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Biotechnology and Biological Transformations

Biotransformation of Food-Derived Saponins, Platycosides, into Deglucosylated Saponins Including Deglucosylated Platycodin D and Their Anti-Inflammatory Activities Su-Hwan Kang, Tae-Hun Kim, Kyung-Chul Shin, Yoon-Joo Ko, and Deok-Kun Oh J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06399 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Journal of Agricultural and Food Chemistry

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Biotransformation of Food-Derived Saponins, Platycosides,

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into Deglucosylated Saponins Including Deglucosylated

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Platycodin D and Their Anti-Inflammatory Activities

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Su-Hwan Kang,† Tae-Hun Kim,† Kyung-Chul Shin,† Yoon-Joo Ko,‡ and Deok-

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Kun Oh*,†

7 8 9 10

†Department

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Korea

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‡National

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Seoul 08826, Republic of Korea

of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of

Center for Inter-University Research Facilities (NCIRF), Seoul National University,

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ABSTRACT: The Platycodon grandiflorum root, Platycodi radix, one of common

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vegetables, and its extract with glycosylated saponins, platycosides, have been used as a food

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item and food health supplements for pulmonary diseases and respiratory disorders. Enzymes

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convert glycosylated saponins into deglycosylated saponins, which exhibit higher biological

19

activity than glycosylated saponins. In this study, β-glucosidase from the hyperthermophilic

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bacterium Dictyoglomus turgidum converted platycosides in the Platycodi radix extract into

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deglucosylated platycosides. In addition, the enzyme completely converted platycoside E

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(PE), platycodin D3 (PD3), and platycodin D (PD) in Platycodi radix extract into

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deglucosylated platycodin D (deglu PD), which was first identified by nuclear magnetic

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resonance. The anti-inflammatory activities of deglu PD and deglucosylated Platycodi radix

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extract were higher than those of PE, PD3, PD, Platycodi radix extract, and baicalein, an anti-

26

inflammatory agent. Therefore, deglucosylated Platycodi radix extract is expected to be used

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as improved functional food supplements.

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KEYWORDS: Platycodi radix, deglucosylated platycodin D, Dictyoglomus turgidum, β-

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glucosidase, biotransformation, anti-inflammatory activity, functional food supplements

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INTRODUCTION

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Platycodon grandiflorum, a species of perennial herbaceous flowering plant, belongs to the

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family Campanulaceae and is commonly known as balloon flower. In Northeast Asia,

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Platycodi radix (root of Platycodon grandiflorum), one of the most common vegetables, has

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been used to make side dish (seasoned balloon flower root), dessert (balloon flower root

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sweet), tea (balloon flower root tea), and flavored liquor. Platycodi radix extract has been

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widely used as dietary supplements for pulmonary diseases and respiratory disorders such as

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cough, asthma, bronchitis, cold, sore throat, tonsillitis, tuberculosis, inflammation, and chest

43

congestion.1

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Recently, saponins in Platycodi radix extract, platycosides, have been shown to have pharmacological

activities,

including

anti-bacterial,2

anti-obesity,3-5

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diverse

anti-

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inflammatory,6 anti-allergy,7 anti-oxidant,8 immune stimulation, and anti-tumor effects.9,10

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Platycosides have two side chains linked to a pentacyclic triterpene aglycon; one side chain is

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the β-glucose residues that are linked by the glycosidic bond at C-3 in the aglycon and the

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other is an oligosaccharide moiety of arabinose, rhamnose, xylose, and apiose that are

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sequentially attached to the ester linkage at C-28 (Figure 1).

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Deglycosylated saponins exhibit higher biological activity than that of glycosylated

52

saponins, and deglucosylated saponins are absorbed in the human body more easily than

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glucosylated saponins.11,12 Several enzymes convert glycosylated platycosides into

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deglycosylated platycosides. β-Glucosidase from Aspergillus usamii13 can convert

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platycoside E (PE) and platycodin D3 (PD3) into platycodin D (PD) and snailase,14

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cellulase,15 and laminarinase16 can convert deapiosylated platycoside E (deapi PE) and PE

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into deapiosylated platycodin D (deapi PD) and PD via deapiosylated platycodin D3 (deapi

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PD3) and PD3, respectively. Crude enzyme from Aspergillus niger convert PD into deapiose3 ACS Paragon Plus Environment


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xylosylated platycodin D (deapi-xyl PD).17 However, these enzymes have not been able to

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convert platycosides to deglucosylated platycodin (deglu PD). Although human intestinal

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bacteria have converted PD in Platycodi radix extract into PD metabolites via deglu PD as an

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intermediate suggested by liquid chromatography-mass spectrometry (LC-MS3),18 the

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biotransformation of glucosylated platycosides into deglu PD has not been attempted and

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deglu PD has never been identified by nuclear magnetic resonance (NMR) to date. Therefore,

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the pharmacological activity of deglu PD has not been investigated.

