Acute and Subacute Toxicities of Thai Silkworm Powder ( Bombyx mori Linn . ) From Three Races in Male Wistar Rats and In vitro Antioxidant Activities

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 DPPH, ABTS and FRAP methods. Results: The acute toxicity in rats by single dose oral administration show that the high dose at a concentration of 2,000 mg/kg b.w. did not have toxicity in rats. The sub-acute toxicity in rats by oral administration showed that the high dose concentration at 2,000 mg/kg body weight for 6 weeks had no toxicity in all group either. In addition, the hematological parameters and lipid profiles of treated group and control groups were not significantly different. The antioxidant study showed that silkworm powders were not significant difference in all groups of samples in three assays (DPPH, ABTS and FRAP). However, significant difference had been found in comparison to ascorbic acid (p < 0.05). Conclusion: Silkworm powder from three races of Thai silkworm did not produce any toxicity in male rats. In addition, the silkworm powder also exhibited antioxidant activities.


INTRODUCTION
The biological activities of silkworm such as antihypertensive, 1 anticancer, 2 antibacterial, 3 antiviral, 4 antioxidant 5 and hepatoprotective activities 6 have been reported.Mulberry silkworm (Bombyx mori L.) has been reared in especially the northeastern part of Thailand for longtime. 7Silkworms are consumed mostly as functional food and pharmaceuticals for diabetes mellitus patient in Korea, China, and Japan. 8The major functional compound of silkworm powder was 1-deoxynojirimycin (DNJ) acting as alpha-glucosidase inhibitor. 9Therefore, the result from previous studies showed that silkworm powders were tended to be potential functional food.The safety of silkworm were still limited that only Korean silkworm races had been reported. 8However, Thai silkworm races had no scientific data regarding the safety for consumption.In order to make Thai silkworm powder widely used as food product, the safety should be investigated.This study was aimed to investigated the acute and sub-acute toxicities in male Wistar rat and in vitro antioxidant activities of three Thai silkworm races.

Chemicals
Reagents of high quality were obtained from Sigma-Aldrich Chemical (St Louis, MO).Other chemical of analytical grade were purchased from Carlo Erba, Italy and Merck, Germany.

Preparation of silkworm powder
Three varieties of Thai mulberry silkworms including Nangnoi, Nanglai, and Samrong were reared at Silk Innovation Center, Mahasarakham University, Thailand.Identifications of silkworm races were performed with regard to the Queen Sirikit Department of Sericulture protocol.The 5 th instar, 3 rd day silk larvae were prepared according to the study of Ryu et al. 10 The silkworms were then freeze-dried and ground in order to obtain fine powders.Silkworm powders were kept in freezer before use.

Animals
Male albino Wistar rats with weight of 150-200 g were purchased from the National Laboratory Animal Centre (NLAC), Mahidol University, Thailand.The rats were housed under standard environmental Pharmacognosy Journal, Vol 9, Issue 4, Jul-Aug, 2017 %inhibition = [(A control -A sample )/A control ] × 100 Where A control was an absorbance of ABTS solution with ethanol.A sample was an absorbance of ABTS solution with sample.The effective concentration of sample requires to scavenge ABTS radical by 50% (EC 50 value) and obtaining by a linear regression analysis of a calibration curve plotting % inhibition versus concentration.FRAP assay (Ferric reducing antioxidant power) -FRAP assay was carried out with some modification from Benzie and Strain. 15The FRAP reagent was prepared from 300 mM acetate buffer (pH 3.6), 20 mM ferric chloride and 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl.All 3 solutions were mixed together in the ratio of 10:1:1 (v/v/v), respectively.The FRAP reagent was freshly prepared daily prior to use.A total of 30 µL sample was added to 270 µL of the FRAP reagent and mixed well.After 30 min at room temperature incubation, the absorbances of final solutions were subsequently measured at 593 nm in 3 replicates using microplate reader.The antioxidant capacity of sample was calculated and compared with trolox calibration curve, and expressed as µmol trolox equivalent (TE) per gram of silkworm powders.

