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0J Ginseng Res xxx (2015) 1—9
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Research article
Microbial conversion of major ginsenosides in ginseng total saponins by Platycodon grandiflorum endophytes
Lei Cui1, Song-quan Wu \ Cheng-ai Zhao 2, Cheng-ri Yin
1 Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University Yanji, China
2 College of Resources and Environmental, Jilin Agricultural University Changchun, China
ARTICLE INFO
Article history: Received 6 February 2015 Received in Revised form 14 November 2015 Accepted 27 November 2015 Available online xxx
Keywords: conversion endophytes minor ginsenosides Platycodon grandiflorum
ABSTRACT
Background: In this study, we screened and identified an endophyte JG09 having strong biocatalytic activity for ginsenosides from Platycodon grandiflorum, converted ginseng total saponins and ginsenoside monomers, determined the source of minor ginsenosides and the transformation pathways, and calculated the maximum production of minor ginsenosides for the conversion of ginsenoside Rb1 to assess the transformation activity of endophyte JG09.
Methods: The transformation of ginseng total saponins and ginsenoside monomers Rb1, Rb2, Rc, Rd, Rg1 into minor ginsenosides F2, C-K and Rh1 using endophyte JG09 isolated by an organizational separation method and Esculin-R2A agar assay, as well as the identification of transformed products via TLC and HPLC, were evaluated. Endophyte JG09 was identified through DNA sequencing and phylogenetic analysis.
Results: A total of 32 ß-glucosidase-producing endophytes were screened out among the isolated 69 endophytes from P. grandiflorum. An endophyte bacteria JG09 identified as Luteibacter sp. effectively converted protopanaxadiol-type ginsenosides Rb1, Rb2, Rc, Rd into minor ginsenosides F2 and C-K, and converted protopanaxatriol-type ginsenoside Rg1 into minor ginsenoside Rh1. The transformation pathways of major ginsenosides by endophyte JG09 were as follows: Rb1 /Rd/F2/C-K; Rb2/C-O/C-Y/C-K; Rc/C-Mc1/C-Mc/C-K; Rg1/Rh1. The maximum production rate of ginsenosides F2 and C-K reached 94.53% and 66.34%, respectively.
Conclusion: This is the first report about conversion of major ginsenosides into minor ginsenosides by fermentation with P. grandiflorum endophytes. The results of the study indicate endophyte JG09 would be a potential microbial source for obtaining minor ginsenosides.
Copyright © 2015, The Korean Society of Ginseng, Published by Elsevier. All rights reserved.
1. Introduction
Ginseng, the root of Panax ginseng Meyer (Araliaceae), has been a greatly valued herb in Asian countries for over 1,000 yr [1]. It consists of ginsenosides, polysaccharides, polyynes, flavonoids, and volatile oils among other components [2]. Ginsenosides are considered the major active components of ginseng species and are responsible for numerous pharmacological properties, such as antitumor, anti-aging, blood vessel softening, anticancer, antiinflammatory, and hepatoprotective effect activities [3—5]. Degly-cosylated ginsenosides (minor ginsenosides Rh1, Rh2, F2, Rg3, C-K,
etc.) are more pharmaceutically active than major glycosylated ginsenosides (Rb1, Rb2, Rc, Rd, Rg1, etc.), because of their smaller size, higher bioavailability, and better permeability across the cell membrane [6]. However, minor ginsenosides are not present in most natural ginseng plants or are present in very low amounts. Therefore, the study of converting major ginsenosides to minor ginsenosides is of great significance [7]. Various approaches including heating [8], acid treatment [9], and biotransformation [10] have been carried out. Among these methods, biotransformation is shown to be the most effective way to produce minor gin-senosides [9,11,12]. Biotransformation is a high specificity, low cost,
* Corresponding author. Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Yanbian University, Gongyuan Road 977, Yanji 133002, China.
