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A Combination of Ultrasound-Assisted Extraction wi

Xiaojing Lin 1, Xikai Shu 1,2, Yan-ling Geng 1, Feng Liu 1, Jian-hua Liu 1, Xiao Wang 1*

 

1 Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China

2 College of Life Science, Shandong Normal University, 88 culture east road, Jinan, Shandong 250014, China

*Correspondence: Dr. Xiao Wang, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, 250014 Jinan, P. R. China. E-mail: wxjn1998@126.com, Fax: +86-531-82964889

 

Abstract An efficient method was built successfully for the rapid separation and purification of 4-trans-10-hydroxy-2-decenoic acid (10-HDA) from royal jelly by ultrasound-assisted extraction (UAE) in combination with high-speed counter-current chromatography (HSCCC). The UAE conditions including percent ethanol, extraction time and solvent ratio were optimized with an orthogonal test. The 10-HDA in the crude extract was separated and purified by HSCCC with a two-phase solvent system composed of petroleum ether–aether–methanol–water (1:1:0.5:1.5, v/v). 98.2 mg of 10-HDA was obtained from 1.0 g of crude extract in one-step separation with the purity of 99.1% as determined by HPLC. The chemical structure of 10-HDA was identified by ESI-MS, 1H-NMR and 13C-NMR.

 

Keyword: Royal jelly / 10-HDA / Ultrasound-assisted extraction / High-speed counter-current chromatography


 

Introduction

Royal jelly (RJ) is secreted from the hypopharyngeal and mandibular glands of worker honeybees (Apis mellifera L.) and is fed to larvae and queen honeybees in the colony. RJ has been used in folk medicine and appreciated as the best natural nutriment all over the world. The chemical compositions of RJ were studied well including proteins, vitamins, minerals, sugars, free amino acid and fatty acids et. al. Among the compositions, 4-trans-10-hydroxy-2-decenoic acid (10-HDA), which was firstly indentified in 1957 by Butenadt and Remboldand, is the major fatty acid in RJ [1].

10-HDA has attracted much attention of researchers due to its various pharmacological effects, such as anti-bacterial [2], anti-inflammatory [3], anti-tumor [4, 5], anti-depression [6], antioxidant effect [7], promoting neurogenesis [8]. 10-HDA is suggested as a freshness parameter for royal jelly [9] and also sex pheromone beyond 9-ODA for communication in the colony [10]. As a unique unsaturated fatty acid, the content of 10-HDA has been adopted as a marker for RJ and is currently used as a means to evaluate bee products containing RJ [11].

The 10-HDA has been obtained by isolating from the RJ with conventional methods, such as shaking extraction, chloroform/methanol extraction and aether extraction, followed by separation and purification of column chromatography [12]. However, these conventional methods consumed longer extraction time, large quantities of organic solvents, and often offered low recoveries of the target products. The application of ultrasound-assisted extraction (UAE) offers many advantages including good extraction efficiency, reduction of temperature and time for extraction, and environmental-friendliness. The cavitation phenomena and high shear forces, induced by propagation of ultrasound pressure waves are involved in the extraction enhancement of ultrasound [13]. UAE has been successfully used for the effective extraction of various natural products such as herbal, oil, protein and bioactive compounds from plant materials [14]. High-speed counter-current chromatography (HSCCC), being as a kind of liquid-liquid partition chromatography, eliminates irreversible adsorption of samples on solid support in conventional column chromatography and offers excellent recovery of target compounds. It has been successfully applied to the separation and purification of different kinds of natural products [15]. In this study, we reported a simple and efficient method for the separation and purification of 10-HDA from RJ by UAE combined with HSCCC directly. The critical parameters, including UAE conditions and solvent system of HSCCC, were optimized.

Experimental

Reagents and materials

Royal jelly was purchased from the local bee keepers and weighted 500 g. Petroleum ether (60-90℃), aether, methanol and ethanol were all of analytical grades (Guangcheng Chemical Factory, Tianjin, China). Methanol used for HPLC analysis was of chromatographic grade and purchased from Tianjin Siyou Special Reagent Factory, Tianjin, China. The water was treated with a Milli-Q water purification system (Millipore, USA) and then was used for all solutions and dilutions throughout the whole experimental process.

