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Integrated utilization of red radish for the efficient production of high purity of procyanidin dimers wen jiang, Xiaohua Zhou, Yang Yang, and Zhiming Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02478 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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Journal of Agricultural and Food Chemistry

Integrated utilization of red radish for the efficient production of high purity of procyanidin dimers Wen Jiang, Xiaohua Zhou*, Yang Yang, Zhiming Zhou Department of Chemical Engineering, School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 401331, PR China

*Corresponding author Xiaohua Zhou Tel: +86-65678925 Fax: (86) 23-65678925 E-mail: [email protected]

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ABSTRACT

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Red radish was extracted by methanol to obtain crude radish procyanidin extracts.

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The purity of procyanidin (PP) and procyanidin dimers (PD) of crude radish

4

procyanidin extracts under different ratio of methanol to radish was optimized to

5

achieve the best extraction performance. Then the crude radish procyanidin extracts

6

was respectively processed six macroporous resins separation to separate radish

7

procyanidin oligomers (RPO) and polymers (RPP). Depolymerization of radish

8

procyanidin polymers (RPP) into oligomers was then conducted. N-acetylneuraminate

9

lyase (NAL) was firstly used as the enzyme to depolymerize RPP. The

10

depolymerization yield (DY) under different depolymerized conditions was also

11

investigated. Results showed the DY of RPP would achieve 53.24 ±0.35% at the best

12

condition. Then high purity of procyanidin dimers was prepared by depolymerized

13

procyanidin oligomers and PRO. Additionally, the chemical structure of the

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preparative radish procyanidin dimers was elucidated by high resolution mass

15

spectrum, one- and two-dimensional NMR.

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Keywords : Procyanidin oligomers; Procyanidin polymers; Depolymerization;

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N-acetylneuraminate lyase; Procyanidin dimers

18 19 20 21

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INTRODUCTION

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Red radish (Raphanus sativus L.) is an important vegetable crop belonging to the

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Cruciferae (or Brassicaceae) family which is widely distributed in China, especially in

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Chongqing Municipality. Radish is regarded as high medicinal and nutritional value

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and recommended as an alternative treatment for various ailment soloplaint because

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of the existence of various phytochemicals, especially polyphenols and flavonoids1-5.

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However, radish is only widely underutilized as raw materials of Sichuan pickles and

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its current applications are limited. Thus, it is desirable to take advantage of the

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phytochemicals, especially polyphenols, of radish to expand its applications. It was

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reported that procyanidins, oligomers and polymers of polyhydroxyflavan-3ol units,

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are a class of polyphenols which accounted for 68% of the total phenols content in

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radish6. Degree of polymerization (DP) was used to describe the size of

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procyanidins7,8. Procyanidins with DP of 1–4 are oligomers, while procyanidins with

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DP≥5 are polymers. Procyanidin oligomers were known to prevent cancers,

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cardiovascular diseases, and other aging related conditions

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intimate knowledge of the bioavailability of pure procyanidin oligomers is still

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exiguous as studies have only used complex procyanidin mixtures due to a lack of

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appropriate reference compounds12-15. Only a few procyanidin oligomers are

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commercially available, such as procyanidin dimers which is the simplest procyanidin

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oligomers16. Furthermore, procyanidin dimers were reported to have high biological

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activities17. Consequently, it is of interest to prepare pure procyanidin dimers from red

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radish. 3

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9-11

. But until now,

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However, the majority of procyanidins in nature including those in red radish are

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polymers18 and a great deal of procyanidin polymers are left after the preparation of

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procyanidins dimer. Procyanidin oligomers are absorbable in vivo whereas polymers

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could not be absorbed in the gastrointestinal tract19,20. Bioavailability of procyanidin

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polymers was relatively lower than oligomers19.

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Depolymerization of procyanidin polymers to oligomers is expected to enhance

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their bioavailability and make for higher bioactivity in vivo21-24. Several strategies

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have been used for the depolymerization of procyanidin polymers. Palm procyanidin

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polymers were depolymerized by Foo and Porter with epicatechin in ethanol-acetic

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acid at 95 oC for 22 h and some dimers were identified25. Sorghum procyanidin

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polymers can also be depolymerized with epicatechin under acidic conditions at 74 oC

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for 61 min and procyanidin monomers and oligomers were recognized26. Grape seeds

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or hazelnut skins procyanidin polymers were depolymerized into dimers with reacted

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with flavan-3-ols under acidic conditions27. Depolymerization under acidic condition

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was convenient and the yield of oligomers is relatively high, but the cost of the

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depolymerization was high with flavan-3-ols as reactant and the reaction apparatus

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would also corrode under acidic conditions. Hydrogenolysis was also applied by Foo

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to the depolymerization of photinia glabrescens procyanidin polymers and

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procyanidin monomers and oligomers were obtained28. It was reported by Li et al.,

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hi-tannin sorghum bran procyanidin polymers hydrogenolyzed with 1MPa hydrogen,

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3 mg Pd/C, at 100 oC for 1-3 h to reach the maximum oligomers yield of 38.3%29.

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However, hydrogenolysis requires high strength equipment and the yield of oligomers 4

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is also low.

