Analysis of Natural Compounds from Silkworm Pupae and Effect of Alcohol Detoxification

Monday, 08 October 2012 09:58

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Amino acid and fatty acid composition of silkworm pupae extracts
  7. Acknowledgments
  8. References

Silkworm pupae have much potential and many applications as a natural medicine to promote human health. However, their chemical components have not been fully characterized or understood. HPLC analysis was conducted to determine the content ratio (%) of individual amino acids in total protein of the pupae. It showed that glutamic acid (18.3%), histidine (14.6%) and alanine (10.2%) are the most common amino acids in silkworm pupae. Fatty acid composition of silkworm pupae oil was revealed by high-pressure liquid chromatography and gas chromatography – mass spectroscopy analyses. They contain a high ratio of essential fatty acids, [α-linolenic acid (ω-3 fatty acid]+ linoleic acid) (49.0%), and also contain non-essential fatty acids, oleic acid (19.9%), palmitoleic acid (2.5%), palmitic acid (19.7%), stearic acid (8.6%), and eicosapentaenoic acid (EPA) (0.3%). In addition, they also contain antioxidants, quercetin diglucoside and nutritionally important riboflavin (vitamin B2). This study suggests that silkworm pupae are a nutritionally valuable food product and are applicable as cosmetic components with essential amino acids, essential fatty acids, antioxidants and vitamins. The animal experiment showed that alcohol dehydrogenase (ADH) activity was significantly higher in the liver of mice orally administered with 0.5 mg/mL of silkworm extract and alcohol than with commercial Dawn808™ and alcohol, indicating that silkworm pupae extracts have alcohol detoxification activity.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Amino acid and fatty acid composition of silkworm pupae extracts
  7. Acknowledgments
  8. References

The pupae of the silkworm (Bombyx mori) are commonly used as a functional food to treat adult diseases such as diabetes and high blood pressure, and to boost the immune system (Yang et al. 1995; Wu et al. 1996; Tamura et al. 2002). Although its safety and clinical effectiveness are well known, its mechanism of action is still unclear. As a natural product, silkworm pupae have few side effects, and their effectiveness in treating adult diseases has been continually proven and validated. Therefore, they are frequently used to develop health food supplements, medicines or cosmetics. However, there has been little research into the purification of their useful chemical components or chemical characterization. Silkworm extracts contain several important chemical compounds. Bombykol (hydroxyhexadecadienol) is one of the key molecular components (Matsumoto 2010). The extracts also contain bombyxin, which is very similar to insulin and several other peptide hormones (Nagasawa 1992; Ishizaki & Suzuki 1994; Satake et al. 1997; Satake et al. 1999). The head of the silkworm contains copious amounts of free nucleotides, and the presence of quercetin glycosides has also been reported. Silkworm extracts contain high levels of arginine, which is a substrate of nitric oxide synthesis, suggesting that their consumption may enhance sexual stamina. Since the contents of these extracts and other silkworm products have not been analyzed, research at the molecular level is necessary to prove their effectiveness. This should involve purification of components of the silkworm extract and analysis of their chemical structures. The results of this analysis will allow us to diversify utilization of silkworm extract and develop new products such as antioxidants, cosmetics or anti-aging therapies (Itoh et al. 1995). Recently, it has been reported that fermented silkworm powder has a protective effect in alcohol-induced hepatotoxicity in a rat model (Cha et al. 2011). In this study, we report the analysis of key compounds (oils, flavone, vitamin B2 and amino acids) purified from the silkworm pupae (Kurioka & Yamazaki 2002; Miura et al. 2002) and the  

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Amino acid and fatty acid composition of silkworm pupae extracts
  7. Acknowledgments
  8. References

Preparation of silkworm pupae extracts

Frozen silkworm pupae (107 g) were ground using a blender, mixed with ethanol (200 mL) and then stirred using an agitator. The sample was filtered to collect particles from the filtrate. The particles were extracted with ethyl acetate (100 mL) three times, and the three extracts were pooled. The extracts were vacuum-concentrated and dissolved in hexane (150 mL). Activated carbon (20 g) was added to the extracts and the mixture was heated for 1 h. The mixture was then cooled and filtered with Celite (Celite Korea Ltd, Seoul, Korea). The filtrate was vacuum-concentrated and viscous oil (3.4 g) was obtained in a colorless and odorless state. Silkworms of different ages were processed using the same procedure.