Apparatus

HSCCC was carried out using the Model TBE-300A commercial instrument (Shanghai, Tauto Biotech, China). The apparatus was equipped with three PTFE preparative coils (internal diameter of tube, 1.6 mm; total volume, 260 mL) and a 20-mL sample loop. The β-values of this preparative column range from 0.47 at the internal to 0.73 at the external (β=r/R, where r is the rotation radius or the distance from the coil to the holder shaft and R (R=7.5 cm) is the revolution radius or the distance between the holder axis and the central axis of the centrifuge). The solvent was pumped into the column with Model NS-1007 constant-flow pump (Beijing Emilion Science & Technology, Beijing, China). Continuous monitoring of the effluent was carried out with Model 8823AUV detector (Beijing Emilion Science & Technology) at 210 nm. Model 3057 portable recorder (Yokogawa, Sichuan Instrument Factory, Chongqing, China) was used to draw the chromatogram. A manual sample injection valve with a 10-mL loop (for the preparative HSCCC) (Tianjin High New Science Technology Company, Tianjin, China) was used to introduce the sample into the column.

HPLC system used throughout this study consisted of Shimadzu SPD M20A photodiode array detection (PDA), Shimadzu CBM-20A system controller, Shimadzu LC-6AD 600 pump, and LC-solution workstation (Shimadzu, Japan).

The ultrasonic cleaning bath with a working frequency of 40 kHz and the power of 300W ( type of SB5200D, NingBo Scientz Biotechnology Co. Ltd, China). The bath was a rectangular container (30 cm × 24 cm × 15 cm).

Optimization of UAE conditions

In order to determine a suitable extraction condition, an orthogonal test design L9 (3)3 was employed where the percent ethanol, extraction time and solvent ratio were considered to be the major factors for effective extraction. Combinations of the three different levels of each factor were listed in Table 1. In each test, 0.2 g of the royal jelly was added to a 10 mL tube and the extractions were carried out under different UAE conditions according to the pre-designed trial. After extraction, sample extracts were collected for HPLC analysis. The crude extract for HSCCC was prepared according to the optimal conditions and then evaporated to dryness under reduced pressure.

Selection of two-phase solvent system

The solvent system was selected according to the partition coefficient (K) of target compound. The K values were determined by HPLC as follows: approximately 2 mg of crude extract was added to the test tube, to which 2 mL of each phase of the two-phase solvent system was added. The test tube was shaken violently for several minutes. Then an equal volume of each phase was analyzed by HPLC to obtain the partition coefficients

HSCCC separation procedure

In the present study, the HSCCC experiments were performed with a two-phase solvent system of petroleum ether–aether–methanol–water (1:1:0.5:1.5, v/v). Solvent mixture was thoroughly equilibrated in a separation funnel by repeatedly vigorously shaking at room temperature. The two phases were separated shortly prior to use. The upper phase was used as the stationary phase, while the lower phase was used as the mobile phase. The sample solution was prepared by dissolving the dried extract in the mixture solution of lower phase and upper phase (1:1, v/v) of the solvent system.

HSCCC separation was performed as follows: the multiplayer coiled column was first filled entirely with the upper organic phase as the stationary phase. The lower aqueous phase was then pumped into the head end of the column at a suitable flow-rate of 2 mL/min, while the apparatus was rotated at a speed of 800 rpm. After hydrodynamic equilibrium was reached, as indicated by a clear mobile phase eluting from the tail outlet, the sample solution was injected into the column through the inject valve. The effluent of the column was continuously monitored with a UV detector at 210 nm and the chromatogram was recorded. Each peak fraction was collected according to the elution profile and determined by HPLC. After the separation was completed, retention of the stationary phase was measured by collecting the column contents by forcing them out of the column with pressurized nitrogen gas.

HPLC analysis and identification of HSCCC fractions

The crude extract and the fractions from the preparative HSCCC separation were analyzed by HPLC. Chromatographic separations were accomplished with a Shim-pack VP-ODS column (250 mm × 4.6 mm, I.D.) at room temperature. The mobile phase was methanol-acetic acid water (55:45, v/v) and performed at a flow-rate of 1.0 mL/min. The effluent was monitored at 209 nm by a photodiode array detector. Routine sample calculations were made by comparison of the peak area with that of the standard.

Results and discussion

Optimization of UAE conditions

The first step using the UAE is to optimize the operating conditions to obtain an efficient extraction of the target compounds. We used an orthogonal L9 (3)3 design to optimize the conditions of extraction including the concentration of solvent, extraction time and solvent ratio which are generally considered to be the most important factors. The results shown in Table 1 indicated that there were great differences in 10-HDA yield among each set of UAE conditions. Based on the analysis in Table 2, the influence on the mean extraction yields of 10-HDA decreases in the order: A > C > B according to the R values. The best UAE condition was A2B2C3 according to the extraction of yields of 10-HDA and the maximum yield of 10-HDA was 20.00 mg/g.