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Meanwhile, an increasing number of attentions were drawn on enzymatic method

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owning to its low cost and high yield. N-acetylneuraminate lyase (NAL, EC 4.1.3.3),

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a (β/α)8-barrel protein, belongs to the dihydrodipicolinate synthase (DHDPS, EC

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4.2.1.52) family of enzymes30. Like the much more closely studied DHDPS, NAL is a

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homotetrameric enzyme, plays an unconnected metabolic role, catalyzing the ultimate

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step in the biosynthesis of sialic acid—the condensation of pyruvate and

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N-acetylmannosamine. Sialic acids, a large family of sugars, derived from the parent

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compound N-acetylneuraminic acid31. N-acetylneuraminic acid is widely acted on the

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surface of cells of animals, where they play important parts in a series of critical

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biological processes, including cellular adhesion and recognition32.

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The degradation mechanism of N-acetylneuraminic acid and procyanidin polymers

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were shown in Figure 1. As shown in Figure 1a, the enzymatic degradation reaction

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of N-acetylneuraminic acid by N-acetylneuraminate lyase (NAL) occurred on the C-C

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bond of pyran ring of N-acetylneuraminic acid. And the structure of procyanidin

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polymers was also shown in Figure 1b. The degradation site of procyanidin polymers

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also occurred on the C-C bond of pyran ring “C” of procyanidin polymers. By

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comparison the degradation of N-acetylneuraminic acid and procyanidin polymers,

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the enzymatic catalytic reactions of N-acetylneuraminic acid by NAL occurred on the

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C-C bond of pyran ring which was similar to the depolymerization of procyanidin

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polymers. Herein, NAL is firstly used as the enzyme catalysts in the depolymerization

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of procyanidin polymers. 5

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In this paper, red radish was firstly processed with the cleavage of radish

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procyanidin polymers and oligomers. The radish procyanidin polymers were

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depolymerized by NAL to yield oligomers which can be used to prepare high purity

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of procyanidin dimers. The factors which affected the depolymerization yield were

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investigated, including the pH, reaction temperature and the mass ratio of NAL to

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RPP. Then the radish procyanidin oligomers were used to prepare high purity of

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procyanidin dimers by preparative HPLC purification method. And the structure of

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the preparative procyanidin dimers was elucidated by high resolution mass spectrum,

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one- and two-dimensional NMR. This study presented a desirable process for efficient

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production of high purity of procyanidin dimers from red radish.

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MATERIAL AND METHODS

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Reagents. Red radish (Raphanus sativus L.) was offered by Haiju Agriculture

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Development Co. Ltd., Chongqing, China. N-acetylneuraminate lyase (35 U/mg) was

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purchased from Xibao Biotechnology Co. Ltd., Shanghai, China. All the reagents

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above were of biological grade. Amilan, D101, AB-8, ADS-17, NAK-7, ADS-7 and

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XAD-4 macroporous resins were purchased from H&E Co., Ltd., Beijing, China.

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(−)-Epicatechin was purchased from Sigma chemical CO. (St. Louis, MO), USA.

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HPLC grade solvents were purchased from Fisher Scientific (Pittsburg, PA), China.

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Formic acid and other chemicals were purchased from Head Chemical Factory,

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Chongqing, China, and all of them were of analytical grade.

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Separation of radish procyanidin oligomers (RPO) and polymers (RPP) from

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red radish. Fresh red radish was cut into pieces (2 × 2 × 2 cm) and then extracted in a 6

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flask with methanol at 50 °C for 2 h, and the ratio of methanol to radish (v/w) was set

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as 20: 1 to 55:1. The extracts were collected and then centrifuged at 5000 g-force for

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10 min. Supernatants was then collected. Residual methanol in the supernatant was

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removed on rotatory evaporation at 40 °C under vacuum by a circulating water

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vacuum pump (SHZ-IIID, Zhixin, Shanghai, CN) to obtain the crude radish

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procyanidin extracts. The crude radish procyanidin extracts was processed further

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separation method, which used macroporous resins (amilan, D101, AB-8, ADS-17,

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NAK-7 and ADS-7). The resins were required to pretreat before use and the method

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used was as Zhang et al33 with some modification. After that, the pretreated resins

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(10g) were dispersed in crude radish procyanidin extracts (100 mL). The mixture was

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rocked at 60 g-force for adsorption under room temperature for 2 h. Then the resin

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loaded radish procyanidin oligomers (RPO) were desorbed with 45% (v/v) methanol

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solution under room temperature, and RPO eluent was collected until the eluent has

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no color. After that, the resin loaded residual radish procyanidins were desorbed with

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80% (v/v) methanol (containing 0.3% (v/v) formic acid) solution under room

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temperature, and radish procyanidin polymers (RPP) eluent was collected until the

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eluent has no color. The methanol of the eluent solutions was removed by a rotary

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evaporator at 40 oC, and the concentrated solution was finally lyophilized by a freeze

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drier (FD-1A-50, Tianling, Jiangsu, CN) to obtain RPO and RPP powder. RPP powder

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could be depolymerized to low molecular weight of RPO which can provide raw

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material for the preparation of high purity of radish procyanidins dimer.