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In the present study, the biotransformation of platycosides in Platycodi radix extract into

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deglucosylated platycosides was performed by β-glucosidase from the hyperthermophilic

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bacterium Dictyoglomus turgidum. After the biotransformation, the chemical structures of

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deglucosylated platycosides were determined. Moreover, the lipoxygenase inhibitory

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activities (anti-inflammatory activities) of deglu PD and deglucosylated platycosides in

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Platycodi radix extract were investigated for the application of deglucosylated Platycodi radix

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extract as improved functional food supplements.

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MATERIALS AND METHODS

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Preparation of Platycoside Standards. The platycoside standards deapi PE (CAS No.

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849758-42-5, with 88% purity), PE (CAS No. 237068-41-6, with 99% purity), deapi PD3

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(CAS No. 67884-05-3, with 93% purity), PD3 (CAS No. 67884-05-3, with 87% purity), and

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PD (CAS No. 58479-68-8, with 99% purity) were purchased from Ambo Institute (Daejeon,

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Republic of Korea). Polygalacin D (CAS No. 66663-91-0, with 95% purity) was kindly

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provided by Doctor Dae Young Lee of the National Institute of Horticultural and Herbal

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Science (Eumseong, Republic of Korea). Dried Platycodi radix was purchased from a

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traditional market (Seoul, Republic of Korea). 4 ACS Paragon Plus Environment

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Deapi PD (CAS No. 78763-58-3, with 89% purity), platycodin A (PA, CAS No. 66779-34-

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8, with 89% purity), 3″-O-acetyl polygalacin D3 (with 90% purity) standards were prepared

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by purification from Platycodi radix extract. Deapi deglu PD, deglu PD, deglu polygalacin D,

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3″-O-acetyl polygalacin D, deglu PA, and deglu 3″-O-acetyl polygalacin D standards were

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made by the purification of deglucosylated platycosides obtained from the biotransformation

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of platycosides in Platycodi radix extract by β-glucosidase from D. turgidum. The

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platycosides in the Platycodi radix extract and deglucosylated platycosides were applied to a

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Preparative high-performance liquid chromatography (Prep-HPLC) (Agilent 1260, Santa

92

Clara, CA, U.S.A.) equipped with a Hydrosphere C18 prep column (10 × 250 mm, 5 µm

93

particle size; YMC, Kyoto, Japan), UV detector at 203 nm, and a fraction collector. The

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column was eluted by water with a flow rate of 4.7 mL/min at 30 °C. The collected samples

95

were purified to approximately 90% purity, which was estimated by comparison between the

96

standards and purified compounds of PE and PD (Figure S1). The purified platycosides were

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used as the substrates and standards.

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Culture Conditions. The gene of β-glucosidase from D. turgidum DSM 6724 (DSMZ,

100

Braunschweig, Germany) was cloned as described previously.19 Recombinant Escherichia

101

coli ER2566 (New England Biolabs, Hertfordshire, UK) containing the β-glucosidase gene of

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D. turgidum (GenBank Accession Number YP_002352162) in pET24a(+) plasmid was

103

cultured at 37 °C in a 2-L flask containing 500 mL Luria−Bertani (LB) medium

104

supplemented with 20 µg/mL kanamycin with agitation at 200 rpm. As the optical density of

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bacterial culture broth at 600 nm was 0.6−0.8, isopropyl-β-D-thiogalactopyranoside at 0.1

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mM was added to the broth to induce expression of β-glucosidase. The strain was then

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cultured at 16 °C with agitation at 150 rpm for a further 14 h to express the enzyme.

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Enzyme Preparation. Recombinant E. coli expressing β-glucosidase from D. turgidum

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was harvested from the culture broth by centrifugation at 6,000 × g at 4 °C for 30 min,

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washed with 0.85% NaCl, and suspended in 50 mM citrate/phosphate buffer (pH 6.0). The

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suspended cells were lysed by sonication on ice for 20 min. The lysate was centrifuged at

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13000 × g at 4 °C for 20 min and the supernatant obtained was heated at 70 °C for 10 min.

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The insoluble proteins aggregated by heating were precipitated and eliminated by

115

centrifugation at 13000 × g for 20 min and the remaining supernatant was filtered with a 0.45

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µm filter. The filtrate was used as the purified enzyme for the biotransformation of

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platycosides. The purified β-glucosidase from D. turgidum was visualized by SDS-PAGE

118

stained with Coomassie blue and its purity was determined from the SDS-PAGE using

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ImageJ program (National Institutes of Health).

