Pharmacogn J. 2017; 9(4): 541-545 A Multifaceted Journal in the field of Natural Products and Pharmacognosy Original Article www.phcogj.com | www.journalonweb.com/pj | www.phcog.net Acute and Sub-acute Toxicities of Thai Silkworm Powder (Bombyx mori Linn.) From Three Races in Male Wistar Rats and In vitro Antioxidant Activities Surapong Rattana1*, Teeraporn Katisart2, Bunleu Sungthong3, Chirapha Butiman4 ABSTRACT Background: The silkworm powder from Japanese and Korean races has a high biological activity. However, there was less scientific evidence regarding bioactivities in Thai silkworm races. Objective: This research was interested in toxicity and antioxidant activities of Thai silkworm races. Material and methods: The 5th instar, 3rd day of growth stage of Thai traditional silkworm (Nanglai, Nangnoi and Samrong races) were lyophilized by freeze dryer and ground as silkworm powder. The acute and sub-acute toxicities studies were carried out in male Wistar rats. The antioxidant capacities of silkworm powder were investigated with Surapong Rattana1*, DPPH, ABTS and FRAP methods. Results: The acute toxicity in rats by single dose oral Teeraporn Katisart2, administration show that the high dose at a concentration of 2,000 mg/kg b.w. did not have Bunleu Sungthong3, toxicity in rats. The sub-acute toxicity in rats by oral administration showed that the high dose 4 concentration at 2,000 mg/kg body weight for 6 weeks had no toxicity in all group either. Chirapha Butiman In addition, the hematological parameters and lipid profiles of treated group and control 1 groups were not significantly different. The antioxidant study showed that silkworm powders Department of Biology, Faculty of were not significant difference in all groups of samples in three assays (DPPH, ABTS and Science, Mahasarakham University, FRAP). However, significant difference had been found in comparison to ascorbic acid Maha Sarakham, 44150, THAILAND. ( < 0.05). Conclusion: Silkworm powder from three races of Thai silkworm did not produce 2Department of Biology, Faculty of p Science, Mahasarakham University, any toxicity in male rats. In addition, the silkworm powder also exhibited antioxidant activities. Maha Sarakham, 44150, THAILAND. Keywords: Silkworm, toxicities, hematological parameters, lipid profile, antioxidant activities. 3Pharmaceutical Chemistry and Natural Products Research Unit, Faculty of Pharmacy, Mahasarakham University, Maha Sarakham, 44150, THAILAND. INTRODUCTION MATERIALS AND METHODS 4Silk Innovation Center, Mahasarakham University, Maha Sarakham, 44150, The biological activities of silkworm such as anti- Chemicals THAILAND. hypertensive,1 anticancer,2 antibacterial,3 antiviral,4 Reagents of high quality were obtained from Sigma- Correspondence antioxidant5 and hepatoprotective activities6 have Aldrich Chemical (St Louis, MO). Other chemical Surapong Rattana, Department of been reported. Mulberry silkworm (Bombyx mori L.) of analytical grade were purchased from Carlo Erba, Biology, Faculty of Science, Mahasarakham has been reared in especially the northeastern part of Italy and Merck, Germany. University, Maha Sarakham, 44150, 7 THAILAND Thailand for longtime. Silkworms are consumed mostly Preparation of silkworm powder as functional food and pharmaceuticals for diabetes Phone number.: +6643-754245 Three varieties of Thai mulberry silkworms including 8 E-mail: [email protected] mellitus patient in Korea, China, and Japan. The Nangnoi, Nanglai, and Samrong were reared at History major functional compound of silkworm powder was Silk Innovation Center, Mahasarakham University, • Submission Date: 17-04-2017; 1-deoxynojirimycin (DNJ) acting as alpha-glucosidase Thailand. Identifications of silkworm races were • Review completed: 13-05-2017; inhibitor.9 Therefore, the result from previous studies performed with regard to the Queen Sirikit Depart- • Accepted Date: 18-05-2017. showed that silkworm powders were tended to be ment of Sericulture protocol. The 5th instar, 3rd day DOI : 10.5530/pj.2017.4.87 potential functional food. The safety of silkworm were silk larvae were prepared according to the study of 10 still limited that only Korean silkworm races had been Ryu et al. The silkworms were then freeze-dried Article Available online and ground in order to obtain fine powders. Silkworm http://www.phcogj.com/v9/i4 reported.8 However, Thai silkworm races had no powders were kept in freezer before use. scientific data regarding the safety for consumption. In Copyright © 2017 Phcog.Net. This is an open- order to make Thai silkworm powder widely used as Animals access article distributed under the terms food product, the safety should be investigated. This Male albino Wistar rats with weight of 150-200 g of the Creative Commons Attribution 4.0 study was aimed to investigated the acute and sub-acute International license. were purchased from the National Laboratory Ani- toxicities in male Wistar rat and in vitro antioxidant mal Centre (NLAC), Mahidol University, Thailand. activities of three Thai silkworm races. The rats were housed