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Determination of the purity of procyanidins. The purity of procyanidins (PP) 7

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was measured using vanillin assay with (−)-epicatechin as standard34. The

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absorbance of the reaction solution of vanillin and (−)-epicatechin at λ=500 nm

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were measured by a UV spectrophotometer (UV-2600, Shimadzu, Japan) to drew

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the standard curve. After that, the content and mass of procyanidins was measured

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by the standard curve obtained above. And the PP was calculated as follows:

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PP (%) =

M Pa M pb

× 100%

Where Mpa is the mass of procyanidins after separation (µg/mL), Mpb is the initial

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mass of procyanidins before separation (µg/mL).

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Depolymerization

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N-acetylneuraminate lyase.

of

radish

procyanidin

polymers

(RPP)

by

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Purification of N-acetylneuraminate lyase. 10 g of N-acetylneuraminate lyase

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(NAL) was firstly poured into 1000 mL aqueous solution. Then the NAL solution was

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purified using the purification method mentioned in the literature with some

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modification35.

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Depolymerization of radish procyanidin polymers by NAL. The radish

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procyanidin polymers (RPP) powder (5 g) was poured into 100 mL distilled water to

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prepare 50mg/mL RPP solution. 5% (w/w) NAL was then added into the above

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solution and the reaction mixture was carried out at 50 oC, pH 8.0 for 4h under gentle

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agitation. After the reaction finished, the pH of the solution was adjusted to 6.4 where

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the isoelectric point of NAL to precipitate NAL. Then the solution was centrifuged for

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10 min at the speed of 5000 g-force, and finally the depolymerization product was

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obtained. 8

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Determination of the depolymerization yield of radish procyanidin polymers.

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The depolymerization yield (DY) of RPP means the content of procyanidin oligomers

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after the depolymerization reaction by NAL. The HPLC-MS method was used to

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verify the depolymerization of procyanidin polymers by NAL and measure the

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content of procyanidin oligomers and polymers. The HPLC equipment was an Agilent

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1200 HPLC system equipped with a high pressure pump and a UV diode array

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detector. The analytical column was a Reverse Phase-C18 column (5 µm particle size,

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250×4.6 mm) (HiQsil, Tokyo, Japan). The analytical HPLC analysis was performed

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as follows: the solvent system consisted of ultra-pure water as fluid phase A and

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methanol as fluid phase B. Gradient conditions: 0-5min, 20% B isocratic; 5-20min,

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20-35% B linear; 20-40min, 35% B isocratic; 40-60 min, 35-55% linear; 60-80 min,

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55% B isocratic. The eluates were detected at λ=280 nm under room temperature. The

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flow rate was set 1.0 mL/min, and 10 µL aliquots were injected into the column. Then

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(−)-epicatechin was used as the external standard to measure the content of

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procyanidin oligomers in the samples according to a standard curve obtained from the

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standard.

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The negative electrospray ionization mode was performed using nebulizer 14 psi,

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drying gas 11 L/min, drying temperature 355 oC, and capillary 4000 V. The full scan

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mass spectra were measured from m/z 150 to 1500. The identification of procyanidin

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monomers, dimers and trimers were based on mass spectra21,23,36. And the

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depolymerization yield (DY) of RPP was determined by the following equation:

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Ca − Ci × 100% C0

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DY =

176

Where Ca is the procyanidins monomer and oligomers content of depolymerization

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product after reaction (µg/mL), Ci is the initial content of procyanidin oligomers in

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RPP (µg/mL), C0 is the initial content of RPP (µg/mL).

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Optimization of radish procyanidin polymers depolymerization. To achieve the

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best depolymerization yield (DY), pH (4, 5, 6, 7, 8, 9), reaction temperature (30, 40,

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50, 60, 70 oC) and the mass ratio of NAL to RPP (0.01, 0.03, 0.05, 0.07, 0.1) were

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optimized.

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Preparation of high purity of radish procyanidin dimers. Radish procyanidin

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dimers, the simplest procyanidin oligomers in red radish, can be separated from radish

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procyanidin oligomers (RPO). Hence, the crude radish procyanidin dimers could be

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obtained by methanol extraction and macroporous resin adsorption. In order to

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preparing high purity of radish procyanidin dimers, the preparative HPLC purification

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method was used. The preparative HPLC equipment and column used agreed with the

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equipment reported in the literature33. And the preparative HPLC separation was

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performed as follows: the solvent system consisted of methanol as fluid phase A and

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ultra-pure water as fluid phase B. Gradient conditions: 0-20 min, 20-35% A linear;

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20-60min, 35% A isocratic. The flow rate was set 20 mL/ min and detected at λ=280

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nm. The injection volume was 5 mL. The peak fraction of radish procyanidin dimers

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was collected manually according to the chromatogram. After that, the residual

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procyanidin oligomers were also prepared. The fraction was concentrated by a rotary

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evaporator, and then processed using a vacuum freeze-drying machine (FD-1A-50, 10

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Tianling, Jiangsu, CN). The purity of radish procyanidin dimers was calculated using

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the following equation.

Mda × 100% M db

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PD(%) =

200

Where Mda is the mass of radish procyanidin dimers after purification which can be

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calculated according to the mentioned in HPLC analysis of radish procyanidin dimers,

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and Mdb is the initial mass of radish procyanidin dimers before purification.