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Preparation of Platycodi Radix Extract. Platycodi radix was prepared according to

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the extraction method of the ginseng root.20 To prevent Maillard reactions between free

123

sugars and enzyme at temperatures above 70 °C, the sugar-free Platycodi radix extract was

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prepared as follows: 100 g of dried Platycodi radix powder was suspended in 1 L of absolute

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methanol (99.8%). After the suspension was incubated at 80 °C for 12 h, the precipitates

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were removed by filtering the mixture through a 0.45 µm filter. The methanol in the filtrate

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was eliminated by evaporation and the methanol-free residue was dissolved in 1 L of distilled

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water. The Platycodi radix extract was loaded onto a column packed with Diaion HP20 resin

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(length × diameter: 500 mm × 12 mm) to adsorb platycosides onto the resin. The

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platycosides-adsorbed resin was eluted with distilled water to eliminate the free sugars and

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other hydrophilic compounds and then sequentially eluted with methanol to extract the

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adsorbed platycosides at a flow rate of 0.5 mL/min. The methanol in the eluent was

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eliminated by evaporation and the residue was then dissolved in 1 L of distilled water. The 6 ACS Paragon Plus Environment

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sugar-free Platycodi radix extract was used for the biotransformation of platycosides in

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Platycodi radix extract into deglu PD.

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Biotransformation. The biotransformation of the reagent-grade PE into deglu PD by β-

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glucosidase from D. turgidum was performed at 80 °C in 50 mM citrate/phosphate buffer (pH

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6.5) containing 0.005 mg/mL enzyme and 0.65 mM PE for 7 h. The biotransformation of PE,

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PD3, and PD in the Platycodi radix extract into deglu PD was performed at 75 °C in 50 mM

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citrate/phosphate buffer (pH 6.5) containing 0.005 mg/mL enzyme and the Platycodi radix

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extract containing 0.65 mM PE, 0.03 mM PD3, and 0.22 mM PD for 20 h.

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Lipoxygenase

Inhibitory Activity. The lipoxygenase inhibitory activity of

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platycosides for presenting anti-inflammatory activity was measured using a lipoxygenase

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inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI, U.S.A.) at 0.4 and 4 µM of

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each sample. Nordihydroguaiaretic acid (NDGA), a standard lipoxygenase inhibitory

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chemical, and baicalein (5,6,7-trihydroxyflavone), an anti-inflammatory agent, were used as

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positive controls. Test samples and positive controls were dissolved in methanol. A test

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sample (10 µL) and 15-lipoxygenase (90 µL) were placed in the testing wells. The reactions

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were initiated by adding arachidonic acid (10 µL) to each well. All wells were placed on a

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shaker and mixed well for 5 min, and chromogen (100 µL) was then added to the wells to

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terminate the enzyme reaction. After the reaction, the hydroperoxide level produced by 15-

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lipoxygenase from arachidonic acid was measured by reading the UV absorbance at 500 nm.

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The lipoxygenase inhibitory activity (%) was calculated as (C−T)/C × 100, where C and T

156

were the values of the UV absorbance at 500 nm without and with the test sample,

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respectively.

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Identification of Platycosides. Liquid chromatography-mass spectrometry2 (LC-MS2)

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analysis was performed to identify the chemical structures of platycosides using a Thermo-

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Finnigan LCQ Deca XP plus ion trap mass spectrometer (Thermo Scientific, Waltham, MA,

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U.S.A.) at the NICEM (Seoul National University, Seoul, Republic of Korea). Ionization of

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the samples was performed using electrospray ionization at 275 °C capillary temperature, 30

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psi nebulizer gas, 5 kV ion source voltage, 46 V capillary voltage in positive mode, 15 V

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fragmentor voltage in negative ionization mode, 0.01 min average scan time, 0.02 min

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average time to change polarity, and 35% abundant precursor ions at collision energy.

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1D (1H, 13C, selective-TOCSY, and 1H homo decoupling) and 2D (COSY, ROESY,

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TOCSY, HSQC, and HMBC) NMR spectra were recorded on a Bruker Avance III HD (850

169

MHz) equipped with a TCI cryoprobe (NCIRF, Seoul National University, Seoul, Republic

170

of Korea) to confirm the structures. Pyridine-D5 was used as a solvent and an internal

171

standard for 1H (8.74 ppm) and 13C (150.35 ppm). All chemical shifts were quoted in δ (ppm).