Amino acid analysis of silkworm pupae extracts

The aqueous phase of the silkworm extract (2 g; 3 mL) was transferred to a vial, and 1 mL of 6 mol hydrochloric acid solution was added. The mixture was hydrolyzed at 110°C for 24 h. After the reaction, the hydrolyzed product was obtained by concentrating the sample in the vial under reduced pressure through removal of residual water. The product was separated by cation exchange chromatography (Biochrom 20; Pharmacia Biotech, Buckinghamshire, UK). Then, the concentration of each amino acid was determined by reaction with ninhydrin to produce colored complexes and by measuring the absorbance (440 and 570 nm).

Hydrolysis and methylation of silkworm oil

Silkworm pupae oil (1 g) was mixed with methanol (25 mL) and water (1 mL) and then sodium hydroxide (300 mg) was added. The mixture was heated under reflux for 1 h. After cooling, the pH of the product was adjusted to 2 with cold hydrochloric acid (1 mol). Water (50 mL) and ethyl acetate (50 mL) were added, and the sample was vigorously shaken and then left to settle until an ethyl acetate layer formed. The ethyl acetate layer was washed with a saturated salt solution. Any residual moisture was removed from the sample with anhydrous magnesium sulfate. The sample was filtered, and the filtrate was concentrated to yield the product (900 mg). A fatty acid mixture (800 mg) was isolated from the product by silica gel chromatography (7:3 hexane/ethyl acetate). It was dissolved in ether (20 mL), and a diazomethane ether solution from a diazomethane generator was added to the sample. The diazomethane-generating method was as follows. A Diazald solution (12.5%) in 40 mL ether was slowly added to 20 mL of a 25% potassium hydroxide solution in 50% ethanol, at 60°C. The diazomethane ether solution was obtained by distillation. The fatty acid/diazomethane ether mixture was agitated for 7 h. Residual diazomethane was eliminated by gradual addition of acetic acid. The mixture was washed with a saturated sodium bicarbonate solution, and then with a saturated salt solution. Anhydrous magnesium sulfate was added, and the mixture was shaken and then filtered. The filtrate was concentrated to obtain a colorless liquid product (750 mg), containing white solids.

Separation of fatty acids on high-pressure liquid chromatography (HPLC) and their structural analysis

The fatty acid mixture was purified by HPLC using a Dynamax C18 column (22.5 mm internal diameter [ID]× 250 mm) (Rainin Instruments, Woburn, MA, USA). The mixture was eluted in acetonitrile of gradually increasing concentrations, from 85% to 100%, at a rate of 7 mL/min for 80 min. Five fractions showing UV absorbance at 208 nm were aliquoted and concentrated. The respective masses of these five aliquots were as follows: 303.1 mg [colorless liquid, Product A: room temperature (RT), 50.8 min], 7.9 mg (colorless liquid, Product B: RT, 62.7 min), 53.7 mg (colorless liquid, Product C: RT, 66.5 min), 176.6 mg (colorless liquid, Product D: RT, 83.7 min) and 174.5 mg (white solid, Product E: RT, 85.5 min). These products were analyzed with infrared light, UV light, 1H NMR (nuclear magnetic resonance), and 13C NMR spectrometers and identified as α-linolenic acid (ω-3 fatty acid), palmitoleic acid, linoleic acid, oleic acid and palmitic acid, respectively, in methyl ester form (Figure 1).

