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Rapid Analysis of Oxyresveratrol-4-3'-β-O-D- Diglycosie in Branch Bark of Mulberry by HPCE-DAD Li-Jun Zhao and Yu-Qing Zhang*

National Engineering Laboratory for Modern Silk, Soochow University; Silk Biotechnology Laboratory, School of Biology and Basic Medical Sciences, Soochow University 702-2303 Room, No. 199, Renai Road, Dushuhu Higher Edu. Town, Suzhou 215123; P. R. China E-mail: sericult@suda.edu.cn Abstract

This study used the branch bark of mulberry (Morus spp. L.) as experimental material. High performance capillary electrophoresis diode array detection (HPCE-DAD), a new, fast and convenient method, was used to rapidly analyze the content of oxyresveratrol-4-3'-β-O-D-diglycoside in samples. Oxyresveratrol diglycoside gave a good linear response for concentrations 0.0025–0.15 mg/mL. The method had good repeatability (RSD: 103.78%), and the recovery rate of the compound was 98.12%. There were significant, nearly 150-fold, differences in oxyresveratrol diglycoside content among branch bark water extracts of 29 mulberry cultivars: the content in cultivar YanBian QiuYu from Korea was highest, with 45.75 mg/g(dry weight), while MaoSang derived from Morus tiliaefolia Makino in Japan was lowest, with only 0.32 mg/g(dry weight). These results indicated that rapid analysis of oxyresveratrol diglycoside in mulberry branch bark by HPCE-DAD was reliable, providing a basic foundation for drug development from mulberry varieties in future and research on characteristics of oxyresveratrol diglycoside.

Keywords

Oxyresveratrol Diglycoside; HPCE-DAD; Mulberry Branch Bark

Introduction

China is rich in natural resources, and mulberry is an important one that has been used and cultivated for a long time. Mulberry leaves are used as the main food of the silkworm; more importantly, the leaf, fruit and root bark of mulberry have many pharmacological functions, such as hypoglycemic1, hypolipidemic2, antioxidative3, 4, anti-inflammatory5, neuroprotective6 and anti-HIV7 properties.

The mulberry branch is the highest yielding part of the tree and over ten million tons are harvested in China every year by summer cutting and winter recutting. However, there is limited utilization of mulberry

branches as a bioresource, and most are burned as firewood, thus wasting resources and causing environmental problems. Recently, secondary metabolites from mulberry branches have attracted much attention in the areas of pharmacology8, cosmetics9 and functional foods10, 11. Recently, colleagues reported a preparation method for the extraction of a novel glycosidase-activity inhibitor from mulberry branches12.

Oxyresveratrol-4-3'-β-O-D-diglycoside is an oxyresveratrol diglycoside where the 4-OH and 3′-OH have been substituted by two glucoses (Fig. 1). It was first isolated from root bark in 198613. Studies have shown that the oxyresveratrol diglycoside has various bio- activities, including anti-tyrosinase14, anti-inflammatory15, hypoglycemic16 and nephroprotective effects17. After oral administration of oxyresveratrol-4-3'-β-O-D-diglycoside, then oxyresveratrol and two oxyresveratrol mono- glycosides were found in rat gastrointestinal contents and feces18. This suggested that in the body the oxyresveratrol-4-3'-β-O-D-diglycoside is first hydrolyzed to oxyresveratrol, thus having similar pharmacological effects on oxyresveratrol.

O

OH

O

OHO

HOHO OH

OH

OHO

HO OH

OH

4

3'

23

16

5

Â

Á

1' 6'5'

4'2'

FIG. 1 THE STRUCTURE OF OXYRESVERATROL-4-3'-β-O-D-

DIGLYCOSIDE High performance capillary electrophoresis (HPCE) is a method that has been rapidly developed over recent years, with many advantages such as high separation performance, short analysis time, small volume of

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sample required and little environmental pollution. It has been used for the separation and determination of various components of Chinese traditional medicines19–23. In this paper, a method was described to separate and detect levels of oxyresveratrol diglycoside in mulberry branches using HPCE-DAD.

Materials and Methods

Materials

Branches of 29 mulberry cultivar including HuSang 32 (Table 3) were harvested in December 2011, from the Experimental Mulberry Garden of the Department of Applied Biology, Soochow University.