E-mail address: cryin@ybu.edu.cn (C.-r. Yin).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
p1226-8453 e2093-4947/$ — see front matter Copyright © 2015, The Korean Society of Ginseng, Published by Elsevier. All rights reserved. http://dx.doi.org/10.1016/jogr.2015.11.004
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1 selective, and environmentally friendly technique, and can be
2 defined as chemical reactions catalyzed by different enzymes of
3 various microbial cells; it can be an alternative for biosynthesis of
4 some important drug metabolites. It also exhibits many advantages
5 over chemical synthesis such as stereo- or region-selectivity, mild
6 reaction conditions, and avoiding complex protection and depro-
7 tection steps [13,14].
8 Endophytes are considered to be bacterial or fungal microor-
9 ganisms that colonize healthy plant tissues without causing any
10 apparent symptoms [15]. Recently, the application of endophytes to
11 biotransformation has also attracted people's attention. Due to the
12 endophytes special living environment and long term coexistence
13 Q2 with their hosts, the endophytes have developed a specific lifestyle
14 to maintain a stable symbiosis, and they can produce various
15 extracellular enzymes for secondary metabolite biosynthesis.
16 Therefore, endophytes have been used for some complex reactions
17 to obtain more active compounds instead of traditional chemical
18 methods [16]. The application of endophytes to biotransformation
19 of region- and stereo-selective production is a suitable method to
20 obtain more active substances; they have been used in natural
21 compound conversion such as flavans, tetrahydrofuran lignin, and
22 astragalosides [17—19].
23 To date, however, no relevant studies have been reported on the
24 conversion of major ginsenosides to minor ginsenosides by endo-
25 phytes isolated from Platycodon grandiflorum. The present study
26 aimed to enhance the content of minor ginsenosides in ginseng
27 total saponins by endophytes from P. grandiflorum. The optimiza-
28 tion of the bioconversion conditions was conducted by single factor
29 tests of the bioconversion procedure.
31 2. Materials and methods
33 2.1. Materials
35 Q3 HPLC-grade methanol and acetonitrile (ACN) were supplied by
36 Merck (Darmstadt, Germany). Ultrapure water was prepared by a
37 Milli-Q purification system (Millipore, Bedford, MA, USA). Ginse-
38 nosides Rb1, Rb2, Rc, Rd, Rg1, Rg3, Rh1, Rh2, Re, F2, C-K and PPD
39 were purchased from Chengdu Mann Stewart Biological Technol-
40 ogy Co., Ltd. (Sichuan, China). The other chemicals used in this
41 study were at least of analytical reagent grade.
43 2.2. Standard solutions and standard curve preparation
45 Stock solutions in methanol were prepared for ginsenosides F2
46 and C-K in concentrations of 2.0 mg/mL and 3.0 mg/mL, respec-
47 tively. The stock solutions were then diluted with methanol to yield
48 a series of standard solutions with different concentrations for
49 linearity validation and standard curve preparation. Working
50 methanolic solutions were prepared daily from stock solutions.
52 2.3. Isolation and screening of b-glucosidase-producing endophytes
54 Plant samples were obtained from a P. grandiflorum field in Yanji,
55 China. The P. grandiflorum was kept refrigerated at 4°C, and endo-
56 phytes were isolated within 48 h.
57 The P. grandiflorum samples were washed adequately under
58 running tap water to remove soil, then surface-disinfected through
59 the following sequence of immersions: 1min in 70% (v/v) ethanol,
60 5—10 min in 5% NaClO, 1 min in 70% (v/v) ethanol, and 1 min in
61 sterile water four times. The outer tissue was removed with a sterile
62 scalpel. Small segments (0.5 cm x 0.5 cm) were cut and pressed on
63 Q4 a potato dextrose agar plates (PDA), R2A agar plates and LB agar
64 plates and incubated at 30° C for 5—7 d. Single colonies from these
65 plates were purified by transferring onto new plates.