Figure 1. Structures of the main fatty acids in silkworm pupae oil: α-linolenic acid (ω-3 fatty acid), linoleic acid, oleic acid, palmitoleic acid, stearic acid, palmitic acid and eicosapentaenoic acid. The omega (ω) number indicates the position of the first double bond counted from methyl ends. α-linolenic acid (ω-3 fatty acid) and linoleic acid are essential fatty acids.

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GC-MS analysis of fatty acid mixture

The methylated fatty acid mixture was analyzed for identification of its contents by gas chromatography (GC), using a HP-1 fused-silica column (30 m, ID 0.25 mm, film thickness 0.25 µm), and mass spectrometry (MS). The oven temperature program of GC was as follows: 50°C (2 min) → 10°C/min → 280°C (10 min). Helium (He, 1 mL/min) was used as a carrier gas. In MS, ionization was at 70 eV. From this analysis, the relative ratios of fatty acid-methyl esters, (α-linolenic acid [ω-3 fatty acid]+ linoleic acid), oleic acid, palmitoleic acid, stearic acid and palmitic acid, were 49.0:19.9:2.5:8.6:19.7, respectively. The mixture of α-linolenic acid (ω-3 fatty acid) and linoleic acid could not be separated by GC-MS. Instead, the mixture was separated by HPLC, giving a ratio of 85 (α-linolenic acid):15 (linoleic acid).

Separation of flavone and vitamin B2 from silkworm pupae extract

Frozen silkworm pupae (2.5 kg) were ground in a blender, mixed with ethanol (2 L) and then stirred on an agitator. The sample was filtered to remove particles from the filtrate. The filtrate was extracted three times with 500 mL ethyl acetate to remove the organic layer. The remaining water-soluble layer (115 g) was subjected to C18 flash column (40–60 µm, 90 mm ID × 100 mm) (Merck, Darmstadt, Germany) chromatography. It was eluted gradually in a series of six water:methanol mixtures in the ratios 10:0, 9:1, 7:3, 5:5, 3:7 and 0:10, at a volume of 2 L each. The fraction (2.3 g) eluted in water: methanol (7:3) was subjected to silica gelchromatography (70–230 mesh, 45 mm ID × 200 mm, eluent 70/27/3 dichloromethane/methanol/water). Vitamin B2 (100 mg) was obtained by concentrating the fluorescent fraction. The eluent (500 mg) in water: methanol (5:5) from the above C18 chromatography was subjected to medium pressure silica gel chromatography (70/27/3 dichloromethane/methanol/water) to obtain nine fractions. The seventh fraction was again subjected to medium pressure silica gel chromatography (70/27/3 methyl chloride/methanol/water) to obtain a quercetin diglucoside (17 mg), the 1H, 13C NMR, correlation spectroscopy (COSY), heteronuclear multiple bond correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and rotating frame nuclear overhauser enhancement spectroscopy (ROESY) spectra of which were analyzed.

Fatty acid and amino acid analyses of extracts from silkworm pupae of different ages

Silkworm pupae samples of different ages were prepared using the aforementioned methods for the generation of extracts, and the preparation of fatty acid and amino acid samples (Kim et al. 2001). Amino acid and fatty acid analyses were performed on these samples. Fatty acids that could not be separated by GC-MS were separated by HPLC, and the ratios of the separated samples were then calculated.