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HPLC Analysis. n-Butanol was added to the reaction solution with the same volume to

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terminate the reaction and extract the product, resulting in separation into n-butanol and water

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fractions. The n-butanol fraction of the extracted solution was obtained and the n-butanol in

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the fraction was evaporated until it was completely dry. After drying, methanol was added to

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the residue. Platycosides dissolved in methanol were analyzed using an HPLC system

178

(Agilent 1100) equipped with an evaporation light scattering detector (ELSD) and a

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hydrosphere C18 column (4.6 × 150 mm, 5 µm particle size, YMC, Kyoto, Japan). The

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column was eluted at 30 °C with a gradient of solvent A (acetonitrile) and solvent B (water)

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from 10:90 to 40:60 for 30 min, from 40:60 to 90:10 for 15 min, from 90:10 to 10:90 for 5

182

min, and constant at 10:90 for 10 min at a flow rate of 1 mL/min. ELSD was set to an

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evaporator and a gas spray nebulizer at 50 °C at a gas flow rate of 1.6 standard liters per min. 8 ACS Paragon Plus Environment

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The linear calibration curves relating the logarithmic value of the peak areas to the

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concentrations of platycosides were constructed using the standard solutions of platycosides

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containing 0.2 to 1.0 mM in triplicate and the curves were used for the determination of the

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concentrations of platycosides. The regression equations for the linear calibration curves of

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15 platycosides are presented in Table S1.

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RESULTS AND DISCUSSION

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Preparation of β-Glucosidase from D. turgidum for the Biotransformation of

193

Platycosides. The gene (2,247 base pairs) encoding β-glucosidase from D. turgidum, with

194

the same sequence as that in GenBank (YP_002352162), was cloned and expressed in E. coli

195

as described previously.19,21,22 The expressed enzyme was purified from the crude extract as a

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soluble protein by heat treatment with a 6.7-fold final purification, 53% yield, and 0.3

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µmol/min/mg specific activity for PE. The purity of the enzyme was estimated to be

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approximately 90.5% (Figure S2). The purified enzyme was used for the biotransformation of

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platycosides.

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β-Glucosidases from hyperthermophilic bacteria have been applied to the deglycosylation

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of glycosylated phytochemicals because they have higher hydrolytic activities than those of

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mesophilic β-glucosidases.23-25 β-Glucosidase from the hyperthermophilic bacterium D.

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turgidum has been used for the production of isoflavone daidzein,19 as well as ginsenosides

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compound Y, compound Mc, aglycone protopanaxadiol,22 and aglycone protopanaxatriol.21

205 206

Identification of an Unknown Product Obtained from the Biotransformation

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of PD by β-Glucosidase from D. turgidum. β-Glucosidase from D. turgidum converted

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reagent-grade PD as a substrate into an unknown product. The total molecular mass of the 9 ACS Paragon Plus Environment


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unknown product was represented by a distinct peak at mass per charge (m/z) 1061.5 as the

210

[M-H]+ ion in the LC-MS spectrum (Figure 2A). This was the same as that of the

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deglucosylated form of PD at C-3. The peaks at m/z 928.5, 796.5, 650.5, and 518.5 in the LC-

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MS2 spectrum resulted from the sequential cleavage of apiose, xylose, rhamnose, and

213

arabinose, respectively (Figure 2B). These results suggest that the unknown product was

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deglu PD.

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For exact identification, the product was analyzed by NMR. The 13C-NMR spectrum of the

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compound showed 51 carbon signals, which were sorted into 6 methyl, 14 methene, 22

217

methine, and 9 quaternary carbons by 2D HSQC and HMBC experiments. There were five

218

sp3 carbons at δ 18.1, 18.1, 25.2, 27.7, and 33.7; two sp2 oleﬁnic carbons at δ 123.5 and 145.0;

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two oxygenated methylene carbons at δ 64.5 and 65.3; three oxygenated methine carbons at δ

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72.4, 74.5, and 75.7; and one carbonyl carbon at δ 176.6 in the

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aglycon (Table S2). The four anomeric carbons of the sugar chain were observed at δ 94.1,

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101.8, 107.2, and 111.8 in the

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protons (δ 5.09, 5.79, 6.22, and 6.45) were also observed in the 1H-NMR spectrum (Figure

224

S3B). The sugar chain sequence was identiﬁed as arabinoses, rhamnose, xylose, and apiose

225

by 2D COSY, ROESY, TOCSY, HSQC, and HMBC NMR experiments (Figure S4). The

226

sugar linkages at C-28 of aglycon were established by HMBC and ROESY NMR as follows:

227

from H-1 (δ 6.22) of terminal apiose to C-3 (δ 83.6) of arabinose, from H-1 (δ 5.09) of xylose

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to C-4 (δ 84.3) of rhamnose, from H-1 (δ 5.79) of rhamnose to C-2 (δ 76.0) of arabinose, and

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from H-1 (δ 6.45) of arabinose to C-28 (δ 176.6). These results indicate that the unknown

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compound is 2β,3β,16α,23,24-pentahydroxyolean-12-en-28-oic acid 28-O-β-D-apiofuranosyl-

231

(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside, deglu

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PD. Although deglu PD has previously been suggested by LC-MS3,18 it has not been

233

identified by NMR to date.

