Extraction of 1-Deoxynojirimycin (DNJ) from Silkworms as

Functional Foods for Lower Postprandial Glucose

Chaluntorn Vichasilp1+

, Pichet Wechavitan1, Onanong Poungchompu

1 and Putthaporn Wiwacharn

2

1 Faculty of Natural Resources, Rajamangala University of Technology Isan, Sakon Nakhon 47160, Thailand

2Queen Sirikit Sericulture center, Sakon Nakhon Province, 47000, Thailand

Abstract. 1-deoxynojirimycin (DNJ) is natural anti -glucosidase found in mulberry leaves and silkworms.

Although, DNJ contents and its activities in a mulberry leaves have been investigated for decades but DNJ

content in silkworms and its application are relative lack of researches. In this study, DNJ content, -

glucosidase inhibitory activity and a potential DNJ extraction were investigated. To extract DNJ, the aqueous

two-phase systems (ATPS) was applied to separate DNJ from other compounds in silkworms with response

surface methodology (RSM). The important 4 factors; ammonium sulfate (X1, 20-50% w/w), ethanol

concentration (X2, 20-80% w/w), ultrasound power (X3, 120-300 watt) and time (X4, 10-20 min) was

examined. It was found the highest extraction recovery (95%) obtained when extracted with ammonium

sulfate of 9.5%, ethanol concentration of 100 %, ultrasonic powder of 390 watt and time of 15 min.

Keywords: 1-deoxynojirimycin, silkworms, extraction, response surface methodology

1. Introduction

DNJ is a piperidine which has potency -glycosidase inhibitor abundant in mulberry leave. For decades,

DNJ have more attention from scientists for its nutraceutical properties. Mulberry DNJ showed a potency to

suppress postprandial hyperglycemic [1]-[4]. In Thailand, most of silkworm production were used for

sericulture industries and their by-products (pupae) were sent to food sector. In general, silkworms were

served as main dishes in some areas in particular people in northeast of Thailand. Due to its unique dish and

special taste, recently they were introduced as amazing dish for tourist. Silkworms could be cooked with fire,

streaming, and hot chilly seasoning. At present, there are high demands of silkworm pupae as a good source

of high protein and fat [5].

For DNJ contain, accumulation of DNJ in silkworms (Bombyx mori L.) was reported [2], [6], [7]. It was

found that silkworms cannot synthesis DNJ by them but could obtain DNJ by feeding mulberry leaves [7].

The high concentration of 2.7-fold was found in the mid-gut silkworms feed with mulberry leaves [2]. The

male larvae showed higher accumulation efficiency of DNJ than the females and DNJ was shown higher

tissue distribution in blood, digestive juice, and alimentary canal but not in silk gland. Silkworm’s DNJ

showed medical effects for lower blood glucose in animal model [8].

Considers DNJ contain and it availability, silkworms is a potential material for use as functional foods.

However, silkworm itself contain high protein and fat, it could be oxidized by oxygen resulting in fatty

oxidation and reduce its qualities. Therefore, purification and the effective extraction method with high DNJ

content is needed. In this study, we investigated DNJ extraction in silkworms with an aqueous two-phase

systems (ATPS) method.

Corresponding author. Tel: +6642771460; fax: +6642771460

E-mail address: c.vichasilp@gmail.com

International Proceedings of Chemical, Biological and Environmental Engineering, Vol. 92 (2016)

DOI: 10.7763/IPCBEE. 2016. V92. 14

81

2. Experimental

2.1. Materials The silkworms was purchased in the markets in Sakon Nakhon province, Thailand. Silkworm pupae used

was in the form of edible whole body as selling for foods. The samples were quickly cleaned and dip into

boiled water to de-active proteinase enzyme. Then, the whole bodies were put in the hot air oven at 60oC for

12 hours. The dried silkworms were ground into the power and kept in -20oC until analysed.

2.2. DNJ determination

Filthy mg of silkworm powder was dissolved in ethanol (50% v/v.) then put in an ultrasonic bath for 5

min. The solution was centrifuged at 1000g/rpm before filling through 0.45 m syringes fillers (Sartorius

Stedim Biotech, Goettingen, Germany). Due to DNJ had no chromophore in the molecules, colour derivative

of DNJ is needed for determination. In this study, silkworm DNJ was treated with 9-fluorenylmethyl

chloroformate (FMOC-Cl) [9]. Briefly, DNJ solution of silkworm was mixed with 10 mL of 0.4 M

potassium borate buffer (pH 8.5). Twenty microliters of 5 mM FMOC-Cl in acetonitrile was added and kept

at 20oC for 20 min in a waterbath. 0.1 M glycine (Sigma, St. Louis, MO, USA) was added to terminate the

reaction. The mixture was diluted with 3. 950 mL of 0.1% (v/v) aqueous acetic acid (17.5 mM) to stabilize

the DNJ-FMOC, and filtered through 0.2-mm nylon syringe filter and 20 l was injected to HPLC. High

Pressure Liquid Chromatography (Shimadzu, Kyoto, Japan) coupled with photo diode array detector (SPD-

M20A, Shimadzu, Kyoto, Japan) at 254 nm with Ultrasphere C18 column were used to detect DNJ in

silkworms.