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HPLC analysis of preparative radish procyanidin dimers. The analytical HPLC

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method was used to analysis the concentration of the preparative radish procyanidin

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dimers. And the analytical HPLC equipment was a LC 3000 system (Chuangxin

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Tongheng Science and Technology Co. Ltd, Beijing, China), which equipped with a

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high pressure pump and a UV–visible diode array detector. The analytical column was

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a reverse phase C18 column (5 µm particle size, 250×4.6 mm) (HiQsil, Tokyo,

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Japan).

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The analytical HPLC analysis was performed as follows: the solvent system

211

consisted of methanol as fluid phase A and ultra-pure water as fluid phase B. Gradient

212

conditions: 0-10min, 20% A isocratic; 10-35min, 20-35% A linear; 35-50min, 35% A

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isocratic. The eluates were detected at 280 nm at 25 °C. The flow rate was set 1.0

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mL/min, and the injected volume was 10 µL. Then the concentration of the

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preparative radish procyanidin dimers could be calculated according to the regression

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line of the preparative procyanidin dimers y = 3.21571E+07 x - 3.29715E+06 (R2 =

217

0.9981), where y is the peak area and x is the concentration of procyanidin dimers 11

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(µg/mL). The working calibration curve of procyanidin dimers standard solution

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revealed good linear correlation over the range of 0.25–2.0 µg/mL.

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Structural elucidation of preparative radish procyanidin dimers. The structural

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elucidation of the preparative radish procyanidin dimers was identified by high

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resolution mass spectrum, one- and two-dimensional NMR.

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The high resolution mass spectrum was conducted using the method as reported by

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Christensen et al37 with some modification. The full spectrum of per- fluorkerosene

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was used for mass calibration across the range of the spectrum collected from m/z

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100–700.

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The one-dimensional 1H and 1

13

C NMR, two-dimensional

1

H-1H correlated

H-1H phase-sensitive nuclear Overhauser enhancement

228

spectroscopy (COSY),

229

spectroscopy (NOESY), heteronuclear single-quantum coherence (HSQC), and

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heteronuclear multiple-bond correlation (HMBC) experiments were performed on a

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Agilent NMR vnmrs600 at 240 K to overcome the atropisomerism which caused

232

signal broadening, using acetone-d6 as solvent, with tetramethylsilane (TMS) as the

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internal standard.

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RESULTS AND DISCUSSION

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Separation of radish procyanidin oligomers and polymers from red radish.

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Red radish was firstly extracted with methanol to extract crude procyanidins. The

237

ratio of methanol to radish (v/m) was studied to achieve the best extraction

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performance. And the influence was revealed in Figure 2. It was indicated that the

239

extraction yield, purity of procyanidins (PP) and purity of procyanidin dimers (PD) 12

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increased rapidly when the ratio of methanol to radish was set to be between 20:1 and

241

40:1 (v/m), but then increased slowly over 40:1 (v/m). The extraction yield, PP and

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PD at the ratio of methanol to radish of 40:1 (v/m) would achieve 91.4 ± 0.7%, 61.8 ±

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1.2% and 22.5 ± 0.5%, respectively. Hence, 40:1 (v/m) was selected as the best ratio

244

of methanol to radish among tested and used for the further study.

245

After that, crude procyanidin extracts was further individually processed six

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macroporous resins (amilan, D101, AB-8, ADS-17, NAK-7 and ADS-7) separation to

247

separate procyanidin oligomers (RPO) and polymers (RPP). The results of adsorption

248

and desorption behavior of the six macroporous resins to total procyanidins and

249

procyanidin dimers were shown in Table 1 and Table 2, respectively. According to

250

the results of PP and PD, one-way ANOVA test was used to the variable. If the

251

differences are statistically significant, then a search for the reason behind the

252

significant result is performed. The results of ANOVA test of PP and PD processed

253

by different resins were given in Tables 3 and Table 4. It can be seen that the values

254

between groups calculated by Fisher–Snedecor functions are higher than the tabulated

255

values of Fisher–Snedecor functions for a significance level α= 0.01. Therefore, the

256

null hypothesis is negated. By negating the null hypothesis (the null hypothesis

257

supposes that the resins have no influence on PP and PD, having values equal to zero),

258

indicate the values of the PP and PD processed by different resins were statistically

259

different for this experiment.

260

As revealed in Table 1 and Table 2, the adsorption and desorption capacity of

261

amilan for procyanidins and procyanidin dimers in crude procyanidin extracts was 13

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highest than that of other five resins. The PP and PD would reach 84.3 ± 0.8% and

263

41.5 ± 1.6% by amilan separation, respectively. Accordingly, amilan was chosen to as

264

the resin for increasing the PP and PD. On the other word, it was also clear that the

265

crude procyanidin extracts separate by amilan macroporous resin is a desirable

266

process to enhance the purity of radish procyanidins and procyanidin dimers. After

267

that, RPP were also separated. And RPP was depolymerized to low molecular weight

268

of RPO which can provide raw material for the preparation of high purity of radish

269

procyanidins dimer.

270

Depolymerization of radish procyanidin polymers.