Acute alcohol induction

Imprinting Control Region (ICR) mice (male aged 8 weeks, weighing 19–22 g) were purchased from Orient Bio Animal Company (Seongnam, Gyeonggi, Korea) and were acclimated in the laboratory environment for 2 weeks. The mice were maintained in an environmentally controlled room (24 ± 2°C; 40–50% humidity; 12 h lighting cycle) with a supply of solid feed and water ad libitum. Silkworm extract used in this study was produced by KunWha (KW) Pharmaceutical Co., Ltd. (Gongju, Korea). Silkworm pupae (1 kg) at pupa period days 12–13 were crushed by homogenizer, followed by incubation in 80% ethanol for 2 h, and then filtered using a 60 mesh. The ethanol extracts of silkworm pupae were concentrated using an evaporator. The extracts were reconstituted with 500 mL Millipore water at 80°C, filtered using a 60 mesh, followed by freeze drying for 7 days. The concentrated extract was stored at −80°C until administration. Alcohol used in this study was “Cham-i-sul” (22% alcohol) manufactured by Jinro Co. (Seoul, Korea). To study the effect of acute alcohol induction, experimental mice were assigned with eight mice per group to five groups; negative control (distilled water [DW]), positive control (DW + alcohol), KW1 (0.01 mg of silkworm extract/mL + alcohol), KW2 (0.05 mg/mL of silkworm extract + alcohol), KW3 (0.1 mg/mL of silkworm extract + alcohol), KW4 (0.5 mg/mL of silkworm extract + alcohol), and positive control (Drink [10-fold dilution of Dawn808TM, commercially available for reducing hangover from Glami, Korea + alcohol] and Alcodex [10-fold dilution of Alcodex from Guju Chemical, Korea + alcohol]). After 24-h fasting, individual samples were orally administrated to the experimental mice 30 min prior to alcohol administration (2 g/kg). At 50 min after alcohol administration, the liver samples were collected, snap frozen, and stored at −80°C until analysis.

Measurement of alcohol dehydrogenase (ADH) activity in liver

Subcellular fractionation of mouse liver was performed according to a previous report (Lodge & Lawrence 2003), with slight modification. Briefly, livers were homogenized in five volumes of 0.25 mol sucrose buffer (pH 7.4) at 4°C. The homogenate was centrifuged at 100 ×g for 10 min and the supernatant further centrifuged at 2000 ×g for 10 min, discarding the nuclear and mitochondrial pellets. The collected supernatant was again centrifuged at 250 000 ×g for 1 h to obtain cytosolic ADH proenzyme. ADH activity was determined using the methods described in previous reports (Lebsack et al. 1976 and Shin et al. 1998). ADH activity was measured spectrophotometrically by the increase in absorbance at 340 nm of nicotinamide adenine dinucleotide (NADH) produced from NAD+. The assay was conducted at 37°C in a total volume of 3.0. The reaction mixture was composed of 0.1 mol Tris-HCl buffer (pH 8.5), 2.6 mL; 0.2 mol ethanol, 0.1 mL as a substrate; 0.05 mol semicarbazide HCl, 0.1 mL; 0.1 mol NAD (in 0.01 mol HCl), 0.02 mL; ADH proenzyme, 0.1 mL; and rotenone as an inhibitor. ADH activity was calculated in U/mg protein as specific activity, considering that 1 unit equals the formation rate of 1 µmol coenzyme for 1 min.

Data analysis

SAS 1998 (SAS Institute Inc., Cary, NC, US) was used for statistical analysis. The means and standard deviation were calculated for all treatment groups. We performed analysis of variance (anova). Any statistical significance between groups was analyzed with Duncan's Multiple Range Test.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Amino acid and fatty acid composition of silkworm pupae extracts
  7. Acknowledgments
  8. References

Amino acid and fatty acid composition of silkworm pupae extracts

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Amino acid and fatty acid composition of silkworm pupae extracts
  7. Acknowledgments
  8. References

Silkworm pupae are a nutritious food with high protein content. To determine their amino acid composition, the total pupae protein extract was hydrolyzed with hydrochloric acid, and free amino acids were analyzed by HPLC (Shiomi et al. 1998). Based on quantitative analysis using standards, the silkworm pupae sample contained relatively high levels of glutamic acid and histidine (Table 1). When the age of the silkworm pupae was considered, older silkworm pupae tended to have increased levels of threonine and serine, and reduced histidine levels (Table 2).