Capillary electrophoresis with UV absorbance detection was performed with a P/ACE MDQ CE system equipped with a diode array detector (DAD) (Beckman Coulter, USA) and 60 cm uncoated fused silica capillaries (Hebei Yongnian Ruifeng Chromatography Devices Co. Ltd. China) of 50 μm i.d., and effective length of 50 cm. The standard oxyresveratrol-4-3'-β -O-D-diglycoside(CASNO.:102841-42-9,>98%, HPLC) was bought from Shanghai Tauto Biotech Co., Ltd (P. R. China). All sample solutions were filtered through 0.22 μm nylon syringe filters.

Sample Preparation

From the cut mulberry branches, leaves were removed and then the bark was stripped by home-made peeling machine. The bark was freeze-dried and then crushed to powder of 30 mesh by machine (JL64, made in Shanghai Jiading Grain and Oil Equipment Co. Ltd, China). Mulberry branch bark powder was extracted for 4 h in an aqueous solution at 80°C24. The obtained aqueous extracts were first filtered with nylon filtering cloth (60 mesh) to remove water-insoluble matter and debris, and then concentrated by rotary concentrators, and sediment removed by centrifuge at 12,000 rpm×15 min. After vacuum freeze-drying, the aqueous extract powder was obtained.

Preparation of Standard Sample Solution

Of standard oxyresveratrol-4-3'-β-O-D-diglycoside, 1.0 mg was dissolved in 1.0 mL of ultrapure water, as the HPCE analysis standard solution, and stored at 4°C. Of mulberry stalk bark aqueous extract powder, 1.0 mg was dissolved in 1.0 mL of ultrapure water made up to 1.0 mg/mL of sample solution, and stored at 4°C.

HPCE Analysis

Electrophoresis conditions: The running buffer was

constituted of a mixture of 10 mM NaH2PO4·2H2O and 20 mM Na2B4O7·10H2O; at pH 8.0–10.0, adjusted to the desired value with 0.5 M NaOH. The detection wavelength was 324 nm. Applied voltage for separation was 15 kV, thermostat controlled at 28°C. Injection time was set at 5 s by applying a pressure of 0.5 psi. New capillaries were pre-treated using methanol for 10 min, ultrapure water for 5 min, 0.1 M hydrochloric acid for 30 min, ultrapure water for 5 min, 0.5 M sodium hydroxide for 30 min, ultrapure water for 15 min, and running buffer for 15 min. Between runs the capillary was rinsed with running buffer for 2 min.

Standard Curve

Of oxyresveratrol-4-3'-β-O-D-diglycoside, 0.0025, 0.01, 0.05, 0.10 and 0.15 mg/mL were respectively injected and treated using the above mentioned experimental conditions. A standard curve was plotted with target peak area integral value in spectra as the ordinate and oxyresveratrol diglycoside concentration as the abscissa.

HPCE Reproducibility for the Determination of Oxyresveratrol Diglycoside

Of biosamples (extract from Husang32), 1.0 mg/mL, 0.5 mg/mL and 0.2 mg/mL solution were diluted with increasing injecting time (5s, 10s and 25s), respectively. After repeated injection four times, the peak area integral values in spectra were compared and HPCE reproducibility was calculated.

Recovery of Oxyresveratrol Diglycoside in Extract

Accurately weighed mulberry branch bark powder (Husang 32, given known contained oxyresveratrol diglycoside 9.10 mg/g)25 0.10g in a 25.0 mL Erlenmeyer flask, add 9.0 mL water and 1.0 mL standard sample solution (concentration of 0.05 mg/mL), and processed for 24 h at 80°C. The obtained supernatant was filtered, injected and treated using the above- mentioned experimental conditions. The recoveries of oxyresveratrol diglycoside in extract were calculated by comparing the target peaks in spectra as the following formula: Recovery (%) = [(measured value)/(content in extract +

addition)]×100

Results

Effect of Buffer pH on Separation

The pH of the electrophoresis running buffer was

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adjusted to 8.0, 8.5, 9.0, 9.5 and 10.0, to determine the optimal pH for the best separating effect. At pH 8.5, the retention time of oxyresveratrol diglycoside in the capillary was 9.1 min, and the signal was strongest (Fig. 2). With increasing buffer pH, the retention time was increased. At pH 10.0, the retention time was extended to 16.7 min, except oxyresveratrol diglycoside peak, there were some small impurity peaks; however, there was a clearly separating effect. Thus, a buffer with pH 10.0 was chosen for capillary electrophoresis analysis in the following tests.