271

HPLC-MS analysis of the depolymerization product of radish procyanidin

272

polymers. Figure 3a was the HPLC chromatogram of radish procyanidin oligomers

273

(RPO). It was shown that our present study exhibited a good separation of RPO. The

274

separation of each compound without peak merging indicated the specificity of the

275

detection method. According to the mass spectrometry (MS) analysis, the peaks at

276

17.1 and 17.8 min had [M-H]- m/z 289 which were identified as monomer (catechin or

277

epicatechin). Peaks at 21.2, 27.6, 31.4 and 37.8 min were recognized as a procyanidin

278

dimers according to [M-H]- m/z 577. Peaks at 40.9 and 45.3 min was confirmed as

279

trimers by [M-H]- m/z of 865. Peaks at 53.3 min was also confirmed as tetramers by

280

[M-H]- m/z of 1153. In addition, a little procyanidin polymers still existed as shown in

281

the peak at 69.8 min identified by [M-H]- m/z over 1440. And the percent of

282

monomers, dimers, trimers, tetramers and polymers were 12.5%, 39.3% and 19.1%,

283

6.7% and 22.4%, respectively. 14

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Figure 3b showed the HPLC chromatogram of radish procyanidin polymers

285

extracted from red radish. Only a main peak was shown in the HPLC chromatograms

286

and the retention time was 70 min. The main peak accounted for 96.2% of the total

287

procyanidins in radish procyanidin polymers. And the percentage of monomers,

288

dimers, trimers and tetramers were 0.5%, 1.6% 1.3% and 0.4%, respectively. The

289

mass spectrometry (MS) analysis moreover demonstrated that radish procyanidins

290

polymer contained exclusively high molecular weight of polymers which identified by

291

[M-H]- m/z over 1440. As shown in Figure 3c, depolymerization resulted in an

292

obviously decrease of this polymer peak and the increase of oligomer peaks, but

293

without tetramers produced. Peak at 18.6 min had [M-H]- m/z 289 which was

294

identified as monomer (catechin or epicatechin). Peak at 25.5 and 32.4 min were

295

recognized as a procyanidin dimer according to [M-H]- m/z 577. Peaks at 41.3 and

296

44.6 min were confirmed as trimers by [M-H]- m/z of 865. The result indicated

297

N-acetylneuraminate lyase (NAL) could depolymerize the carbon-carbon bonds in

298

radish procyanidin polymers to liberate monomer and dimer of lower molecular

299

weight. And radish procyanidins polymer was successfully depolymerized by NAL.

300

Optimization of the condition parameters on the depolymerization.

301

The effect of pH on the depolymerization. Figure 4a showed that the

302

depolymerization yield (DY) of 100 mL 50 mg/mL radish procyanidin polymers (RPP)

303

solution was depolymerized by 0.25 g NAL, at different pH, 50 oC and 20 g-force for

304

4 h. It was observed that DY increased gradually with pH increased from 4.0 to 8.0.

305

However, DY decreased when the pH over 8.0. The phenomenon might be explained 15

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as follows: at the beginning, the increase of pH accelerated radish procyanidin

307

polymers depolymerized into oligomers. And the content of procyanidin oligomers

308

increased which resulted in the increase of DY. But when pH increased to 9.0, the

309

intense alkaline condition affected the enzyme activity of NAL which resulting in the

310

decrease of DY. Accordingly, the optimal pH was 8.0.

311

The effect of reaction temperature on the depolymerization. Figure 4b showed

312

the depolymerization yield (DY) of 100 mL 50 mg/mL RPP solution was

313

depolymerized by 0.25g NAL at different temperature, pH 8.0 and 20 g-force for 4 h.

314

With the reaction temperature increased from 30 to 50 oC, DY increased from 23.12%

315

to 52.34%, but then decreased over the range, which could be seen that the reaction

316

temperature affected the movement of the molecules. Therefore, the activity of the

317

reactants and the rate of reaction were promoted gradually with the increase of the

318

temperature. However, the high temperature would make enzyme inactivation and

319

resulted in the decrease of DY. In addition, the reversible reaction was also

320

accelerated because the enzyme-catalyzed reaction was an exothermic reaction38. To

321

conclude, the optimal reaction temperature was 50 oC.

322

The effect of the mass ratio of NAL to RPP on the depolymerization. Figure 4c

323

showed the depolymerization yield (DY) of 100 mL 50 mg/mL RPP solution was

324

depolymerized by different mass ratio of NAL to RPP at pH 8.0, 50 oC and 20 g-force

325

for 4 h. When the mass ratio of NAL to RPP was set between 0.01-0.05, DY increased

326

gradually. But the further increase of mass ratio of NAL to RPP did not show any

327

clear influence on DY. That might be explained as that NAL catalyzed the 16

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depolymerized of carbon-carbon bonds in RPP to liberate monomer and dimer of

329

lower molecular weight. But after the binding of enzyme and the substrate reached

330

saturation, continued to increase the content of enzyme, DY could not significantly

331

increased. In terms of the depolymerization efficiencies, the optimal mass ratio of

332

NAL to RPP was determined at 0.05.

