroid profile of rats. Methods . Acute toxicity test was performed following up and down procedure and the analysis of thyroid profile was performed following chemiluminescence assay method. Results . Acute toxicity test showed no mortality and no behavioral changes in rats treated with 2000 mg kg –1 of SNPs. High dose (400 mg kg –1 ) of SNPs showed high hyper thyroid activity by increasing T3 = 38.20±4.06 ng dL –1 ; T4 = 4.40±0.32 μg dL –1 ; and by decreasing TSH = 0.73±0.06 μIU mL –1 compared to control group (T3 = 30.20±1.02 ng dL –1 ; T4 = 2.94±0.08 μg dL –1 ; TSH = 0.87±0.01 μIU mL –1 ) and animal group treated with 200 mg kg –1 dose of SNPs (T3 = 35.69±1.13 ng dL –1 ; T4 = 3.82±0.24 μg dL –1 ; TSH = 0.80±0.02 μIU mL –1 ). Conclusions . Pleurous tuber-regium extract loaded silver nanoparticles are non toxic and can be used in drug delivery as well as in the formulation of drugs used in hypothyroidism.

In recent decades a great attention has been paid for preparation of materials of small size and the technology. Synthesis of nanomaterials especially metal nanoparticles and their application in different field of biological sciences and medicine has provided new hope for development of nanodrugs and their effective delivery in the biological system [1]. In recent decade application of nanoparticles have been increasing due to their unique properties such as ultra-small size, large surface to volume ratio, high reactivity and unique interactions with structural components which improve the pharmacokinetics as well as therapeutic index of the drugs [2]. Among metals, application of silver and its compound in the field of medicine has been experiencing for over 2000 years and they fund nontoxic but safe bactericidal agent [3]. Recently several physical and chemical methods of synthesis of silver nanoparticles have been developed but biological methods such as using plant and fungal material are quite easy because of less expensive, safe and eco-friendly nature [4].
Thyroid hormones are directly associated with metabolic regulation of body and modulate a lot of physiological status of health such as body weight, oxygen requirement, normal growth and development during childhood [5] and patho-physiological dysfunction of thyroid gland is the most common endocrine disorder and in India, and about 42 million people suffer from thyroid associated diseases [6]. It has also been reported that serum cholesterol level increase in hypothyroidism and associated diseases and enhance the risks of coronary artery disease, atherosclerosis and heart failure [7].
Mushrooms of Pleurotus genus are popularly consumed by the peoples of all over the world due to their high nutritional values and some medicinal properties. The Pleurotus mushrooms are rich in proteins, essential amino acids, polysaccharides and essential fatty acids, dietary fibers, minerals, some vitamins etc. [8]. Plurotus tuber-regium has been used as medicinal supplement for headache, stomach ailments, cold and fever, asthma, small pox, high blood pressure as well as for weight gain and malnourishment [9]. The aim of this work was to synthesis silver nanoparticles loaded with aqueous extract of P. tuber-regium and to study their impacts of extract loaded silver nanoparticles on profile of rat because the impacts of silver nanoparticles loaded with extract of P. tuber-regium has not been explored till dated.

Materials and Methods
Study of impact of silver nanoparticles on thyroid profile on rat Animals: Wistar albino rats of 175 to 200 g were obtained from the National Institute of Nutrition, Hyderabad, India. They were kept in cage and maintained under standard laboratory conditions at ambient room temperature (22 ± 3 °C) and relative humidity (30-65%), with dark-light cycle of 12 h for 5 days. 15 rats were fed with a commercial pallet diet (Sadguru Shri Shri Industries Pvt. Ltd. Pune, India) and water. The experiment was performed after prior approval of the Ethics committee of Ranchi University, Ranchi (Proceeding no. 46, page no. 137). Acute toxicity study of P. tuber-regium extract loaded SNPs on rat was also done according to OECD [10] test guideline 425 (Up and Down procedure), limited test was performed for at the test dose 2 000 mg kg -1 on rats and dose was fed according to the body weight.
For the study of impacts of P. tuber-regium extract loaded SNPs on thyroid profile of rats seven days experimental period was chosen followed previous method of Garba et al [11]. For the treatment fifteen rats were distributed into three groups and each group contain 5 rats. Two doses (high dose: 400 mg kg -1 and low dose: 200 mg kg -1 ) of SNPs (average 71.36 nm diameter with -11.2 mV zeta potential) were taken and the doses were feed according to the body weight of the animals [12, 13]. The experiment designed for the study is described below: Group-1: Rats were served as control and they received single dose (1 mL) of distilled water (vehicle) daily orally for 7 days.
Group 2: Rats of this group received daily single dose (200 mg kg -1 ) of nanoparticles orally for 7 days.
Group 3: Rats of this group were received daily single dose (400 mg kg -1 ) of nanoparticles orally for 7 days.