Table 1.  Amino acid content of silkworm pupae obtained from pupa period day 12–13 (Unit: mass %)
Amino acidMass (%)
Aspartic acid 1.5
Threonine 3.3
Serine 5.2
Glutamic acid 18.3
Proline 4.1
Glycine 7.3
Alanine 10.2
Cysteine 1.5
Valine 5.7
Methionine 5.0
Isoleucine 2.7
Leucine 3.5
Tyrosine 6.2
Phenylalanine 3.0
Histidine 14.6
Lysine 4.5
Arginine 3.6
Table 2.  Amino acid content changes depending on age of silkworm pupae (Unit: mass %)
Amino acidDay 7Day 10Day 13
  1.  

    The ratio of each amino acid to total amino acids in mass was calculated to determine content ratio (%) of individual amino acids in total protein.

Aspartic acid 1.5 2.2 6.0
Threonine 4.6 8.5 9.7
Serine 3.4 9.0 10.6
Glutamic acid 18.9 21.5 18.1
Proline 6.1 4.4 3.4
Glycine 5.2 7.0 6.1
Alanine 6.4 3.8 5.1
Cysteine 0.2 0.6 0.4
Valine 4.2 4.0 2.8
Methionine 6.5 6.4 5.0
Isoleucine 2.3 1.8 2.1
Leucine 2.4 2.1 2.5
Tyrosine 6.2 6.8 6.7
Phenylalanine 2.6 1.4 1.6
Histidine 25.1 16.0 13.8
Lysine 2.9 3.1 3.9
Arginine 1.3 1.5 2.5

Physical and chemical data of fatty acid products obtained from pupae using NMR analysis

The following are the physical and chemical data of the materials of products A, B, C, D and E obtained from pupae as described in Materials and Methods:

Material A (α-linolenic acid methyl ester): colorless liquid: UV (MeOH) lmax < 200 nm; IR (KBr plate) nmax 3011, 2930, 2855, 1742, 1652, 1462, 1436, 1197, 1172, 722 cm−1; 1H NMR δ 5.43–5.29 (6H, m), 3.67 (3H, s), 2.81 (4H, br t, J= 6.4 Hz), 2.31 (2H, t, J= 7.6 Hz), 2.09 (2H, q, J= 7.2 Hz), 2.07 (2H, quin, J= 7.2 Hz), 1.63 (2H, m), 1.37–1.30 ((8H, m), 0.98 (3H, t, J= 7.2 Hz) ppm; 13C NMR δ 174.2, 132.0, 130.3, 128.34, 128.31, 127.8, 127.2, 51.7, 34.4, 29.9, 29.5, 29.43, 29.40, 27.5, 25.93, 25.85, 25.3, 20.9, 14.6 ppm.

Material B (palmitoleic acid methyl ester): colorless liquid, UV (MeOH) lmax <200 nm; IR (KBr plate) nmax 3005, 2926, 2855, 1743, 1462, 1435, 1197, 1171, 724 cm−1; 1H NMR δ 5.34–5.31 (2H, m), 3.65 (3H, s), 2.28 (2H, t, J= 7.2 Hz), 2.02–1.97 (4H, br q, J= 5.6 Hz), 1.62–1.55 (2H, m), 1.33–1.24 (16H, m), 0.87 (3H, t, J= 7.2 Hz) ppm; 13C NMR δ 174.3, 130.1, 129.8, 51.7, 34.4, 32.1, 30.1, 30.0, 29.5, 29.46, 29.42, 29.3, 27.6, 27.5, 25.3, 23.0, 14.5 ppm.