13C-NMR

13C-NMR

spectrum of the

spectrum (Figure S3A). The four sugar anomeric


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Biotransformation of PE and Platycodi Radix Extract into Deglu PD by β-

236

Glucosidase from D. turgidum. β-Glucosidase from D. turgidum at 0.005 mg/mL

237

completely converted 0.65 mM (1 mg/mL) reagent-grade PE to 0.65 mM deglu PD via PD3

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and PD within 7 h (Figure 3A). Laminarinase from Trichoderma sp. converted PE in

239

Platycodi radix extract into PD via PD3.16 The crude enzyme from A. niger converted PD in

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Platycodi radix extract into deapi-xyl PD (hydrolysate of apiose-xylose disaccharide in PD).17

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Most of the enzymes converted PE (three glucose residues) into PD (one glucose) via PD3

242

(two glucose residues), but the hydrolysis of the remaining last glucose has not been reported

243

to date except for human intestinal bacteria. Human intestinal bacteria metabolized PD to

244

arabinosyl platycodigenin via deglu PD and deapi deglu PD with sequentially hydrolyzing

245

glucose, apiose, and xylose-rhamnose disaccharide.18 Although the quantitative enzymatic

246

biotransformation of platycoside in Platycodi radix extract has been reported, the

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biotransformation of single pure platycoside has never been attempted to date.

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The total concentration of platycosides in 10% (w/v) Platycodi radix extract was 3.33

249

mg/mL and the concentration of PE, the main compound, was 1.35 mg/mL, which was 40.54%

250

(w/w) of total platycosides (Table 1). The other platycosides were polygalacin D (0.99

251

mg/mL), PD (0.36 mg/mL), PA (0.23 mg/mL), and 3″-O-acetyl polygalacin D3 (0.21 mg/mL).

252

The main compound in Platycodi radix extract was PE, PD, or other platycoside in other

253

reports because it depended on the cultivation area and solvent extraction method.26,27 The

254

time course reactions for the biotransformation of platycosides in the Platycodi radix extract

255

into deglu PD were performed (Figure 3B). The enzyme at 0.005 mg/mL completely

256

converted 0.65 mM PE, 0.03 mM PD3, and 0.22 mM PD in the Platycodi radix extract into

257

0.9 mM deglu PD after 20 h. The reaction time for the complete biotransformation of

258

platycosides in the Platycodi radix extract to deglu PD was 2.9-fold longer than that of 11 ACS Paragon Plus Environment


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reagent-grade PE. The retardation might be due to the inhibition of enzyme activity by other

260

saponins in the extract.28

261 262

Identification of Platycosides in Platycodi Radix Extract and Deglucosylated

263

Platycosides Obtained from their Biotransformation by β-Glucosidase from D.

264

turgidum. Six platycosides (peak numbers of 1, 2, 3, 4, 6, and 7) in the Platycodi radix

265

extract in the HPLC profile and total ion chromatography (TIC) were identified by detecting

266

them with the same retention times as deapi PE, PE, deapi PD3, PD3, PD, and polygalacin D

267

standards, respectively (Figure S5A, S5C). The other three platycosides in the Platycodi radix

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extract (Figure S5A, S5C) and six deglucosylated platycosides (Figure S5B, S5D) in the

269

HPLC profile and TIC were unknown compounds. These unknown compounds were

270

analyzed by LC-MS and LC-MS2. The total molecular masses were represented by the main

271

peaks in the LC-MS spectra. The fragment peaks of the other three platycosides in the

272

Platycodi radix extract in the LC-MS2 spectra resulted from the cleavage of apiose, xylose,

273

rhamnose or acetyl rhamnose, and arabinose at C-28 and the glucose at C-3 (Figure S6A-

274

S6C). These results suggested that the platycosides (peak numbers of 5, 8, and 9) in the

275

Platycodi radix extract were deapi PD, 3″-O-acetyl polygalacin D3, and PA, respectively

276

(Table S3A). The acetyl residue in acetyl polygalacin D3, was not identified by LC-MS2.

277

Moreover, 2″-O-acetyl polygalacin D3 was converted into 3″-O-acetyl polygalacin D3 by

278

inter-conversion because it was unstable in the polar solvent such as water used for the

279

purification process of acetyl polygalacin D3.29 Therefore, we expected the acetyl polygalacin

280

D3 to be 3″-O-acetyl polygalacin D3. The fragment peaks of the deglucosylated platycosides

281

resulted from the cleavage of the apiose, xylose, rhamnose, or acetyl rhamnose, and arabinose

282

at C-28 without (Figure S6D–S6F, S6H, and S6I) and with (Figure S6G) the glucose at C-3.