6 8 10 12 14 16 18 20 22 24

0

2

4

6

8

10

12

14

16

18 pH10.0 pH9.5 pH9.0 pH8.5 pH8.0

ABS 32

4nm (m

AU)

Retention Time (min) FIG. 2 EFFECT OF BUFFER pH ON SAMPLE PREPARITION

Standard Curve of Oxyresveratrol Diglycoside

The mother solution of standard oxyresveratrol diglycoside was analyzed by HPCE using the abovementioned experimental conditions. The data was processed and plotted into a standard curve using Origin7.5 software, (Fig. 3), with the target peak area integral value in spectra as the ordinate and oxyresveratrol diglycoside concentration as the abscissa. The regression equation of oxyresveratrol diglycoside was y = 661.66523 + 225171.27583x, with correlation coefficient (r) = 0.99756.

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.160.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

ABS 32

4nm

(AU)

Oxyresveratrol diglycoside concentration (mg/mL) FIG. 3 STANDARD CURVE OF OXYRESVEROTRAL

DDIGLYCOSIDE

Identification of Oxyresveratrol Diglycoside in the Extract

Mulberry branch bark extract samples, standard oxyresveratrol diglycoside, and a mixture of both samples were respectively analyzed by capillary electrophoresis (Fig. 4). Fig. 4 represents the electrophoresis pattern of mulberry branch bark extract (red line) by HPCE-DAD with a retention time of 16.7 min and signal strength of 5 mAU. When standard oxyresveratrol diglycoside was added, the peak retention time (blue line) was still 16.7 min, and there was no other electrophoretic peak–the superposition of two signals resulted in the signal strength reaching 27.5 mAU. This demonstrated that the peak in the mulberry branch bark extract must be due to oxyresveratrol diglycoside.

2 4 6 8 10 12 14 16 18 20

0

1

2

3

4

5 Extraction Extraction+Standard

Retention Time (min)

ABS

324n

m(m

AU

)

0

5

10

15

20

25

30

ABS

324n

m(m

AU

)

FIG. 4 MULBERRY BRANCH BARK EXTRACTION AND

STANDARD BY HPCE-DAD SPETRA

In addition, we also used an U3000 Spectrophotometer (Hitachi, Japan) to make a comparative analysis of UV absorption spectrum between mulberry branch bark extract and the standard sample (Fig. 5)–the UV absorption spectra of both were almost identical. This indicated that the peak at retention time of 16.7 min was the oxyresveratrol diglycoside signal indeed. It also showed that the main ingredients in the mulberry branch bark extract was oxyresveratrol diglycoside.

250 300 350 400

0.0

0.5

1.0

1.5 Sample Standard sample

ABS

324n

m(A

U)

Wavelength (nm) FIG. 5 UV ABSORPTION SPETRUM OF EXTRACTION SAMPLE

AND STANDARD SAMPLE

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TABLE 2 REPRODUCIBILITY OF HPCE DETERMINATION OF STANDARD SOLUTION

Sampling mode & volume Repeated injection times (n=4) Mean ± SD RSD% 25 nL (1.0 mg/mL× 5 s) 3198 3005 3172 2966 3085.30 ± 116.80 103.78

50 nL (0.5 mg/mL × 10 s) 3542 3867 3569 3620 3649.50 ± 148.60 104.07 125 nL (0.2 mg/mL × 25 s) 3852 4066 4223 3637 3944.50 ± 255.20 106.47

SD is standard deviation. RSD is relative standard deviation

TABLE 1 THE RECOVERY OF OXYRESVERATROL DIGLYCOSIDE

Sample (g) Content(mg/g) Addition(mg) Theoretical value (mg) Measured value(mg) Recovery(%) 0.100±0.003 9.12±0.37 0.05 0.962 0.944±0.002 98.12

Sample is the powder of mulberry branch bark. Addition means added standard oxyresveratrol diglycoside. Theoretical value means the total content of mulberry branch bark powder and added standard sample in theory