Material C (linoleic acid methyl ester): colorless liquid, UV (MeOH) lmax < 200 nm; IR (KBr plate) nmax 3009, 2927, 2855, 1743, 1436, 1196, 1171, 724 cm−1; 1H NMR δ 5.43–5.30 (4H, m), 3.68 (3H, s), 2.78 (2H, br t, J= 6.8 Hz), 2.31 (2H, t, J= 7.2 Hz), 2.06 (4H, br q, J= 6.8 Hz), 1.63 (2H, m), 1.40–1.28 (14H, m), 0.90 (3H, t, J= 7.6 Hz) ppm; 13C NMR δ 174.3, 130.3, 130.1, 128.1, 128.0, 51.7, 34.4, 31.9, 29.9, 29.7, 29.50, 29.46, 29.44, 27.55, 27.53, 26.0, 25.3, 22.9, 14.5 ppm.

Material D (oleic acid methyl ester): colorless liquid, UV (MeOH) lmax < 200 nm; IR (KBr plate) nmax 3004, 2925, 2854, 1744, 1464, 1436, 1196, 1171, 723 cm−1; 1H NMR δ 5.38–5.29 (2H, m), 3.66 (3H, s), 2.30 (2H, t, J= 7.2 Hz), 2.03–1.99 (4H, br q, J= 6.0 Hz), 1.62 (2H, m), 1.36–1.26 (20H, m), 0.88 (3H, t, J= 7.2 Hz) ppm; 13C NMR δ 174.2, 130.0, 129.8, 51.7, 34.4, 32.2, 30.1, 30.0, 29.8, 29.64, 29.48, 29.45, 29.4, 27.53, 27.5, 26.0, 25.3, 23.0, 14.5 ppm.

Material E (palmitic acid methyl ester): white solids, mp 26–27°C; UV (MeOH) lmax <200 nm; IR (KBr pellet) nmax 2952, 2917, 2849, 1742, 1473, 1463, 1175, 730 cm−1; 1H NMR δ 3.67 (3H, s), 2.30 (2H, t, J= 7.6 Hz), 1.62 (2H, m), 1.33–1.25 (24H, m), 0.88 (3H, t, J= 6.8 Hz) ppm; 13C NMR δ 174.3, 51.7, 34.4, 32.2, 30.01 (2C), 30.00, 29.98 (2C), 29.9, 29.8, 29.7, 29.6, 29.5, 25.3, 23.0, 14.5 ppm.

Fatty acid content of silkworm oil

Silkworm oil extracts were decolorized and deodorized with activated carbon. The oil thus obtained was almost colorless, transparent and viscous. The oil was free from the innate smell of the silkworm pupae. NMR analysis of this oil revealed that the omega-3 fatty acid content was 41.2%. To analyze the structures and relative ratios of the fatty acids in the silkworm pupae oil, it was hydrolyzed with sodium hydroxide, methylated and different fatty acids were separated by HPLC. The structures of the separated and methylated fatty acids were analyzed using spectroscopic methods such as infrared ray and NMR. The fatty acid content ratio was determined by individually weighing the separated, methylated fatty acids after HLPC. The main fatty acids contained in silkworm pupae oil were α-linolenic acid (ω-3 fatty acid), linoleic acid, oleic acid, palmitoleic acid and palmitic acid, and their mass ratios were 42.3:7.5:24.7:1.2:24.3, respectively. In addition, GC-MS was used for the further separation of fatty acids in methyl ester form, and their content ratios were determined. Using this method, stearic acid and eicosapentaenoic acid (EPA) were additionally detected, comprising 8.6% and 0.3% of total fatty acids, respectively. Overall, the fatty acid content of silkworm pupae oil is as follows: (α-linolenic acid [ω-3 fatty acid]+ linoleic acid) (49.0%), oleic acid (19.9%), palmitoleic acid (2.5%), palmitic acid (19.7%), stearic acid (8.6%) and EPA (0.3%). Interestingly, the presence of EPA in our sample distinguishes it from the fatty acid contents of the Brazilian silkworm (Pereira et al. 2003). In addition, the percentage of α-linolenic acid (ω-3 fatty acid) in our sample was 41.2%, which was higher than that of the Brazilian silkworm. Therefore, based on HPLC and GC-MS analysis, our silkworm pupae samples contain 0.3% EPA, and predominant fatty acids such as stearic acid, α-linolenic acid (ω-3 fatty acid), linoleic acid, oleic acid, palmitoleic acid and palmitic acid. These main fatty acids are bound to glycerol in the form of fatty acid glycerol esters. The content ratio of unsaturated to saturated fatty acids is 71.7:28.3 (Table 3). The structures of these main fatty acids and EPA are shown in Figure 1. Although pupae age does not greatly affect the overall fatty acid content, there is an increase in EPA content with age. The amount of α-linolenic acid (ω-3 fatty acid) ranges from 36% to 39% (Table 4).