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Based on this analysis, deglucosylated platycosides (peak numbers of 10−15) were identified 12 ACS Paragon Plus Environment

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as the deglucosylated platycosides such as deapi deglu PD, deglu PD, 3″-O-acetyl

285

polygalacin D, deglu PA, and deglu 3″-O-acetyl polygalacin D, respectively (Table S3B). To

286

the best of our knowledge, deglu PA and deglu 3″-O-acetyl polygalacin D are first reported in

287

the present study. β-Glucosidase from D. turgidum converted all platycosides, including not

288

only PE, PD3, and PD in the Platycodi radix extract but also other platycosides into

289

deglucosylated platycosides via the hydrolysis of glucose residues at C-3. The HPLC profiles

290

and pathways during the biotransformation of platycosides into deglucosylated platycosides

291

by the enzyme are shown in Figure S7 and Figure 4, respectively.

292 293

Lipoxygenase Inhibitory Activities of Platycosides and Deglucosylated

294

Platycosides in Platycodi Radix. The lipoxygenase inhibitory activities being the anti-

295

inflammatory activities of platycosides were evaluated using a lipoxygenase inhibitory

296

screening assay kit. The lipoxygenase inhibitory activities of the positive controls, NDGA

297

and baicalein, were 59% and 44% at 4 μM, respectively (Figure 5). Those of PE, PD3, PD,

298

deglu PD, Platycodi radix extract containing platycosides, and deglucosylated Platycodi radix

299

extract containing platycosides were 41%, 44%, 49%, 55%, 42%, and 60% at 4 μM,

300

respectively. The lipoxygenase inhibitory activities of platycosides followed the order no

301

glucose (deglu PD) > one glucose (PD) > two glucose (PD3) > three glucose residues (PE) at

302

C-3 in platycosides. The reason why the biotransformed extract had higher activities than

303

deglu PD was due to the higher activity of deglu PA than deglu PD (Figure S8). The

304

lipoxygenase inhibitory activity of deglucosylated Platycodi radix extract was higher than

305

those of Platycodi radix extract and baicalein as an anti-inflammatory agent, suggesting that

306

deglucosylated Platycodi radix extract can be used as improved functional food supplements.

307

Lipoxygenases catalyze the oxidation of arachidonic acid as an initial enzyme in the

308

arachidonic acid pathway.30,31 The oxygenation products catalyzed by lipoxygenases in the 13 ACS Paragon Plus Environment


309

pathway are involved in inflammation, and thus the inhibition of lipoxygenase is helpful in

310

anti-inflammation. The anti-inflammatory activity of platycoside increased with decreasing

311

numbers of glucose residues linked to C-3. Although many pharmacological activities of

312

Platycodi radix extract or platycosides have been reported, information on these in

313

deglucosylated platycosides is limited to only deapi-xyl PD, which is produced by the crude

314

enzyme from A. niger. The platycoside demonstrated reduced toxicity and improved anti-

315

oxidation and sensory values compared with PD.17

316

In conclusion, β-glucosidase from D. turgidum with high specific activity converted

317

platycosides in the Platycodi radix extract into deglucosylated platycosides that specifically

318

hydrolyzed the glucoses at C-3 and the chemical structures were identified by LC-MS2. In

319

addition, the enzyme completely converted PE and PE, PD3, and PD in the Platycodi radix

320

extract into deglu PD, which has anti-inflammatory activity as identified by NMR. To the

321

best of our knowledge, this is the first quantitative enzymatic production and

322

pharmacological activity determination of deglu PD. The lipoxygenase inhibitory activity of

323

deglucosylated Platycodi radix extract was higher than that of baicalein as an anti-

324

inflammatory agent. Thus, the biotransformation of platycosides in the Platycodi radix extract

325

into bioactive platycosides can be applied to the preparation of Platycodi radix extract with

326

higher biological activity as improved functional food supplements.

327 328

ASSOCIATED CONTENT

329

Supporting Information

330

Table S1: Regression equations for linear calibration curves of platycosides in Platycodi

331

radix extract (numbers 1–9) and deglucosylated platycosides obtained from their

332

biotransformation by β-glucosidase from D. turgidum (numbers 10–15). Table S2: 13C and

333

1H

data of deglucosylated platycodin D. Table S3: Suggestion of (A) platycosides in 14 ACS Paragon Plus Environment

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Page 15 of 30


334

Platycodi radix extract and (B) deglucosylated platycosides obtained from their

335

biotransformation by β-glucosidase from D. turgidum. Figure S1: HPLC chromatograms

336

for the standards and purified compounds of (A) PE and (B) PD. Figure S2: Purity

337

determination of the purified β-glucosidase from D. turgidum in SDS-PAGE stained with

338

Coomassie blue using ImageJ program. Lanes: M, marker protein; 1, plasmid pET 24a(+);

339

2, crude extract; 3, purified enzyme. Figure S3: 1D NMR data of deglu PD. (A) 13C NMR

340

and (B) 1H NMR peaks of deglu PD. Figure S4: 2D NMR of deglu PD. (A) COSY, (B)

341

ROESY, (C) TOCSY, (D) HSQC, and (E) HMBC of deglu PD. Figure S5: HPLC profiles

342

of (A) platycosides in Platycodi radix extract and (B) deglucosylated platycosides