Analysis of Thyroid Hormone
Study the impacts of P. tuber-regium extract loaded SNPs on thyroid profile of rats were done by chemiluminescence immune assay [14].
Statistical analysis Entire statistical works were done using full proof statistical software WinSTAT (R. Fitch Software, Canal Park, Cambridge, Massachusetts, USA). Data were taken n = 5 and results were expressed as a mean ± standard error of mean. Statistical analysis was performed by one-way ANOVA with posthoc Student's t-test, P ≤ 0.05 was considered as statistically significant.

Results and Discussion
Although P. tuber-regium have valuable pharmacological effects and lack toxicity. Therefore, the current study was conducted to assess the acute toxicity of synthesized SNPs loaded with P. tuber-regium by following OECD guidelines 425 [10] before study of impact of P. tuber-regium extract mediated nanoparticles on thyroid profile of rats. It has been reported that, as the acute oral toxicity study is necessary to determine the safer dose range to manage the clinical signs and symptoms of the drugs [15] and the toxic outcomes of drugs such as decrease body weight, clinical signs and symptoms which are principal observations among various toxicity indicators [16]. In the present extract and SNPs did not show deleterious acute toxicity symptoms such as increased somatomotor activity, convulsion, tremor and itching etc. after till 14 days.
The result of impacts of SNPs loaded with P. tuber-regium extract on thyroid profile of rats is presented in the Table. The result of impacts of SNPs loaded with P. tuber-regium extract on thyroid profile of rats is presented in Table 3. The result shows both low and high doses of SNPs act on the thyroid-pituitary axis and significantly increases T3 and T4 level and decreases TSH level compared to control group. T3 most active form of thyroid hormone than T4, and both are synthesized in the thyroid follicles in low and high amount respectively. T4 is synthesized by monoiodination of T3 in liver [17]. Serum TSH level is inversely correlated with serum T3 and T4 level [18]. It has been reported in hypothyroidism significant decrease in serum T3 and T4 level occurs with increase in TSH level [19]. Sublethal exposure of silver oxide nanoparticles lack capping agent of bioactive substances from medicinal plants or fungi, disrupt signalling of thyroid hormone during metamorphosis of Xenopus laevis [20] but no recent work has been reported on impact of SNPs loaded with P. tuber-regium extract on thyroid profile of rats. Biochemicals of medicinal plants and mushrooms origin such as flavonoids, coumarins, alkaloids, minerals, essential oilcomponents, terpinene, other antioxidant compounds etc. and they directly influence the pituitary-thyroid axis and elevate or decrease the THS level and directly or indirectly elevate or decrease the serum thyroid hormone level [21]. In the present study 400mg kg -1 dose of SNPs significantly (P < 0.05) increased T3 and T4 concentration and decrease TSH concentration compare to control and low dose treatment groups (Table). Thus, present study can be correlated with the previous studies done for study the hyper thyroid effect of natural plant and mushroom extract and can also be said that, SNPs loaded with P. tuber-regium extract act on pituitarythyroid axis and increased the synthesis of T3 and T4.

Conclusions
P. tuber-regium extract can be used for synthesis of silver nanoparticles and the synthesized nanoparticles loaded with P. tuber-regium extract showed increased thyroid level. Thus, P. tuber-regium extract loaded nanoparticles can be used for the formulation of pharmaceuticals related to hypo thyroidism.

Acknowledgement
The authors acknowledge the financial assistance received from DBT, NER-BPMC, New Delhi (BT/462/NE/TBP/2013) under the Twinning Project (EAEBCEPMA) to Department of Zoology, Ranchi University, Jharkhand.
Authors declared there is no conflict of interest among them.

Impact of silver nanoparticles loaded with P. tuber-regium extract on thyroid profile of rat
Note: "*" -P ≤ 0.05 when compared the values of Group-2 (Low dose) and Group-3 (High dose) with Group-1 (Control).