2.3. -Glucosidase inhibitory activity

-Glucosidase inhibitory activities in silkworm were analysed by method of Ma et al. [10]. Briefly, the

crude rat-intestine acetone powder (1g) was dissolved in 100 mM potassium phosphate buffer (pH 7.0) and

put in ultrasonic bath for 10 min. The obtained solution was centrifuged at 1000 rpm for 5 min and

supernatant was used as crude a glucosidase. To determine the activity, 10 l of 2 mM 4-nitrophenyl--D-

glucopyranoside in 100 mM potassium phosphate buffer was used as substrate in 96-well plates.

Consequently, 10 l of the silkworm solution and control (no silkworm solution) was added following

addition of crude enzyme. The reaction was done by incubation at 37oC for 20 min and then added 10 l of

2.0 M maleate-Tris-NaOH buffer to stop the reaction and absorbance (A) was measured at absorbance of 405

nm. The -Glucosidase inhibitory activity was calculated following equation.

-Glucosidase inhibitory activity = (Acontrol-Asample) × 100/Acontrol (1)

2.4. DNJ extraction

Due to silkworms containing high fat, aqueous two-phase systems (ATPS) was used for DNJ extraction.

ATPSs is potential method to extract bioactive compounds from matrices [11] with low expense and suitable

of scale up suitable for industrial [12]. The response surface methodology (RSM) was done by following

factors; ammonium sulfate (X1, 20-50% w/w), ethanol concentration (X2, 20-80% w/w), ultrasound power

(X3, 120-300 watt) and time (X4, 10-20 min). DNJ concentration and powder yield was investigated after

dried extract solution by freeze drier.

3. Research Results

3.1. DNJ determination With this condition, standard DNJ and in silkworm was clearly observed as shown in Fig. 1. DNJ content

in silkworm extracted powder was 75-105 mg/100g. Comparing to DNJ content in mulberry leaves (50-150

mg/100g) [13], DNJ in silkworms was considered as a good sources for functional food enriched DNJ.

3.2. -Glucosidase inhibitory activity DNJ content and -Glucosidase inhibitory activity were correlated by Pearson correlation. It was found

82

that lower correlation with R2 = 0.76 comparing to DNJ in mulberry leaves (R

2 = 0.84 of DNJ in mulberry

leaves [13]). The inhibitory activity involved the concentration of target compounds to its activities. In this

study, the lower activity of DNJ in silkworms could be affected by other compositions in silkworms such as

protein or fat. Therefore, to get high therapeutic effects, the effective extraction should be considered.

�(A)

�(B)

y = �.5081x + �.

R² .

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20

Silkworm DNJ (l/mg)

-Glu

cosi

da

sein

hib

ito

ry a

ctiv

ity (

%)

Fig. 1: Chromatogram of DNJ standard (A) and DNJ found in silkworms (B) and correlation of DNJ content and -

Glucosidase inhibitory activities in extracts of silkworm (n=30)

3.3. DNJ extraction The silkworm pupae having high DNJ content of (105mg/100g) was used to study with APTs extraction

with 4 important factors; ammonium sulfate concentration (X1) ethanol concentration (X2), ultrasonic power

(X3) and extraction time (X4). The code and level of each factor were shown in Table 1. After extraction

process, 2 phases of lipid layer and water layer were clearly observed. For DNJ contain, the water layer was

used and then concentrated in a rotary evaporator, dried in freeze drier and bound into powder. The DNJ

concentration and extraction yield were measured. The treatments and the results were shown in Table 2

Table 1: Code and real value of each factors.

As results in Table 3, the coefficient that have significantly difference (p≤0.05) of each factor was used

to conduct a regression model as following;

DNJ concentration (Y1) = 45.25-5.36X4+4.86X22-11.61X3X4 R

2 = 0.78 (2)

Extraction yields (Y2) = 23.07-2.61X12-4.09X2

2 R

2 = 0.64 (3)

The statistic results showed that the model with moderated accuracy could be obtained for Y1 (DNJ

concentration model). As shown in Fig. 2, concentration of ethanol was highly affected to purification of

DNJ extraction following by effects of ultrasonic power and extraction time.

In Fig. 3, it was found that the concentration of ammonium sulfate and ethanol concentration was

83

affected to extraction yields. To find the optimum points of DNJ concentration and extract yields, Response

optimizer in Minitab software was used with siting parameters as shown in Table 4. The results showed that

the highest DNJ concentration (100mg/100g; 95.2% extraction recovery) was obtained when extraction

condition was ammonium sulfate concentration of 9.5%, ethanol concentration of 100%, ultrasonic power of

390 watt and extraction time of 15 min. For extraction yield, the high yield (23g/100g) was received when

extraction conditions of ammonium sulfate of 36%, ethanol concentration of 53%, ultrasonic power of 250

watt and extraction time of 50 min, respectively.