343

obtained from their biotransformation by β-glucosidase from D. turgidum after 20 h. The

344

total ion chromatograms (TICs) of (C) platycosides in Platycodi radix extract and (D)

345

deglucosylated platycosides. Figure S6: LC-MS2 analysis of (A) 5, deapi PD, (B) 8, 3″-O-

346

acetyl polygalacin D3, (C) 9, PA, (D) 10, deapi deglu PD, (E) 11, deglu PD, (F) 12, deglu

347

polygalacin D, (G) 13, 3″-O-acetyl polygalacin D, (H) 14, deglu PA, and (I) 15, deglu 3″-

348

O-acetyl polygalacin D. Figure S7: HPLC profiles during the biotransformation of

349

platycosides in Platycodi radix extract by β-glucosidase from D. turgidum. Figure S8:

350

Lipoxygenase inhibitory activities of deglucosylated platycosides. (PDF)

351 352 353

AUTHOR INFORMATION

354

Corresponding Author

355

* Phone: (822) 454-3118. Fax: (822) 444-5518. E-mail: [email protected]

356

ORCID

357

Deok-Kun Oh: 0000-0002-6886-7589

358

Funding 15 ACS Paragon Plus Environment


359

This study was supported by Konkuk University in 2016.

360

Notes

361

The authors declare no competing financial interest.


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modification of saponins from Platycodon grandifiorum with Aspergillus niger. Journal of

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Agricultural and Food Chemistry 2007, 55, 8908-8913.

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spectrometry-based structural analysis of new platycoside metabolites transformed by human

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(19) Kim, Y. S.; Yeom, S. J.; Oh, D. K., Characterization of a GH3 family -glucosidase

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from Dictyoglomus turgidum and its application to the hydrolysis of isoflavone glycosides in

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spent coffee grounds. Journal of Agricultural and Food Chemistry 2011, 59, 11812-11818.

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(20) Kim, S. K.; Kwak, Y. S.; Kim, S. W.; Hwang, S. Y.; Ko, Y. S.; Yoo, C. M.,

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Improved method for the preparation of crude ginseng saponin. J. Ginseng. Res. 1998, 22,

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(21) Lee, H. J.; Shin, K. C.; Lee, G. W.; Oh, D. K., Production of aglycone

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protopanaxatriol from ginseng root extract using Dictyoglomus turgidum -glycosidase that

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specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-

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type ginsenosides. Applied Microbiology and Biotechnology 2014, 98, 3659-3667.

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(22) Lee, G. W.; Kim, K. R.; Oh, D. K., Production of rare ginsenosides (compound Mc,

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compound Y and aglycon protopanaxadiol) by -glucosidase from Dictyoglomus turgidum

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that hydrolyzes linked, but not -linked, sugars in ginsenosides. Biotechnology Letters

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2012, 34, 1679-1686.

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(23) Shin, K. C.; Kim, T. H.; Choi, J. H.; Oh, D. K., Complete biotransformation of

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protopanaxadiol-type ginsenosides to 20-O--glucopyranosyl-20(S)-protopanaxadiol using a

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novel and thermostable -glucosidase. Journal of Agricultural and Food Chemistry 2018, 66,



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2822-2829. (24) Shin, K. C.; Nam, H. K.; Oh, D. K., Hydrolysis of flavanone glycosides by -

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glucosidase from Pyrococcus furiosus and its application to the production of flavanone

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aglycones from citrus extracts. Journal of Agricultural and Food Chemistry 2013, 61, 11532-

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11540.

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(25) Yeom, S. J.; Kim, B. N.; Kim, Y. S.; Oh, D. K., Hydrolysis of isoflavone glycosides

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by a thermostable -glucosidase from Pyrococcus furiosus. Journal of Agricultural and Food

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Chemistry 2012, 60, 1535-1541.

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(26) Yoo, D. S.; Choi, Y. H.; Cha, M. R.; Lee, B. H.; Kim, S. J.; Yon, G. H.; Hong, K. S.;

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Jang, Y. S.; Lee, H. S.; Kim, Y. S.; Ryu, S. Y.; Kang, J. S., HPLC-ELSD analysis of 18

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platycosides from balloon flower roots (Platycodi Radix) sourced from various regions in

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Korea and geographical clustering of the cultivation areas. Food Chem 2011, 129, 645-651.

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(27) Ha, Y. W.; Na, Y. C.; Seo, J. J.; Kim, S. N.; Linhardt, R. J.; Kim, Y. S., Qualitative

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and quantitative determination of ten major saponins in Platycodi Radix by high performance

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liquid chromatography with evaporative light scattering detection and mass spectrometry.

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Journal of Chromatography A 2006, 1135, 27-35.