Statistical analysis
All data were expressed as mean ± standard error of mean (SEM).The investigated parameters of each sample were evaluated in triplicate.The statistical analyses of the data were carried out by analysis of variance (ANOVA), using the statistical package SPSS 15 and the Duncan's multiple range test (DMRT) to indicate the statistical difference at P-value <0.05.

The acute and sub-acute toxicity
For acute toxicity study, the rats were single administered silkworm powder from 3 races at dose of 1,000, 1,500 and 2,000 mg/kg b.w.The mortality and behavioral sign and symptoms of toxicity were observed within 24 h and further for 14 days.During this experiment, rats body weights were measured as shown in Table 1.The result indicated that no dead animals and toxicity symptoms were found.In additions, the body weights of control and treated rats tended to increase week by week without significant difference among groups.For sub-acute toxicity, the rats were administered every two days with 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 weekly for 6 weeks (Table 2 and 3).At the end of the experiments, blood chemistry parameters and hematological values were analyzed (Table 4  and 5).The results showed that body weights of control and treated rats were not different but tend to be increased week by week.The body weights among control and silkworm powder treated rats were not significantly different.On the other hands, blood glucose levels and blood chemistry parameters of the control and silkworm powder treated rats were not different.This finding suggested that silkworm powder did not alter organ system function in male rats and did not exhibit any toxicity in rats.

Antioxidant activities
The results of antioxidant activities of three races silkworm powders tested by DPPH, ABTS and FRAP methods were showed in Table 6.The antioxidant study indicated that the capacities of silkworm powders were not significant difference in all groups of samples in three assays (DPPH, ABTS and FRAP).However, significant difference has been found comparing with ascorbic acid (p>0.05). it was found that Thai silkworm from three races have less antioxidant capacity than those of ascorbic acid.conditions (at 25 ± 2°C, 40-60 % humidity with 12 h light/12 h dark cycle). 11All animals were given a standard laboratory diet with access to water ad libitum.The experimental protocol and the experiments performed on the rats were approved by the Institutional Animal Care and Use Ethic Committee, Mahasarakham University, Thailand (License No. 0015/2011).

Acute toxicity test
The rats were divided into 10 groups, 6 rats per group, were fasted overnight (12 h) and weighed.The doses of silkworm powder from 3 races were calculated according to the body weight of fasted animals and administered at doses of 1000, 1500 and 2000 mg/kg body weight.The animals were regularly and individually observed for behavioral changes and general toxicity signs after dosing for the first 24 h, with special attention being given during the first 4 h.Thereafter, observation was continued daily for a total of 14 days.Mortality, body weights, bizarre behavior and abnormal sign including seizures, urination, vomiting, anorexia or depression were recorded. 12

Sub-acute toxicity test
The rats were divided into 10 groups, 6 rats per group, were treated orally with silkworm powder (two days per dose) for 6 weeks.Silkworm powders were administered at 2000 mg/kg body weight in each silkworm races.Mortality, body weights and blood glucose level were recorded for every week.Hematological values and lipid profile were recorded at last week. 12

Antioxidant activity
DPPH radical scavenging assay -The test was carried out using the 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging assay.The free radical scavenging activity of the silkworm powder and standard ascorbic acid solution in absolute ethanol were determined based on their ability to react with the DPPH free radical. 13A 750 µL aliquot of silkworm powder (10 -1,000 µg/mL, dissolved in 10% DMSO and filtered) was added into 750 µL of DPPH in absolute ethanol (152 µM).After incubation at room temperature in the dark condition for 20 min, the absorbance of each solution was determined at 517 nm using 96-well plates with microtiter plate reader (BMG LABTECH GmbH, Ortenberg, Germany).The results were presented as a percentage of inhibition as follows:

%inhibition = [(A control -A sample )/A control ] × 100
Where A control was an absorbance of DPPH solution without sample.A sample was an absorbance of DPPH solution with sample.The inhibitory concentration of the sample requires scavenging DPPH radical by 50% (IC 50 value) and obtaining a linear regression analysis of a calibration curve plotting, %inhibition versus concentration.ABTS radical scavenging assay -2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) diammonium salt radical cationic decolorization test was performed using spectrophotometric method from the previous studied 14 with slight modification.The ABTS stock solution was prepared by mixing 88 µL of 140mM potassium persulfate (K 2 S 2 O 8 ) with 5 mL of 7mM of ABTS stock solution (pH 7.4).The working ABTS reagent was prepared by diluting the stock solution with ethanol to obtain absorbance of 0.7 ±0.05 at 734 nm.The test concentrations of standard ascorbic acid, silkworm powders were prepared in the same manner as previously described in DPPH assay.The ABTS working solution (100 µL) with equal volume of ethanol served as a control.The reaction mixtures were incubated at room temperature for 30 min.The absorbance of each solution was determined at 734 nm using 96-well plates with microtiter plate reader (BMG LABTECH GmbH, Ortenberg, Germany).The results were expressed as percentage of inhibition as follows:

DISCUSSION
The Acute and Sub-Acute Toxicity For the acute toxicity study, rats were administered with single dose of 5 th instar, 3 rd day silkworm powder of 3 races at doses of 1,000, 1,500 and 2,000 mg/kg body weight.The behavior and body weight were recorded at 0, 7, and 14 days, respectively.For the sub-acute toxicity study, rats were administered with silkworm powders from three races at dose of 2,000 mg/kg body weight, every 2 days for 6 weeks.The result showed that no signs of toxicity or abnormalities in rats, such as seizures, vomiting, ataxia, lethargy, loss of appetite had been observed.This study is the first report on acute and sub-acute toxicities of Thai silkworms.In addition, there were only studies on the nutritional values of silkworm powder, reporting the main components of a protein about 40% in Indian silkworm. 16The protein components were not different from normal food resulting to low toxicity.Similar to the studies of Mondal et al, 17 the substances in the silk powder were about 16-18 amino acids.The study of Ninpetch et al 18

The Antioxidant Activity
The antioxidant study indicated that silkworm powders were not significant difference in all groups of samples in three assays (DPPH, ABTS and FRAP).However, significant difference had been observed comparing with ascorbic acid.Antioxidant activities of mulberry silkworm may derive from some pigments in cocoon shell sericin.The coloring components are associated with phenolic compounds in the leaves of mulberry (Morus alba L.), the major feed for mulberry silkworm larvae.0] The studies of Tamura et al 19 isolated quercetin 5-glucoside, quercetin 5,7,4'-triglucoside and quercetin derivatives from the yellow-green cocoon shell of Multi-Bi race.The previous study showed that there are several native races of silkworm in Thailand with various color of cocoon shell.The most common native Thai silkworm is Nangnoi with intent yellow, while the color of cocoon shell from other races are varied from white to green yellow.Flavonoid compounds are also responsible for antioxidant properties of mulberry silk cocoon.The studies of Butkhup et al 21 showed that the strong correlation between total active ingredients (phenolics and flavonoids) and antioxidant activities (DPPH, ABTS and FRAP assay) had been observed in Thai silk sericin.

CONCLUSION
Acute and sub-acute toxicities of silkworms on hematological parameters and lipid profiles had not been observed.Silkworms also present antioxidant capacities.

Table 5 : Hematological parameters of rats in sub-acute toxicity study (n=6) at 6 th week Treatment 2,000 mg/kg WBC (cells/mm 3 ) RBC (10 6 cell/mm 3 )
analyzed the protein and lipid contents of the fifth instar mulberry silkworm (Bombyx mori L.) for substitution of protein from meat.Three silkworm races (Jim thompson, Nanglay and Thai hybrid) showed a rich source of protein.Jim Thompson race had 68.97 % of protein, Nanglay race had 55.55 % of protein and Thai hybrid race had 54.19 % of protein.