Esculin-R2A agar was used to isolate ß-glucosidase-producing 66
endophytes. The endophytes producing ß-glucosidase that hydro- 67
lyze esculin appeared as colonies surrounded by a reddish-brown 68
to dark brown zone. Esculin-R2A agar contains (per 1 L): esculin 69
1 g and ferric citrate 0.5 g with 15.2 g R2A agar, autoclaved at 121°C 70
for 15 min. 71
2.4. Bioconversion of major ginsenosides in ginseng total saponins 73
2.4.1. Transformation of ginseng total saponins 75
The ß-glucosidase-producing endophytes were cultured in LL 76
medium consisting of 0.5 g/L NH4Cl, 1.0 g/L K2HPO4,0.5 g/L KH2PO4, 77
0.25 g/L MgSO4, and 1.0 g/L yeast extract. The medium was steril- Q5 78
ized at 121°C for 15 min, and the initial pH was 7.0. The suspension 79
of the strain was incubated until it reached logarithmic phase 80
growth, then mixed with the same volume of 15 mg/L ginseng total 81
saponins solution and incubated in a shaking incubator (30° C, 82
150 rpm). The culture was extracted with the same volume of 83
aqueous saturated n-BuOH and concentrated in a vacuum to dry- 84
ness, then the residue was dissolved in methanol and analysis of 85
the biotransformation ability of ginsenosides was carried out by 86
TLC. 87
In order to explore the source of minor ginsenosides and the 88
conversion pathway by endophytes, we mixed the suspension 89
culture of endophytes and ginsenosides Rb1, Rb2, Rc, Rd, and Rg1 90
and confirmed at regular intervals by TLC and HPLC analysis. 91
2.5. Optimization of fermentation conditions 93
Microbial fermentation requires a suitable fermentation me- 95
dium, temperature and pH. The optimum fermentation condition is 96
the key to getting a high yield of fermentation products success- 97
fully; in order to obtain the best conversion for ginsenosides, 98
single-factor experiments were performed to optimize the medium 99
and pH value. Six culture mediums (H2O, R2A, PDB, LB, NB, LL, initial Q6 100
pH of 7.0) were tested one by one. Subsequently, fermentation 101
conditions, on the basis of the optimum medium, at pH values of 102
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 were investigated. 103
2.6. Analytic methods 105
2.6.1. TLC analysis 107 TLC was carried out using 60F254 silica gel plates (Merck) with 108
CHCl3-CH3OH-H2O (10:5:1, v/v/v, lower phase) as the solvent. The 109
spots on the TLC plates were visualized by spraying with 10% (v/v) 110
H2SO4 solution, followed by heating at 110° C for 5 min. 111
2.6.2. HPLC analysis 113 HPLC analysis was performed using an HPLC system with 114
automatic injector and single wavelength UV detector. The sepa- 115
ration was carried out on a BDS HYPERSIL C18 (5 mm, 116
250 mm x 4.6 mm; Agilent 1100, USA) with a column temperature Q7 117
of 25°C. The mobile phase was A (water) and B (acetonitrile). 118
Gradient elution started with 77% solvent A and 23% solvent B and 119
was then changed to: A from 77% to 54%, 0-13 min; A from 54% to 120
32%, 13-33 min; A from 32% to 0%, 45-55 min; A from 0% to 77%, 121
60-63 min. The flow rate was 1.0 mL/min, and samples were 122
detected by absorption at 203 nm, with an inject volume of 10 mL. 123
2.7. Phylogenetic analysis 125
The endophyte genomic DNA was extracted by the method of 127
CTAB [20]. The 16S ribosomal DNA was amplified using universal Q8 128
bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R 129
(5'-GGTTACCTTGTTACGACTT-3') [21]. The PCR reaction system was 130
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Q16 Fig. 1. Gel electrophoresis of recombinant plasmid DNA of endophyte JG09.
carried out in volumes of 20 pL, containing 2 pL of template DNA, 2 mL of each primer, 2 mL of dNTPs, 2 mL of 10 x PCR buffer (containing Plus Mg2+), 0.4 mL of TaKaRa Taq enzyme, and 10 mL of ddH2O. The amplification program was as follows: an initial denaturation at 95°C for 3 min, 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 90 s, and a final extension at 72°C for 7 min.