333

Preparative HPLC purification of radish procyanidin dimers. The high purity

334

of radish procyanidin dimers can be obtained from the radish procyanidin oligomers

335

(RPO) and the depolymerization production of RPP by the preparative HPLC system.

336

The result of preparative liquid chromatography was shown in Figure 5a. And Figure

337

5b was the analytical HPLC of the preparative radish procyanidin dimers. As shown

338

in Figure 5b, the purity of radish procyanidin dimers was 95.3 ± 0.2% in this process.

339

It was found that preparative HPLC purification would obviously improve the purity

340

of radish procyanidin dimers. In this study, every one gram of radish procyanidin

341

oligomers could get 8.12 ± 0.11 mg procyanidin dimers.

342

Structural elucidation of preparative radish procyanidin dimers. After the

343

purification of preparative HPLC, The structural elucidation of the high purity of

344

preparative radish procyanidin dimers was characterized by high resolution mass

345

spectrometry, one- and two-dimensional NMR.

346

The high resolution mass spectrometry analysis of the preparative radish

347

procyanidin dimers was shown in Figure 6a. It was observed that the characteristic

348

pseudo molecular ions [M-H]- at m/z 577 for procyanidin dimers with fragment ions

349

at m/z 577, 559, 451, 424, 289 and 150, which indicated the molecular formula of 17

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C30H25O12. Procyanidins are composed of the flavan-3-ol monomers (+)-catechin and

351

(―)-epicatechin units linked mainly through C4–C8 and/or C4–C6 (B-type). The

352

flavan-3-ols can also be doubly linked by an additional ether bond between C2 of the

353

upper unit and the oxygen at C7 or C5 of the lower unit (A-type)39. Procyanidin

354

dimers are the simplest procyanidin oligomers which polymerized by (+)-catechin or

355

(―)-epicatechin through A- or B-type as shown in Figure 6b. The postulated

356

fragmentation of the preparative radish procyanidin dimers was shown in Figure 6c

357

which taken B-type procyanidin dimers as example. The molecular weight of

358

procyanidin dimers is 578 which corresponded to the [M-H]- m/z of 577 (M Da,

359

Figure 6c)40. On the basis of the fragment ion with m/z 559 could be derived from the

360

ion with m/z 577 via the loss of water (M-H-18 Da, Figure 6c). The ion with m/z 451

361

could be formed from the ion with m/z 577 via heterocyclic ring fission (HRF) with

362

the loss of phloroglucinol of procyanidin dimers (M-126 Da, Figure 6c). The ion with

363

m/z 424 can result from retro-Diels-Alder reaction (RDA) of procyanidin dimers of

364

ring C of the lower unit and absence of m/z 150 (M-154 Da, Figure 6c). The ion with

365

m/z 289 was derived from quinone methide (QM) fission of procyanidin dimers with

366

the loss of (+)-catechin or (―)-epicatechin (M-289 Da, Figure 6c)41.

367

The spectrum of one-dimensional 1H NMR and 13C NMR of the preparative radish

368

procyanidin dimers were shown in Figure 7a and Figure 7b, respectively. In 1H

369

NMR (600 MHZ, acetone-d6, 240K), δ 2.38 (d, 1H, J = 13.8 Hz, H4t), 2.69 (d, 1H, J

370

= 13.8 Hz, H4t), 3.62 (s, 1OH, OH11u), 3.79 (s, 1OH, OH11t), 4.16 (s, 1H, H3t),

371

4.32* (d, 1H, J=67.8 Hz, H4u), 4.48 (d, 1H, J=27.6 Hz, H4u), 4.74 (s, 1H, H3u), 4.94 18

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(d, 1H, J=22.2 Hz, H2t, H2u), 5.71 (s, 1H, H6u), 5.81 (d, 1H, J=16.2, H8u, H6t), 6.52

373

(d, 1H, J=6.6 Hz, H2’t ), 6.64 (t, 1H, J=19.2 Hz, H6’t ), 6.85 (d, 1H, J=33.6 Hz, H3’u,

374

H3’t), 6.99 (s, 2H, H2’u, H6’u). (* rotamer B).

375

In

13

C NMR (600 MHZ, acetone-d6, 240K), δ 27.2* (C4t), 27.5 (C4t), 35.6 (C4u),

376

64.6* (C3t), 71.4 (C3t), 75.3* (C3u), 77.8 (C3u), 94.4 (C6t), 94.5 (C6u), 95.6 (C8u),

377

95.7* (C6u), 98.6 (C6t), 101.8 (C10u), 102.1* (C10u), 106.1 (C10t), 106.4* (C10t),

378

107.1 (C8t), 107.3* (C8t), 114.4* (C2’u, C6’u), 114.6 (C2’u, C6’u), 114.8* (C2’t,

379

C6’t), 114.8 (C2’t, C6’t), 117.7 (C3’u), 118.1 (C3’t), 130.2 (C1’t), 131.2 (C1’u), 144.0

380

(C4’t, C5’t) , 144.2* (C4’t, C5’t) , 144.5* (C4’u, C5’u), 144.5 (C4’u, C5’u), 152.8*

381

(C9t), 153.8 (C9t), 154.0* (C9u), 154.0 (C9u), 155.8* (C5t, C7t), 155.8 (C5t, C7t),

382

156.5* (C5u, C7u), 156.5 (C5u, C7u). (* rotamer B).

