Silkworms (Bombyx mori L.) grow by ingesting the leaves of Morus alba L. (white mulberry); a portion of the M. alba L. proteins are digested, but the majority are discharged. Feces from B. mori L. are called silkworm feces or silkworm manure and they contain organic acids (84%), ash (9-16%), and nitrogenous content (2-4%). Silkworm feces also contain a variety of amino acids such as histidine, leucine, lysine, and various sterols such as beta-sitosterols and cholesterols. Furthermore, they are composed of uric acid, phosphoric acid, potassium, calcium, vitamin A and B groups, plant growth hormones, and chlorophylls. In addition, β-carotene (Tong and Xu 2005), 1-deoxynojirimycin, fagomine, and 3-epifagomine (Zhou et al. 2007) have also been reported as components of silkworm feces.
Silkworm feces are nontoxic, and are purported to prevent paralysis of the body and limbs, and strengthen organs. They are also effective in the treatment of skin diseases and possess anti-inflammatory and analgesic properties. Studies have shown that they are effective in the treatment of diabetes and are rich in various nutritional components (Kim et al. 2007). Traditionally, silkworm feces have been used as a therapeutic agent in China, Korea, and other Eastern Asian countries to treat infectious diseases, headache and abdominal pain (Tulp and Bohlin, 2004). Moreover, silkworm feces are a good source of natural colorant for the food industry. Silkworm excreta are also available in the form of an instant tea, which is claimed to be a nutraceutical product. Studies of silkworm feces from the 1950-60s focused mainly on the value of livestock manure containing nutritional components. After 1990, a study regarding anticancer drugs derived from silkworm feces was reported, and another study, wherein porphyrin was separated from silkworm feces, was designed to treat photosensitive cancer. Very few antiviral substances are isolated and purified from silkworm feces. Hirayama et al., (1993) purified an antiviral protein from silkworm fecal matter and reported it to be a glycoprotein. Hiraki et al., (1996) obtained antiviral substances from silkworm fecal matter extract, which were shown to have marked antiviral effect on the growth of enveloped viruses, such as HVJ (Sendai virus), HSV-1 (herpes simplex virus type-1), and HIV-1 (human immunodeficiency virus type-1).
The value of utilizing silkworm feces obtained from B. mori L. breeding is underestimated. It is easy to obtain bulk amounts of silkworm feces through one-time breeding of B. mori L., with a yield of 104 kg per box (20,000 individuals). However, there are a lack of studies with regard to the compositions and bioactive compounds found in silkworm feces; studies of novel medicines, cosmetics, and plant compositions have yet to be performed as well. Therefore, along with studies on the main products of the sericulture industry including B. mori L powder, Cordyceps production, and male moth production, strategic studies investigating the effective active components and functional materialization of silkworm feces are necessary in order to promote the benefits of sericulture products.
Utilizing various solvents and extraction methods, this study evaluated the effects of physiologically active components in dried silkworm feces. A database of effective methods to extract silkworm feces was also formed. In conclusion, the use of silkworm feces could potentially increase the value of an otherwise useless by-product of the industrial sericulture industry and raise the income of local farmers.
The silkworm feces used in this study were obtained from B. mori L. (3 days 5th instar) from the Department of Agricultural Biology, National Academy of Agricultural science of the Rural Development Administration (RDA, Korea) in 2010. Small pieces of plant leaves and dead silkworm bodies were manually removed. Samples were dried at 50°C for 12h and then ground into fine powder. Fresh samples were kept at -70°C if extraction was not performed on the day of delivery.
The fecal extracts were extracted in accordance with each individual extraction protocol (listed below), the extracts were then filtered and concentrated using a vacuum evaporator. The concentrate was freeze-dried and ground into a powder. In the final step, the yield of the concentrate was investigated.