Table 3.  Main fatty acids and their content ratios in silkworm pupae obtained from pupal period days 12–13 of different ages (GC-MS analysis)
Fatty acidContent ratio of fatty acid (%)
  • Due to overlaps in GC analysis, the relative ratios of α-linolenic acid and linoleic acid were calculated based on their masses after separation by high-pressure liquid chromatography. GC-MS, gas chromatography – mass spectroscopy.

α-linolenic acid (ω-3 fatty acid) 41.6
Linoleic acid 7.4
Oleic acid 19.9
Palmitoleic acid 2.5
Stearic acid 8.6
Palmitic acid 19.7
Eicosapentaenoic acid (EPA) 0.3
Table 4.  Main fatty acids and their content ratios in silkworm pupae of different ages (GC-MS analysis)
Fatty acidFatty acid content at different ages (%)
Day 7Day 10Day 13
  • Due to overlaps in GC analysis, the relative ratios of α-linolenic acid, linoleic acid, and oleic acid were calculated based on their masses after separation by high-pressure liquid chromatography. GC-MS, gas chromatography – mass spectroscopy.

α-linolenic acid (ω-3 fatty acid) 36.4 38.9 38.6
Linoleic acid 8.2 5.7 8.7
Oleic acid 21.1 24.3 21.8
Palmitoleic acid 2.0 1.7 1.8
Stearic acid 9.6 8.3 8.8
Palmitic acid 22.6 21.0 20.0
Eicosapentaenoic acid (EPA) 0.1 0.3

Purification of flavone and vitamin from silkworm pupae extracts

The aqueous phase of the silkworm pupae extract (2.5 kg) was purified using C18 chromatography, and then subjected to silica gel MPLC. A quercetin diglucoside in the form of a flavonol (17 mg) was obtained from the sample. The chemical structure was analyzed by NMR spectroscopy such as 1H NMR, 13C NMR, COSY, HSQC, HMBC and ROESY. The structure derived from the NMR data is illustrated in Figure 2. This substance is a glucoside of quercetin, which is known to have a strong antioxidant effect. It is not found in the mulberry tree, the main food source of silkworms, but is present in other plants such as red onion.

Figure 2. Structure of quercetin diglucoside (a) and vitamin B2 (b) identified from silkworm extracts, and their nuclear magnetic resonance data.

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The following description relates to a fluorescent material obtained in the 5:5 H2O/MeOH elution from C18 chromatography of the aqueous phase. It is a yellowish solid, and is strongly fluorescent in solvents such as methanol. The structure of this substance was analyzed as described above, using NMR spectroscopy such as 1H NMR, 13C NMR, COSY, HSQC, HMBC and ROESY. It was identified as riboflavin (vitamin B2) (Fig. 3). Vitamin B2 is essential for the synthesis of the oxidoreductase enzyme, flavin adenine dinucleotide (FAD), in the human body (Fig. 4). Its deficiency is associated with dermatitis, dizziness, hair loss, growth retardation, indigestion, anemia, diarrhea, fatigue, insomnia, mental disorders, cataract, corneitis, eye redness, excess tear production, and light sensitivity. In addition, vitamin B2 levels are severely depleted by excessive alcohol consumption, antibiotic administration and stress. This is the first study to identify vitamin B2 in silkworm pupae, which suggests that the silkworm pupae may have high nutritional value as a natural product. The recommended daily vitamin B2 requirement for adults is 1.6 mg and 1.2 mg for men and women, respectively. Therefore, consumption of silkworm pupae would provide a convenient source of vitamin B2 in a natural form.