383

To further elucidate the structural of the preparative radish procyanidin dimers, the

384

two-dimensional homonuclear (1H-1H COSY),

385

Overhauser enhancement spectroscopy (NOESY), and heteronuclear (HSQC, HMBC)

386

were also employed. The results were shown in Figure 8.

387

1

1

H−1H phasesensitive nuclear

H-1H COSY spectrum afforded the assignment of the B- and C-rings protons.

388

From the H2’t and H3’t protons it was possible to determine the H2’ and H3’ protons

389

of the B-ring of the terminal unit. From the correlation of H2/H3 and H3/H4 of the

390

terminal unit it was possible to determine the H2, H3 and H4 protons of the C-ring of

391

the terminal unit. The addition correlations of H2’t/H6’t in the 1H-1H NOESY

392

spectrum and C2’/H2’, C3’/H3’and C6’/H6’ of the terminal unit in the HSQC-spectra

393

further verify the configuration of B-ring. Similarly, the configuration of A-ring was 19

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also identified by the correlation of C6/H6 and C8/H8 of the terminal unit in the

395

HSQC spectra and C8t/C6t in the HMBC spectrum. The correlations of C2/H2,

396

C3/H3 and C4/H4 of the terminal unit further verify the configuration of C-ring from

397

the HSQC-spectra. NOE correlations between H2’t and H6’t (B-ring) as well as H2t

398

(C-ring) allowed the identification of B- and C-rings of both units. The correlations of

399

C1’t/H2t and C10t/H4t in the HMBC-spectra further determine that the configuration

400

of preparative radish procyanidin dimers was flavan-3-ol unit. In addition, due to the

401

absence of NOE correlation of H2t and H4t with H3t, the terminal unit of flavan-3-ol

402

unit is identified as (―)-epicatechin (2, 3-cis configuration). Due to the absence of

403

these correlations, the upper unit was determined readily. The configuration of the

404

upper unit was also flavan-3-ol (2, 3-cis configuration). In the spectrum of HMBC,

405

the correlation of C8 of the terminal unit with H4 of the upper unit was characteristic

406

of a 4 → 8 interflavanoid linkages. In other words, this compound are linked C4 →

407

C8. Accordingly, the preparative radish procyanidin dimers was composed of

408

(―)-epicatechin units which were linked via a C4 → C8 bond (B-type), and this

409

preparative radish procyanidin dimers was (―)-epicatechin-4β → 8-(―)-epicatechin

410

(procyanidin B2).

411

In Conclusions, the present study showed that red radish was successively treated

412

with methanol extraction and macroporous resins separation to separate radish

413

procyanidin oligomers (RPO) and polymers (RPP). After that, the purity of

414

procyanidins (PP) and procyanidin dimers (PD) would achieve 41.5 ± 1.6% and 84.3

415

± 0.8% by 40:1 (v/m) methanol extraction and amilan macroporous resins separation. 20

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Depolymerization of radish procyanidin polymers (RPP) into oligomers was then

417

conducted by N-acetylneuraminate lyase (NAL). The HPLC-MS analysis of the

418

depolymerization product indicated that procyanidin polymers were successfully

419

depolymerized by NAL. Then high purity of procyanidin dimers was prepared by the

420

depolymerized procyanidin oligomers and PRO. The high purity of procyanidin

421

dimers (95.3 ± 0.2%) was prepared by preparative high performance liquid

422

chromatography. The complete structures of the preparative radish procyanidin dimers

423

were elucidated using high resolution mass spectrum, one- and two-dimensional

424

NMR.

425

(―)-epicatechin-4β → 8-(―)-epicatechin (procyanidin B2). This study presented a

426

desirable process for efficient production of high purity of procyanidin dimers from

427

red radish.

428

ACKONWLEDGEMENT

This

preparative

radish

procyanidin

dimers

was

identified

as

429

I would like to express my gratitude to all those who have helped me during the

430

writing of this manuscript. I gratefully acknowledge the help of my supervisor

431

professor Zhou Xiaohua. I do appreciate his patience, encouragement and professional

432

instruction for my manuscript. And I also appreciate the help of Dr. Yang Yang and

433

professional Zhou Zhiming. All authors approved the final manuscript.

434

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L. Screening of foods containing proanthocyanidins and their structural characterization

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using LC-MS/MS and thiolytic degradation, J. Agric. Food Chem. 2003, 51, 7513-7521.

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Anal. Chem. 2007, 79, 1739-1748. 26

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FUNDING SOURCES

549

This work was supported by the National Natural Science Foundation of China

550

(21206175 and 315014682), the Industrial Biotechnology Program of Tianjin

551

Municipal

552

Fundamental

553

106112017CDJXFLX0014), and the Henan Provincial Science and technology Open

554

cooperation projects (162106000014). This work is also partially supported by Open

555

Funding Project of the State Key Laboratory of Bioreactor Engineering, Shanghai,

556

China.

Science

and

Research

Technology Funds

for

Commission the

Central

(14ZCZDSY00066), Universities

27

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(Project

the No.