(1) Ethanol ultrasonification extraction (EUE)
The precisely weighed 100 g of silkworm fece powder was mixed with 1 L of 80% ethanol in a flask. The flask was not heated, and the extract was prepared for 2 h using 40 KHz ultrasound. This step was repeated 4 times.
(2) Ethanol stirrer extraction (ESE)
The precisely weighed 25 g of silkworm feces powder was mixed with 500 ml of 80% ethanol in a flask. Then, the contents were heated to 25±2℃ for 24 h and extracted twice.
(3) Ethanol reflux extraction (ERE)
The precisely weighed 100 g of silkworm feces powder was mixed with 1 L of 80% ethanol in a flask and then attached to a cooling tube. The extract was prepared at 60°C for 3 h in a water bath; this step was repeated 4 times.
(4) Ethanol reflux extraction after ultrasonification extraction (ERUE)
The precisely weighed 100 g silkworm feces powder was mixed with 1 L of 80% ethanol in a flask and then attached to a cooling tube. The extract was prepared at 60℃ for 3 h in water bath and then the extract was prepared for 2 h using a 40 KHz ultrasound 4 times.
(5) Water ultrasonification extraction (WUE)
The precisely weighed 100 g silkworm fece powder were mixed with 1 L of water in a flask. The flask was not heated, and the extract was prepared for 2 h using a 40 KHz ultrasound. This step was repeated 4 times.
(6) Butanol ultrasonification extraction (BUE)
The precisely weighed 100 g of silkworm feces powder was mixed with 1 L of n-butanol in a flask. The flask was not heated, and the extract was prepared for 2 h with 40 KHz ultrasound. It was repeated 4 times.
Total polyphenol content was determined according to the Folin-denis method (Kim et al., 2009). The reaction mixture was composed of 100 μL of the fecal extracts, 2 mL of 2% Na2CO3 and 100 μL of 50% Folin-Ciocalteu reagent. The mixture was left to sit for 30 min at room temperature, and the absorbance was measured at 720 nm. The total polyphenol content was measured from standard curve using galic acid.
To measure the total flavonoid content, 0.25 mL of 0.1% (w/ v) extract was mixed with 1.25 mL H2O and 5 mL of 5% NaNO2, and allowed to react for 5 min. Then, 0.15 mL of 10% AlCl3∙6H2O was mixed into the sample, and absorbance was measured at 510 nm (Hairi et al., 1991). The total flavonoid content was measured from the standard curve using (+)-catechin hydrate.
Different concentrations (1.25, 2.5, 5, and 10 mg/mL) of the silkworm extracts, prepared in 96 well plates, were added to 80 μL. The resulting samples were then added to 120 μL DPPH and 6 mg ethanol, and kept in for 30 min at 37℃. Ascorbic acid was used as positive control (Kim et al., 2012)
Superoxide dismutase activity was measured with an aliquot of silkworm feces extract using potassium phosphate buffer in the reaction mixture. The reaction mixture consisted of 50 mM sodium/potassium phosphate buffer (pH 7.5), 58 mM nitroblue tetrazolium (NBT), 9.9 mM methionine and 0.025% (v/v) Triton X-100. Finally, 2.45 mM riboflavin was added to the mixture and allowed to react for 8 min. The color intensity was then determined at 560 nm with a UV-spectrophotometer. One unit of SOD was expressed as the quantity of enzyme required to inhibit the reduction of NBT by 50% (Almansa et al., 1989).
Sterilized nutrient broth was inoculated with test bacteria (Staphylococcus aureus and Staphylococcus mutans) and incubated at 37℃ overnight. The obtained inoculum was diluted as follows: S. aureus (1:200) and S. mutans (1:200). Sterile paper discs 6 mm in diameter were impregnated with 25 μL of 10 mg/mL of S. aureus and 25 mg/mL on S. mutans.
We assayed α-glucosidase inhibitory activity using 4-nitrophenyl- α-D-glucopyranoside as a substrate. After reacting at pH 6.4 and 37℃ for 60 min, we measured the increase in absorbance at 405 nm to determine enzyme activity and to calculate inhibition.