The concentration of ethanol was found to be the most important factor for yields of 10-HDA. The results in Table 2 showed that the yield of 10-HDA is higher with 95% ethanol than with 80% ethanol. However, the extraction yield decreased sharply when extracted with anhydrous alcohol. From these results, it is clear that the proper amount of water in alcohol improved the extraction efficiency, probably due to the proper effects on the absorption, transferability of ultrasonic energy and good solubility of 10-HDA. Therefore, 95% ethanol showed the best extraction solvent and was chosen as the isolation solvent in the following experiments. The solvent ratio also had significant influence on the extraction yields of 10-HDA. It is seen in Table 2 that the yield of 10-HDA increased with the solvent ratio because 10-HDA could be easily dissolved into extraction solvent. Extraction time showed slight influence on the yields of 10-HDA in comparison with the other two factors. The yield increased with extraction time in some extent. But the increasing ultrasonic energy of long extraction time may destroy or change the structure of 10-HDA. Therefore, extraction time of 10 min was adopted in the present study.

These results indicated that the optimal conditions for extraction of 10-HDA by UAE were 95% ethanol, 10 min of extraction time and 30/1 (mL/g) of solvent ratio. Under the optimum UAE conditions, the extraction yields of 10-HDA were 20.00 mg/g. Therefore, UAE can be used as a rapid, efficient and reliable method for extraction of 10-HDA from royal jelly.

Selection of HSCCC separation conditions

The choice of a suitable two-phase solvent system is the first and critical step in a HSCCC experiment. To achieve a successful separation using HSCCC, the suitable solvent system should provide an ideal range of partition coefficient (K, 0.5–2) for 10-HDA. In our research, the K values were 0.11 and 0.27 respectively when the volume ratios of petroleum ether-aether-methanol-water were 1:1:1.5:0.5 and 1:1:1:1. The K value was 1.05 when petroleum ether-aether-methanol-water with the volume ratio of 1:1:0.5:1.5 was used as the two-phase solvent system and good separation results could be achieved.

Purification of 10-HDA by HSCCC

The crude extract (1g) was separated and purified in one step by the preparative HSCCC with petroleum ether-aether-methanol-water (1:1:0.5:1.5, v/v) as a solvent system (Fig. 1). The retention of the stationary phase was 60.0% and the separation time was within 4 h in each separation run. The HSCCC fractions were analyzed by HPLC and the HPLC chromatograms of the collected fractions are shown Fig. 2. The separation produced 98.2 mg of 10-HDA at 99.1% purity according to HPLC analysis. These results demonstrated the high resolving power of HSCCC.

Identification of 10-HDA

The structural identification of 10-HDA was carried out by ESI-MS, 1H-NMR and 13C-NMR spectra. The ESI-MS data is m/z 187.3 [M+H]+. 1H-NMR (600 MHz, CDCl3) δ: 7.06 (2H, m, H-3), 5.83 (1H, d, J=15.6 Hz, H-2), 3.64 (2H, m,H-10), 2.22 (2H, m, H-4), 1.56 (2H, m, H-9), 1.48 (2H, m, H-5), 1.37 (6H, m, H-6, H-7, H-8). 13C-NMR (150 MHz, CDCl3) δ: 171.6 (C-1), 152.1 (C-3), 120.7 (C-2), 62.9 (C-10), 32.6 (C-9), 32.2 (C-4), 29.1 (C-7), 29.0 (C-6), 27.7 (C-5), 25.6 (C-8). The 1H-NMR and 13C-NMR data were accorded with the literature [16], indicating the structural identification of 10-HDA.

Conclusions

The optimal conditions to extract 10-HDA by UAE from royal jelly were 95% ethanol, 10 min of extraction time and 30/1 (mL/g) of solvent ratio and under the optimal conditions the yield of 10-HDA was 20.0 mg/g. The UAE crude extract was separated and purified by HSCCC with a two-phase solvent system composed of petroleum ether–aether–methanol–water (1:1:0.5:1.5, v/v). 98.2 mg of 10-HDA was obtained from 1.0 g of crude extract in one-step separation with the purity of 99.1% as determined by HPLC. The chemical structure of 10-HDA was identified by ESI-MS, 1H-NMR and 13C-NMR.

Acknowledgements

This work was supported by grants from the Natural Science Foundation of China sponsored by the Ministry of Science and Technology of China and financial supports of Shandong Province.