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(28) Son, J. W.; Kim, H. J.; Oh, D. K., Ginsenoside Rd production from the major

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ginsenoside Rb1 by -glucosidase from Thermus caldophilus. Biotechnology Letters 2008,

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(29) Zeng, L. F.; Zhu, M.; Zhong, J. L.; Yan, W. D., Structural stability of acetyl saponins in different solvents and separation materials. Phytochemistry Letters 2015, 11, 368-372.

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(30) Wei, A.; Shibamoto, T., Antioxidant/lipoxygenase inhibitory activities and chemical

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D.; Liu, R. H., The inhibitory activities of the components of Huang-Lian-Jie-Du-Tang

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(HLJDT)

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Ethnopharmacology 2011, 135, 561-568.

on

eicosanoid

generation

via

lipoxygenase

465


pathway.

Journal

of


467

Figure captions

468 469

Figure 1. Chemical structures of platycosides in Platycodi radix and deglucosylated

470

platycosides obtained from their biotransformation by β-glucosidase from D. turgidum.

471

Platycosides in Platycodi radix and deglucosylated platycosides by β-glucosidase from D.

472

turgidum are numbers 1 to 9 and numbers 10 to 15, respectively. Deapi PE, deapiosylated

473

platycoside E; PE, platycoside E; deapi PD3, deapiosylated platycodin D3; PD3, platycodin D3;

474

deapi PD, deapiosylated platycodin D; PD, platycodin D; PA, platycodin A; deapi deglu PD,

475

deapiosylated deglucosylated platycodin D; deglu PD, deglucosylated platycodin D; and

476

deglu PA, deglucosylated platycodin A. Platycosides contain glycosides at C-3 and C-28. In

477

platycosides, glycosides at C-3 are Glc, Glc-Glc, and Glc-Glc-Glc and glycosides at C-28 are

478

Ara-Rham (or Rham(Ac))-Xyl-Api. Glc, β-D-glucopyranose-; Ara, α-L-arabinopyranose-;

479

Rham, α-L-rhamnopyranose-; Xyl, β-D-xylopyranose-; Api, β-D-apiosefuranose-; and Ac,

480

acetyl.

481 482

Figure 2. (A) LC-MS and (B) LC-MS2 analysis of deglu PD. The red asterisk in the LC-MS

483

indicates the total molecular mass of deglu PD.

484 485

Figure 3. (A) Biotransformation of reagent-grade PE into deglu PD. The reactions were

486

performed in 50 mM citrate/phosphate buffer (pH 6.5) containing 0.005 mg/mL enzyme and

487

0.65 mM PE at 80 °C for 7 h. (B) Biotransformation of PE, PD3, and PD in the Platycodi

488

radix extract into deglu PD. The reactions were performed in 50 mM citrate/phosphate buffer

489

(pH 6.5) containing 0.005 mg/mL enzyme and 0.65 mM PE, 0.03 mM PD3, and 0.22 mM PD

490

in Platycodi radix at 75 °C for 20 h. The symbols ▲, △, ○, and ● represent PE, PD3, PD, and

491

deglu PD, respectively. Data represent the means of three experiments and error bars 22 ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30

492


represent the standard deviations.

493 494

Figure 4. Biotransformation pathways of platycosides into deglucosylated platycosides by β-

495

glucosidase from D. turgidum.

496 497

Figure 5. Lipoxygenase inhibitory activities of PE, PD3, PD, and deglu PD, Platycodi radix

498

extract containing platycosides, and deglucosylated Platycodi radix extract containing

499

platycosides. The black and gray bars represent 0.4 and 4 µM, respectively.

500

Nordihydroguaiaretic acid (NDGA) and baicalein (5,6,7-trihydroxyflavone) were used as

501

positive controls. NDGA and baicalein are a standard lipoxygenase inhibitory chemical and

502

an anti-inflammatory agent, respectively.



Page 24 of 30

Table 1. Contents of platycosides in 10% (w/v) Platycodi radix extract no.

platycoside

1

Deapi PE

2

PE

3

content (%, w/w)

concentration (mg/mL)

2.70

0.09

40.54

1.35

Deapi PD3

0.30

0.01

4

PD3

1.80

0.06

5

Deapi PD

0.90

0.03

6

PD

10.81

0.36

7

Polygalacin D

29.73

0.99

8

3"-O-Acetyl polygalacin D3

6.31

0.21

9

PA

6.91

0.23

Total

100

3.33


Page 25 of 30


Figure 1



A

B

Figure 2 26 ACS Paragon Plus Environment

Page 26 of 30

Page 27 of 30


A

Concentration (mM)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

0

1

2

3

4

5

6

7

Time (h)

B

Concentration (mM)

1.0

0.8

0.6

0.4

0.2

0.0

0

4

8

12

Time (h)

Figure 3


16

20


Figure 4 28

ACS Paragon Plus Environment

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Figure 5

29



For Table of Contents Only


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