under standard environmental Cite this article : Rattana S, Katisart T, Sungthong B, Butiman C. Acute and Sub-acute Toxicities of Thai Silkworm Powder (Bombyx mori Linn.) From Three Races in Male Wistar Rats and In vitro Antioxidant Activities. Pharmacog J. 2017;9(4):541-5. Pharmacognosy Journal, Vol 9, Issue 4, Jul-Aug, 2017 541 Toxicity and Antioxidant Effect of Silkworm conditions (at 25 ± 2°C, 40-60 % humidity with 12 h light/12 h dark %inhibition = [(Acontrol-Asample)/Acontrol] × 100 cycle).11 All animals were given a standard laboratory diet with access to water ad libitum. The experimental protocol and the experiments per- Where Acontrol was an absorbance of ABTS solution with ethanol. Asample formed on the rats were approved by the Institutional Animal Care and was an absorbance of ABTS solution with sample. Use Ethic Committee, Mahasarakham University, Thailand (License No. The effective concentration of sample requires to scavenge ABTS radical 0015/2011). by 50% (EC50 value) and obtaining by a linear regression analysis of a calibration curve plotting % inhibition versus concentration. Acute toxicity test FRAP assay (Ferric reducing antioxidant power) - FRAP assay was carried The rats were divided into 10 groups, 6 rats per group, were fasted over- out with some modification from Benzie and Strain.15 The FRAP reagent night (12 h) and weighed. The doses of silkworm powder from 3 races was prepared from 300 mM acetate buffer (pH 3.6), 20 mM ferric were calculated according to the body weight of fasted animals and chloride and 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM administered at doses of 1000, 1500 and 2000 mg/kg body weight. The HCl. All 3 solutions were mixed together in the ratio of 10:1:1 (v/v/v), animals were regularly and individually observed for behavioral changes respectively. The FRAP reagent was freshly prepared daily prior to use. and general toxicity signs after dosing for the first 24 h, with special A total of 30 µL sample was added to 270 µL of the FRAP reagent and attention being given during the first 4 h. Thereafter, observation was mixed well. After 30 min at room temperature incubation, the absorbances continued daily for a total of 14 days. Mortality, body weights, bizarre of final solutions were subsequently measured at 593 nm in 3 replicates behavior and abnormal sign including seizures, urination, vomiting, using microplate reader. The antioxidant capacity of sample was calcu- 12 anorexia or depression were recorded. lated and compared with trolox calibration curve, and expressed as µmol trolox equivalent (TE) per gram of silkworm powders. Sub-acute toxicity test The rats were divided into 10 groups, 6 rats per group, were treated orally Statistical analysis with silkworm powder (two days per dose) for 6 weeks. Silkworm powders All data were expressed as mean ± standard error of mean (SEM). The were administered at 2000 mg/kg body weight in each silkworm races. investigated parameters of each sample were evaluated in triplicate. The Mortality, body weights and blood glucose level were recorded for every statistical analyses of the data were carried out by analysis of variance 12 week. Hematological values and lipid profile were recorded at last week. (ANOVA), using the statistical package SPSS 15 and the Duncan’s Antioxidant activity multiple range test (DMRT) to indicate the statistical difference at P- value <0.05. DPPH radical scavenging assay - The test was carried out using the 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging assay. RESULTS The free radical scavenging activity of the silkworm powder and stan- The acute and sub-acute toxicity dard ascorbic acid solution in absolute ethanol were determined based on their ability to react with the DPPH free radical.13 A 750 µL aliquot For acute toxicity study, the rats were single administered silkworm of silkworm powder (10 - 1,000 µg/mL, dissolved in 10% DMSO and powder from 3 races at dose of 1,000, 1,500 and 2,000 mg/kg b.w. filtered) was added into 750 µL of DPPH in absolute ethanol (152 µM). The mortality and behavioral sign and symptoms of toxicity were After incubation at room temperature in the dark condition for 20 min, observed within 24 h and further for 14 days. During this experiment, the absorbance of each solution was determined at 517 nm using 96-well rats body weights were measured as shown in Table 1. The result indicated plates with microtiter plate reader (BMG LABTECH GmbH, Ortenberg, that no dead animals and toxicity symptoms were found. In additions, Germany). The results were presented as a percentage of inhibition as the body weights of control and treated rats tended to increase week by follows: week without significant difference among groups. For sub-acute toxicity, the rats were administered every two days with %inhibition = [(A -A )/A ] × 100 control sample control silkworm powder from three races at a dose of 2,000 mg/kg b.w. During the experiment, body weight and blood glucose levels were measured Where Acontrol was an absorbance of DPPH solution without sample.