TABLE 3 THE LEVEL DIFFERENCE OF OXYRESVERATROL DIGLYCOSIDE AMONG MULBERRY CULTIVARS

No. Cultivated mulberry Source (Morus spp. L.) Contents (mg/g, n = 3) ± SD 1 79×201 Hybrid 2.99 2.18 2 QingPi GuaRang — 8.82 0.46 3 XiaoJiGuan Morus multicaulis P 31.29 1.54 4 Japan MaoSang Morus tiliaefolia Makino 0.32 0.23 5 YanBian QiuYu From Korea 45.75 5.10 6 HuangLuTou — 10.39 0.91 7 Kenmochi Morus bombycis Koidz 10.39 1.21 8 FuYang Morus multicaulis Perr. 24.98 0.040 9 843 Octoploid 33.57 1.37 10 Yu71-1 Hybrid 16.91 1.12 11 HuYe JingSang — 39.70 1.81 12 XinAn ChunSang Morus multicaulis Perr. 26.33 0.38 13 Lu-5 — 22.52 0.84 14 LiuYeSang Morus alba Linn. 17.65 1.14 15 HuSang1 — 10.43 1.78 16 Yin6 From Japan 1.59 0.23 17 HuaSang Morus wittiorum Hand-Mazz 8.01 0.39 18 BaiTiaoSang — 18.14 1.77 19 Husang 13 — 19.63 1.10 20 Lu-9 — 1.65 0.53 21 Sha2 Morus atropurpurea Roxb. 3.69 0.041 22 TongXiangQing Morus multicaulis Perr. 20.76 0.17 23 RiZhouJiu — 21.64 0.67 24 HongPi MaoSang Morus tiliaefolia Makino 13.45 0.36 25 DaHongPi Morus alba Linn. 11.70 2.31 26 MaHaiSang — 11.49 0.54 27 HeiYe MoDou — 2.22 0.31 28 HongDingSang — 7.38 0.72 29 HuSang 32 Morus multicaulis Perr. 7.00 0.97

Recovery Trial of Oxyresveratrol Diglycoside in the Extract

A specific amount of standard oxyresveratrol diglycoside was added into mulberry branch bark powder and then extracts for HPCE analysis, and then detected using the same method of HPCE-DAD (Table 1). The measured recovery of 98.12% indicated that detecting content of oxyresveratrol diglycoside in mulberry branch bark samples had high recovery rates.

The Reproducibility Analysis of the HPCE for the Determination of Oxyresveratrol Diglycoside

In this experiment, three kinds of biosample solution were analyzed by varying the injection time and

repeating the analysis four times, respectively. The response signal of the target peak gradually increased with prolongation of the injection time by capillary electrophoresis (Table 2). Although the peak signal response of third sampling mode was the strongest of all, the repeatability error was the highest. In the first injection mode, the response signal of the peak was slightly lower, the relative standard deviation was a minimum of 103.78%, and so an injection time of 5 s was selected for all capillary electrophoresis analysis.

Different Levels of Oxyresveratrol Diglycoside among Cultivars

In this study, comparative analysis of branch bark showed that the contents of oxyresveratrol diglycoside

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significantly differed among the 29 mulberry cultivars (Table 3). Oxyresveratrol diglycoside content in Yanbian Qiuyu was highest, up to 45.75 mg/g, while the Japan MaoSang content was lowest, only about 0.32 mg/g–a 143-fold difference between cultivars. These experimental data may have major significance for cultivation of mulberry cultivars and the development of mulberry active ingredients in future.

Conclusion

In this experiment the branch bark of HuSang 32 and 28 other mulberry cultivars was used as test material. At first we used water for extraction, and then determined the content of oxyresveratrol diglycoside in the mulberry branch bark water extract by the HPCE method. Standard oxyresveratrol diglycoside was used to construct a linear equation to determine oxyresveratrol diglycoside contents. The study showed that the oxyresveratrol diglycoside content of branch bark of different mulberry cultivars varied greatly. The content in YanBian QiuYu branch bark water extract was highest, about 45.75 mg/g(dry weight), while the content in Japan MaoSang was lowest, about 0.32mg/g(dry weight), 143-fold difference. Using HPCE to determine the content of oxyresveratrol diglycoside showed good repeatability (103.78%) and high recovery rate (98.12%). Thus, branch bark of YanBian QiuYu could be used to extract higher amounts of oxyresveratrol diglycoside, and so a foundation was built for the full use and efficient development of mulberry branch bark. In addition, the results also showed that rapid analysis of oxyresveratrol diglycoside from mulberry branch bark by HPCE-DAD was reliable, which will provide a good foundation for drug development of mulberry varieties and study on the characteristics of oxyresveratrol diglycoside in future.

ACKNOWLEDGMENT

The authors gratefully acknowledged the earmarked fund for China Agriculture Research System (CARS) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China.

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