Quantitative analysis of rutin content using silkworm genetic resources

Abstract

Rutin is an important bioflavonoid that is consumed in the daily diet. This study compared the functional components of rutin from various silkworm species using a gene database with those of rutin produced by silkworms selectively bred through cross-combinations. We made comparisons between the geographical origin and species of silkworm using a gene database and discovered that rutin activity was ranked in the following order by species, Chinese (C5)> miscellaneous varieties (Jamsaeng 1 Ho) >Japanese (Jam 115) > European (E58) >Korean (Sun 3 ho). However, rutin levels with respect to various genetic traits (blood color, silk color, and egg color) were consistent. In order to study rutin changes that occurred during the cross breeding of the silkworm gene, we bred cross-combinations utilizing Jam 115 and the 4051 silkworms. In conclusion, in order to provide information about the constituents of functional materials contained in silkworm powder, it is imperative that silkworm cross breeding occurs so that the database of functional materials extracted from silkworms will expand.

Introduction

Flavonoids exhibit a wide variety of biological activities, including antiviral, antibacterial, anti-inflammatory, and antioxidant actions (Javed et al., 2012; Richetti et al., 2011; Nassiri-Asl et al., 2010). Among flavonoids, rutin (2-phenyl-3,5,7,3’,4’-pentahydroxybenzopyrone), is a non-toxic flavonoid glycoside with P vitamin activity and a bioflavonoid and antioxidant (Schwedhelm et al., 2003; Janbaz et al., 2002). Rutin can be broadly extracted from vegetables, fruits, herbs, leaves, seeds, red wine and several plantssuch as buckwheat, passion flower, apple and tea (Harborne, 1986; Havsteen, 1983). It has been demonstrated that rutin scavenges superoxide radicals and can chelate metal ions such as ferrous cations. More importantly, many studies have been conducted to prove the efficiency of rutin’s pharmacological functions as an antioxidant (Gao et al., 2002; Nagasawa et al., 2003; Kamalakkannan and Prince, 2006).

The silkworm, Bombyx mori L. has long been used in China and Korea as a folk remedy for the treatment of diabetes and is an economically important insect that converts mulberry leaf (Morus spp.) protein into silk. Mulberry leaves have been used to feed silkworms (Bombyx mori). These leaves are rich in flavonoids, alkaloids, and polysaccharides components that have been identified as some of the most potent major active compounds by chemical constituent research. The flavonoids in mulberry leaves were rutin, quercetin, isoquercitrin, and quercetin 3-(6-malonylglucoside) (Lee et al., 2007). A previous study indicated that mulberry leaf extracts could significantly reduce blood glucose, high blood pressure, high cholesterol, neutral fat and prevent thrombus formation and ageing. Mulberry is one of the plants that contain the highest levels of flavonoids in their dried leaves with levels of 1-3% (Chen et al., 2008). These flavonoids have found applications in food and pharmaceutical industries for their valuable properties, and adsorbent resins have been used to separate and concentrate these products from the natural matrixes (Fu et al., 2005; Qi et al., 2007). In particular, 1-deoxynojirimycin (DNJ), an intestinal α-glucosidase inhibitor, lowers blood glucose levels and is present in mulberry leaves and sericulture products such as silkworm powder (Asano et al., 2001; Asano, 2003). A previous study showed that in silkworms that eat only mulberry leaves, DNJ concentrations in the blood of the silkworms are 3 times higher than that in the mulberry leaves.

Similarly, silkworm powder is also rich in useful components and has long been favored as an antidiabetic agent, and for its various amino acids and flavonoids. However, the flavonoid contents of silkworm powder have not been tested using scientific methods. To date, to our knowledge, no studies have shown the extraction of bioactive compounds (including rutin) from silkworm powder except for DNJ. The aim of this study is to determine the main flavonoid (rutin) using high performance liquid chromatography (HPLC). In addition, we wanted to provide a basis for understanding rutin content in various silkworm species. Furthermore, our goal was to create a database for silkworm genetic resources utilizing morphological and, genetic characteristics data.

 

Materials and Methods

Materials

Silkworm resources were collected from the Sericulture and Apiculture Division for the Department of Agricultural Biology, RDA in Suwon, Republic of Korea. Both spring and autumn reared silkworms of 168 varieties were used for the rutin analysis (Japanese 64; Chinese 69; European 28; miscellaneous species 5; Korean 2). The freeze-dried 5th instar, 3-day-old silkworms (Ilshin Lab Co., Ltd) were ground and used within 48 h.

Sample preparation procedures

About 1 g of larva powder was mixed with 20 mL methanol, followed by vigorous shaking at 80℃ for 1 h, and then treated with sonication for 30 min. These extracts were cooled to 4℃ and subjected to preliminary filtration. The pellet was treated again by repeating the above steps, and the extracts were diluted to 50 mL methanol. The extracts were filtered using a 0.45 mm Millipore filter and used for HPLC analysis.