Each experiment was carried out in triplicate, all data were the average of three independent experiments and analyzed by SPSS (version 18.0), and expressed as mean ± standard deviation (SD). Results were considered significant at p < 0.05.
The dry matter mass was measured after freeze-drying each of extracts, and then shown as a percentage of the total material. As shown in Table 1, the yield of ethanol reflux extraction after ultrasonification extraction (ERUE) was higher than the other extraction methods. Total polyphenol contents were, analyzed by using Folin-denis method with results shown in Fig. 1. Butanol ultrasonification extraction (BUE) had the lowest results with 3.3 mg TAE/g, whereas water ultrasonification extraction (WUE) had the highest results with 51.6 mg TAE/g. The distribution of flavonoids, using different extraction methods are presented in Fig. 2. Total flavonoid contents, according WUE and BUE, were 158.3 and 151.3 mg QRE/g, respectively. On the other hand, ERE yielded a significantly high concentration (266.8 mg QRE/g). Flavonids, act as natural antioxidants and, function as anti-aging and immunity-enhancing compounds (Huang et al., 2007). According to previous research, silkworm feces sre rich in flavonoids, chlorophyll, alkaloids, carotenoids, pectin, and lutein compounds (Liu et al., 2007). Through these results, we could potentially identify the various flavonoid contents from silkworm feces.
The results of DPPH radical scavenging activity showed that, there was no significant difference among the extraction methods with the exception of the WUE method (Fig. 3). Xu et al. (2014) reported that the solubility of silkworm excrement using different solvents increased with increasing concentration. The capability of scavenging DPPH free radical of silkworm excrement extracts from different solvents was the highest in an aqueous extract. In SOD-like activity, the extract from WUE showed significant anti-oxidant effects (Fig. 4.). Free radicals occur in everyday life, and most are removed by enzymes like superoxide dismutase (SOD) (Kim et al. 2007, Kedziora and Bartosz, 1988, Cross et al., 1987). The free radicals, reactive oxygen species and peroxides that cause aging, cancer, various diseases are fatal to humans. Currently, synthetic antioxidants were developed to inhibit these mechanisms. However, due to toxicity usage limitations and low activity of synthetic antioxidants, research on safer and stronger natural antioxidants is needed. Therefore, it can be concluded that the capability of antioxidants in silkworm feces extracted from various solvents and methods were markedly different, and that aqueous extracts exhibited the strongest antioxidant ability.
This experiment compared the effects of each of the extracts on S. aureus and S. mutans growing on agar plates. The antibacterial activity was evaluated utilizing traditional antibiotic susceptibility testing using the disc diffusion method. As shown in Fig. 5., the antimicrobial effects and thrombolysis could not be confirmed.
For decades, researchers have shown that rat and human α-glucosidases are strongly inhibited by mulberry leaf extract (Anno et al., 2004, Miyahara et al., 2004, Oku et al., 2006). Mulberry DNJ binds to the active center of α-glucosidase and is a potent inhibitor of this enzyme in the small intestine (Junge et al., 1996). The results of α-glucosidase inhibitory studies are summarized in Fig. 6. All other extracts, except for BUE, had α-glucosidase inhibition at around 60%. The methanolic extract of C. calcitrapa showed a concentration-dependent inhibition of the enzyme. The highest concentration tested, 100 mg/mL, showed a maximum inhibition of nearly 78.97% (Raad, 2013). Also, Lee and Lee (2001) isolated and identified genistein, an aglycon form of isoflavone found in soybeans, as a candidate for α-glucosidase inhibition extracted from fermentation broths of Streptomyces sp. Genistein was shown to be a reversible, slow-binding, and non-competitive inhibitor of yeast α-glucosidase (Lee and Lee, 2001). Therefore, according to these results, we have assembled the base data of effective methods to extract silkworm feces.