The authors have declared no conflict of interest.

Reference

[1] Sabatini AG, Marcazzan GL, Caboni MF, Bogdanov S, Almeida-Muradian LB (2009) J ApiPro ApiMed Sci 1:16-21.

[2] Blum MS, Novak AF, Taber S3rd (1959) Science 130:452-453.

[3] Takahashi K, Sugiyama T, Tokoro S, Neri P, Mori H (2012) Cell Immunol 273:73-78.

[4] Townsend GF, Morgan JF, Tolnai S, Hazlett B, Morton HJ, Shuel RW (1960) Cancer Res 20:503-510.

[5] Yang XY, Yang DS, Zhang W, Wang JM, Li CY, Ye H, Lei KF, Chen XF, Shen NH, Jin LQ, Wang JG (2010) J Ethnopharmacol 128:314-321.

[6] Ito S, Nitta Y, Fukumitsu H, Soumiya H, Ikeno K, Nakamura T, Furukawa S (2012) Evid Based Complement Alternat Med 139140. [Epub 2011 Jul 24]

[7] Liu JR, Yang YC, Shi LS, Peng CC (2008) J Agric Food Chem 56:11447-11452.

[8] Hattori N, Nomoto H, Fukumitsu H, Mishima S, Furukawa S (2007) Biomed Res 28: 261-266.

[9] Antinelli JF, Zeggane S, Davico R, Rognone C, Faucon JP, Lizzani L (2003) Food Chemistry 80:85-89.

[10] Brockmann A, Dietz D, Spaethe J, Tautz J (2006) J Chem Ecol 32:657-667.

[11] Genc M, Aslan A (1999) J Chromatogr A 839:265-268.

[12] Ferioli F, Marcazzan GL, Carbon MF (2007) J Sep Sci 30:1061-1069.

[13] Ji JB, Lu XH, Cai MQ, Xu ZC (2006) Ultrason Sonochem 13:455-462.

[14] Vilkhu K, Mawson R, Simons L, Bates D (2008) Innov Food Sci Emerg 9:161-169.

[15] Fang L, Liu YQ, Zhuang HY, Liu W, Wang X, Huang LQ (2011) J Chromatogr B 879:3023-3027.

[16] Noda N, Umebayashi K, Nakatani T, Miyahara K, Ishiyama K (2005) Lipids 40:833–838.


 

Figures

Fig. 1. HSCCC chromatogram of the crude sample of 10-HDA. Solvent system: petroleum ether-aether-methanol-water (1:1:0.5:1.5, v/v); revolution speed: 800 r/min; flow rate: 2.0 mL/min; sample size: 1g; UV detection wavelength: 210 nm; retention of stationary phase: 60%.

 

 

 

 

 

 


 

Fig. 2. (A) HPLC chromatogram of the crude extract from preparative UAE; (B) HPLC analysis and UV spectrum of 10-HDA purified by HSCCC. Conditions: Shim-pack VP-ODS column (250 mm × 4.6 mm i.d.); column temperature: 25℃; mobile phase: methanol-acetic acid water (55:45, v/v); flow-rate: 1.0 mL/min; detection wavelength: 209 nm; injection volume, 10 μL.

Table 1

Results of L9 (3)3 orthogonal test design.

 

Test No.

Factors

Yield (mg/g)a

Percent ethanol (%)

Extraction time (min)

Solvent ratio (mL/g)

1

A1

80

B1

5

C1

10:1

13.90

2

A1

80

B2

10

C2

20:1

17.35

3

A1

80

B3

15

C3

30:1

13.45

4

A2

95

B1

5

C2

20:1

18.50

5

A2

95

B2

10

C3

30:1

20.00

6

A2

95

B3

15

C1

10:1

17.50

7

A3

100

B1

5

C3

30:1

20.70

8

A3

100

B2

10

C1

10:1

16.10

9

A3

100

B3

15

C2

20:1

18.10

a Extraction yield (mg/g) = the amount of 10-HDA /sample mass.

Table 2

Analysis of L9 (3)3 orthogonal test results.

 

 

10-HDA yield (mg/g)

 

A

B

C

K1

44.700

53.100

47.500

K2

56.001

53.450

53.950

K3

54.900

49.050

54.150

k1

14.900

17.700

15.833

k2

18.667

17.817

17.983

k3

18.300

16.350

18.050

R

3.767

1.467

2.217

Optimal Level

A2

B2

C3

KiA = Σextraction yield at Ai; kiA =KiA/3; RiA = max{ kiA }-min{ kiA}.

 

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