Rutin measurement

In order to measure rutin within the analytes, HPLC (Waters e2695), Waters 486 Tunable Absorbance UV/visible detector (Em 254, EX 322), and Waters Nova-Pak C18 column were used. The absorbance of the effluent was monitored at 355 nm and flow rate was 1 mL/min. The mobile phase used was on 2.5% acetic acid : methanol : acetonitrile = 70:10:20 (V/V/V). The flow rate was 0.6 mL/min. The standard material was rutin (SIGMA), and the rutin content was calculated (Fig. 1.).

Fig. 1.Analysis chromatogram of rutin standard (A) and silkworm variety C5 containing the best rutin content (B).

 

Results and discussion

Rutin contents of 168 silkworm varieties

There are many reports on rutin in Morus, and it was found that rutin contents in mulberry leaves were affected by many factors, such as varying Morus species and seasons. However, almost no studies were performed on the silkworm, and rutin accumulation and excretion in the larva are still not clear.

By using data acquired from this study, we built a database of silkworm and mulberry resources with morphological and genetic characteristics data. Moreover, certain bioactive compounds have been receiving increasing attention. We analyzed rutin content in addition to the base genetic information by using 168 varieties of silkworms. All silkworms were freezedried 5th instar species that were 3 d old.

Results showed that overall, Jam 115 had the highest rutin content with 0.446% (Table 1). In contrast, the 4051 had the lowest rutin content with 0.069%. Of the Chinese silkworm types, C5 had the highest rutin content (0.662%) while C61 had the lowest of all varieties with 0.056% (Table 2).

Table 1.Rutin contents for Japanese silkworm varieties

Table 2.Rutin contents for Chinese silkworm varieties

Of the European breeds, E58 had the highest rutin content (Table 3). The rutin content of the Korean varieties was relatively 0.319% (Sun 3 ho) and 0.209% (Sammyeonhonghoibaek) (Table 3). On the other hand, the highest rutin levels of the miscellaneous varieties were found in Jamsaeng 1 Ho (0.613%). For comparison of geographical origins of the silkworm gene resource, rutin concentration ranking was as follows: Chinese (C5) > miscellaneous varieties (Jamsaeng 1 Ho) >Japanese (Jam 115) > European (E58) > Korean (Sun 3 ho) (Table 3). In addition, no discernable difference in rutin content was noted with regard to blood, cocoon, or egg color of the silkworm.

Table 3.Rutin contents for various of silkworm varieties

Many flavonoids have been identified from the root bark of the mulberry tree. The leaves of M. alba L. contain quercetin derivatives such as isoquercitrin and rutin (Naito, 1968). Fujimoto and Hayashiya (1972) reported that the flavonoids in the cocoon shell of B. mori were also made from an artificial diet containing quercetin, rutin or isoquercitrin. Rutin was also found to be similarly distributed within caper plant, common buckwheat (Sofic et al., 2010), and amaranth plants, where the highest amount of rutin was found in the leaves (Kalinova and Dadakova, 2009). This was established from levels of 4% to 9% of rutin per dry weight depending upon the stage of development of the plant (Kalinova et al., 2006). In addition, Atanassova and Bagdassarian (2009) found that in the case of dry fruits and vegetables, the rutin content show little variation in range (from 0.15% to 0.18%), with the exception of the red hot chili pepper (0.22%) and aronia (0.34%).

Rutin contents produced by crossbreeding

The purpose of the cross-breeding experiment was to optimize hybrid combinations that yielded rutin concentrations higher than that by the basic silkworm larvae. The rutin contents of various silkworm varieties were investigated with results indicating a difference among the various silkworm varieties. When the varieties Jam 115 and 4051 were hybridized, the rutin content of the offspring was lower than the parent Jam 115. Likewise, in a cross between the Chinese breeds C5 and C61, the rutin content was lower in the hybrid than in the C5 parent (Table 4). These results show that the crossbreeding tests were not effective in the production of offspring with sustained or improved rutin concentrations. The breeding objectives employed in different countries where sericulture is practiced remained quite different for many of the traits and also to provide productive hybrid strains specifically for commercial exploitation (Nagaraju, 2002). In addition, hybrid silkworm strains are recommended for large scale laboratory and intensive, commercially viable sericulture operations. Regarding the concentration of DNJ in silkworm powder, the DNJ contents produced by crossbreeding silkworm were higher than those in non-crossbreeding silkworm (Ju et al., 2014). However, these results indicate that the rutin content in silkworm powder was affected by the variety of silkworm. We conclude that it is the most effective and economic strategy to use nonhybrid silkworms by crossing the 5th instar parent variety larvae of 3 d of age.

Table 4.Rutin contents by silkworm cross-breeding

In conclusion, obtaining information about the concentration of functional materials in silkworm powder could contribute to the development and promotion of processed, functional products derived from silkworm.