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FIGURE CAPTIONS Figure 1. Degradation mechanism of N-acetylneuraminic acid and procyanidin polymers. Figure 2. Influence of the ratio of methanol to radish on extraction yield, purity of procyanidin and purity of procyanidin dimers. Extraction temperature 50

o

C,

extraction time=2 h. Figure 3. HPLC chromatograms of (a) RPO, (b) RPP and (c) the depolymerization product of RPP at λ 280 nm. Figure 4. The effects of the condition parameters on the radish procyanidin polymers depolymerization (a. pH, t=50 oC, NAL/RPP=0.05, depolymerization reaction time=4 h; b. Reaction temperature, pH=8.0, NAL/RPP=0.05, depolymerization reaction time=4 h; c. The mass ratio of NAL to RPP, pH=8.0, t=50 oC, depolymerization reaction time=4 h). Figure 5. Preparative HPLC purification of radish procyanidin dimers (a. Preparative HPLC analysis of radish procyanidin dimers; b. HPLC analysis of the preparative radish procyanidin dimers). Figure 6. High resolution mass spectrum (a), structure of A-type and B-type (u: upper unit; t: terminal unit) (b) and postulated fragmentation (c) of the preparative radish procyanidin dimers. Figure 7. One-dimensional 1H NMR (a) and 13C NMR (b) spectra of the preparative radish procyanidin dimers. Figure 8. The two-dimensional NMR of 1H-1H COSY (a), 1H−1H NOESY (b), HSQC 28

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(c) and HMBC (d) spectra of the preparative radish procyanidin dimers.

29

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Table 1. The Adsorption and Desorption Behavior of The Resins to Total Procyanidins.

Resins

Adsorption capacity(mg/g)

Adsorption ratio(%)

Amilan

7.43 ± 0.07

93.3 ± 0.9

6.84 ± 0.06

92.1 ± 0.9

83.5

84.3

85.1

D-101

4.47 ± 0.05

53.8 ±0.6

4.13 ± 0.07

92.4 ± 1.4

68.4

69.5

70.7

AB-8

4.63 ± 0.10

57.6 ± 1.2

4.24 ± 0.07

91.6 ± 0.7

67.4

68.1

68.8

ADS-17

5.01 ± 0.09

62.1 ± 0.5

4.67 ± 0.09

93.2 ± 1.0

70.5

71.9

73.3

NKA-9

5.46 ± 0.12

67.8 ± 0.8

4.63 ± 0.10

84.8 ± 0.8

72.4

74.1

72.4

ADS-7

5.29 ± 0.09

64.5 ± 1.2

4.35 ± 0.07

82.2 ± 0.7

78.4

79.7

81.1

Desorption capacity(mg/g) Desorption ratio(%)

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Purity of procyanidins(%)

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Table 2. The Adsorption and Desorption Behavior of The Resins to Procyanidin Dimers.

Resins

Adsorption capacity(mg/g)

Adsorption ratio(%)

Amilan

3.79 ± 0.05

94.7 ± 1.3

3.46 ± 0.04

91.3 ± 1.1

39.9

41.5

43.1

D-101

1.31 ± 0.01

32.8 ±0.3

1.25 ± 0.02

95.4 ± 1.5

28.6

29.3

30.1

AB-8

1.32 ± 0.03

33.0 ± 0.7

1.22 ± 0.02

92.5 ± 1.5

27.3

28.5

29.7

ADS-17

1.68 ± 0.03

42.1 ± 0.7

1.58 ± 0.03

94.0 ± 1.7

30.1

31.5

32.9

NKA-9

2.23 ± 0.05

55.8 ± 1.3

1.91 ± 0.04

85.6 ± 1.7

30.7

32.8

34.9

ADS-7

2.42 ± 0.04

60.5 ± 1.0

2.03 ± 0.03

83.9 ± 1.1

34.2

35.7

37.2

Desorption capacity(mg/g) Desorption ratio(%) Purity of procyanidin dimer(%)

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Table 3. One-way ANOVA Result of The Purity of Procyanidins Processed By Different Resins.

Variation source

Freedom degrees number

Sum of Squares

Mean Square

F function calculated

F0.05

F0.01

Between Groups

2

16.8

8.40

59.31

4.10

7.56

Within Groups

5

587.7

117.54

829.69

3.33

5.64

Error

10

1.42

0.14

Total

17

605.92

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Journal of Agricultural and Food Chemistry

Table 4. One-way ANOVA Result of The Purity of Procyanidin Dimers Processed By Different Resins.

Variation source

Freedom degrees number

Sum of Squares

Mean Square

F function calculated

F0.05

F0.01

Between Groups

2

24.65

12.33

57.42

4.10

7.56

Within Groups

5

346.46

69.29

322.79

3.33

5.64

Error

10

2.15

0.21

Total

17

373.26

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Figure 1

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Figure 2

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Figure 3

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Figure 4

60

b

50

40

40

30 20 10

30 20

4

6

8

10

10

pH

c

60

50 DY (%)

DY (%)

a

30

40

50 DY (%)

60

Reaction temperature (oC)

60

40 30 20 10 0.00

50

0.02 0.04 0.06 0.08 0.10 The mass ratio of NAL to PPC

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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TOC Graphic

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