Figure 3. Association between vitamin B2 and the oxidation-reduction enzyme cofactor flavin adenine dinucleotide (FAD). FAD is derived from riboflavin and also contains adenosine monophosphate and a pyrophosphate linkage. FAD is involved in energy metabolism by delivering two hydrogens to electron transport chain.

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Figure 4. Effect of silkworm extracts, Drink and Alcodex on hepatic alcohol dehydrogenase (ADH) activities in acute alcohol-treated mice. Alcohol was orally administered at 30 min after oral administration of samples in all groups except for NC. The liver was harvested 50 min after ethanol treatment for hepatic ADH activity. Each bar represents the mean ± SEM. Abbreviations: NC (negative control); distilled water (DW), PC (positive control); DW + alcohol, Alcodex 10-fold dilution of Alcodex + alcohol, Drink; 10-fold dilution of Dawn808TM+ alcohol, KW1-KW4: Silkworm extract (KW1: 0.01 mg/mL, KW2: 0.05 mg/mL, KW3: 0.1 mg/mL, KW4: 0.5 mg/mL) + alcohol. *P < 0.05 versus PC, **P < 0.001 versus PC.

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The nutritional and medicinal value of silkworm pupae has been known for a long time. Nevertheless, no previous studies have been undertaken to identify the substances responsible for these effects. This study established effective purification methods for silkworm pupae oil and determined its fatty acid composition by GC-MS. This oil was found to comprise more than 40% α-linolenic acid, which is effective in the prevention of cardiovascular disease, cancer, and dementia, and reduction of cholesterol levels. This is the first report of these results. In addition, amino acid analysis confirmed that silkworm pupae are a high-protein food with high nutritional value. We found that silkworm contains high levels of both the antioxidant quercetin diglucoside and the nutritionally important vitamin, vitamin B2. Interestingly, these nutrients do not exist in white mulberry, the main diet for silkworms. Consumption of silkworm pupae could supplement vitamin B2 intake, which can be important to prevent the serious effects of vitamin B2 deficiency. Vitamin B2 supplementation is especially important, because it can be easily lost through alcohol consumption, antibiotic administration and stress, as mentioned previously. This is the first time that vitamin B2 has been found in silkworm pupae, a finding which suggests that it may have high nutritional value as a natural product. Because 100 mg of vitamin B2 was purified from 2.5 kg silkworm pupae, moderate consumption of silkworm pupae would be sufficient to meet the daily vitamin B2 requirement, which is 1.6 mg and 1.2 mg for adult men and women, respectively.

Effect of silkworm extract on ADH activity in mouse liver

Figure 4 showed effect of silkworm extract on ADH activity in the liver of alcohol-treated mice. ADH activity in treatment groups was presented as the optical density (OD) value of the positive control (PC0 group. The OD values were measured 50 min after alcohol administration. The ADH activity of the Drink group was shown to be significantly higher than that of the PC (P < 0.05). The ADH activity of KW1, KW2, KW3 and Alcodex was not significantly different from that of the PC. However, KW1 showed the highest ADH activity, which is significantly higher than that of PC (P < 0.001) (Fig. 1). Effect of silkworm, Dwan808 and Alcodex on ADH activity was diminished at 500 min after alcohol treatment (Data not shown). When alcohol is orally exposed into the body, alcohol is converted into acetaldehyde by ADH, which is the first step for alcohol metabolism. Taken together, the present study demonstrated that the silkworm pupae extracts have beneficial effect on alcohol detoxification in the animal indicating that the extracts can be used as a medicinal material to reduce hangover

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