Table 2: Experimental design and results of DNJ concentration and extraction yields

> 100

< 100

< 96

< 92

< 88

< 84

< 80

> 120

< 120

< 100

< 80

< 60

< 40

< 20

X2

X3X3

X4

Fig. 2: Response surface graph of DNJ concentration; X2: concentration of ethanol aqueous, X3: ultrasonic power and

X4: extraction time

84

Table 3: ANOVA of 2nd

order model for DNJ concentration (Y1) and extraction yield (Y2)

> 22

< 22

< 18

< 14

< 10

< 6

< 2

< -2

X1X2

Fig. 3: Response surface graph of extraction yields; X1: ammonium sulfate, X2: ethanol aqueous

Table 4: Parameters for response optimizer to get optimum point

4. Conclusion

Silkworm pupae contained high DNJ and have high correlation to -Glucosidase inhibitory activities.

The aqueous two-phase systems showed as a potential method to extract DNJ from silkworm pupae. Ethanol

concentration, ultrasonic power and extraction time affected DNJ purification whilst ammonium sulfate and

ethanol concentration affected to extraction yields. The highest DNJ concentration (95.2% extraction

recovery) was obtained when using ammonium sulfate concentration of 9.5%, ethanol of 100%, extraction

power of 390 watt and extraction time of 15 min. This study could be benefit to develop silkworm pupae as

functional foods for lower postprandial glucose.

5. Acknowledgements

This work was financial supported by National research council of Thailand year 2014 (Rajamangala

University of Technology Isan)

85

6. References

[1] M. Yagi, T. Kouno, Y. Aoyagi and H. Murai. The structure of moraoline, a piperidine alkaloid from Morus species.

Nippon Nougei Kagaku Kaishi. 1976, 50:571-572.

[2] N. Asano, T. Yamashita, K. Yasuda, K. Ikeda, H. Kizu, Y. Kameda, A. Kato, R. J. Nash, H. S. Lee and K. S. Ryu.

Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.).

Journal of Agricultural and Food Chemistry. 2001, 49:4208-4213.

[3] T. Kimura, K. Nakagawa, H. Kubota, Y. Kojima, Y. Goto, K. Yamagishi, S. Oita, S. Oikawa and T. Miyazawa.

Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood

glucose in humans. Journal of Agricultural and Food Chemistry. 2007, 55(14):5869-5874.

[4] W.H. Kong, S.H. Oh, Y. R. Ahn, K.W. Kim, J.H. Kim and S.W. Seo. Antiobesity effects and improvement of

insulin sensitivity by 1-deoxynojirimycin in animal models. Journal of Agricultural and Food Chemistry. 2008,

56:2613-2619.

[5] T. Hiroyuki, K. Mitsuaki and Y. Masayuki. Silkworm pupae (Bmobyx mori) are new source of high quality protein

and lipid. Journal of nutritional science and vitaminology. 2010, 56:446-448.

[6] K. Nakagawa, K. Ogawa, O. Higuchi, T. Kimura, T. Miyazawa and M. Hori. Determination of iminosugars in

mulberry leaves and silkworms using hydrophilic interaction chromatography-tandem mass spectrometry.

Analytical Biochemistry. 2010, 15 (2):217-222.

[7] Y. Hao, S. Xin-qin, Bo Sun, Y. Jing-jing, D. Zu-an, Z. Xiao-ling, C. Wei-zheng and W. Xiao-feng. Accumulation

of 1-deoxynojirimycin in silkworm, Bombyx mori L. Journal of Zhejiang University Science B. 2010, 11(4):286–

291.

[8] K. S. Ryu, H. S. Lee, S.H. Chung and P. D. Kang. An activity of lowering blood-glucose levels according to

preparative condition of silkworm powder. Korean journal of sericultural science. 1997, 39(1):79-85.

[9] J. Kim, S. Kim, H. S. Lee, I. Kim, M.Y. Ahn and K.S. Ryu. Determination of 1-deoxynojirimycin in Morus alba L.

leaves by derivatization with 9-fluorenylmethyl chloroformate followed by reversed-phase highperformance liquid

chromatography. Journal of Chromatography A. 2003, 1002:1611-1617.

[10] C. M. Ma, M. Hattori, M. Daneshtalab and L. Wang, Chlorogenic acid derivatives with alkyl chains of different

lengths and orientations: potent aglucosidase inhibitors. Journal of Medicinal Chemistry. 2008, 51:6188-6194.

[11] S. F. Shen, Z. D. Chang, J. Liu, X. H. Sun, X. Hu and H.Z. Liu. Separation of glycyrrhizic acid and liquiritin from

Glycyrrhiza uralensis Fisch extract by three-liquidphase extraction systems. Separation and Purification

Technology. 2007, 53:216-223.

[12] A. Greve and M. R. Kula, Cost structure and estimation for the recycling of salt in a protein extraction process

system, Bioprocess and Biosystems Engineering. 1990, 6:173–177.

[13] C. Vichasilp, K. Nakagawa, P. Sookwong, O. Higuchi, S. Loumoungkong and T. Miyazawa. Development of 1-

deoxynojirimicin (DNJ)-enriched mulberry tea and use of response surface methodology to optimize tea-making

conditions for highest DNJ extraction. LWT - Food Science and Technology. 2012, 45 (2):226-232.

86