The PCR products were purified using a Silica Bead DNA Gel Q9 Extraction Kit (Fermentas Life Sciences, China), in order to obtain more complete sequence information, then ligated into a pGEM-Teasy Vector (Biofine, China), and subsequently used to transform JM109 competent cells. Transformed cells carrying inserts were identified with blue/white colony selection. Recombinant plasmids in transformed cells were cloned and then sequenced (Shanghai Biotechnology Co., Ltd. Megiddo).
The obtained sequencing results were checked for homology using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/ BLAST), and compared with available data from GenBank sequence databases, getting the closest strain's rDNA sequence. The resulting alignment was used to construct a Neighbor-joining (NJ) qio phylogenetic tree using MEGA 4.0 software [22].
3. Results and discussion
3.1. Isolation and phylogenetic analysis of b-glucosidase-producing endophytes
A total of 69 endophytes were isolated from P. grandiflorum by the method of organizational separation. After the Esculin-R2A agar assay, 32 р-glucosidase-producing microorganisms were assayed to verify their activity for converting ginseng total sapo-nins. Among these endophytes, strain JG09 showed the strongest activity for converting ginseng total saponins into minor ginseno-sides C-K, F2 and Rh1.
The genomic DNA of strain JG09 was extracted and amplified, recombined and sequenced. Strain JG09 was an endophytic bacte-qii ria; after a series of operations with the extracted DNA, such as PCR and recombinant plasmid generation, the resulting gel electro-phoresis figure showed the DNA length to be approximately
Table 1
Similarity between the isolates and closest species in GenBank
Strain ID Accession number
Closest known species
Similarity (%)
JG09 KC342052 Luteibacter sp. NORREL-Li2
FR727720 Luteibacter sp. IMP04
JF778702 Luteibacter rhizovicinus strain HRT22
1,600 bp (Fig. 1). The obtained sequences were compared with those in the GenBank database, and the results are shown in Table 1.
Phylogenetic analysis based on 16S rDNA gene sequences indicated that the strain JG09 belongs to the genus Lucteibacter, and endophyte JG09 is most closely related to Luteibacter sp. NORREL-Li2 (99%), as seen in Fig. 2.
3.2. Effect of pH and culture medium on bioconversion of major ginsenosides
The culture medium must meet the demand of microbial cell growth and reproduction, because a large number of cells are needed to generate large amounts of product. However, excessive growth of cells will consume a large amount of nutrients; this sometimes can affect the production capacity of the cell, resulting in a decreased yield of product. So an appropriate media is essential to a microbial strain.
In order to compare the effect of various media, strain JG09 was cultured in the broths of H2O, R2A, PDB, LB, NB and LL, at an initial pH of 7.0, mixing with ginseng total saponins for 7 d. The suspension were analyzed by TLC (Fig. 3). The cultures in PDB broth and LB broth couldn't convert major ginsenosides into C-K, but LL broth fermented byJG09 efficiently converted major ginsenosides into F2 and C-K (Fig. 3), with the maximum degree of conversion of major ginsenosides. This suggested that LL broth was not only suitable for the growth of the strain JG09, but also for the production of F2 and C-K.
The pH value of the culture medium mainly causes the change in the charge of the microorganism's cytomembrane, and impacts the ionization degree of nutrients, thereby affecting absorption of nutrients by the microbial cells. The pH also affects the biologically active substances, such as enzyme activity. Different microbes have different optimum pH values to adapt to the environment. The optimum pH for microbial growth ranges from 4.0 to 9.0 in general. Therefore, fermentation conditions, on the basis of the optimum medium, at pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 were investigated. The results indicated that the optimum pH of strain JG09 was 4.0 (Fig. 4), which could maximize the content of generated minor ginsenosides F2 and C-K.
3.3. Bioconversion of major ginsenosides in ginseng total saponins
Under optimized fermention conditions, endophyte JG09 was inoculated (3%, v/v) into a 250 mL Erlenmeyer's flask containing 100 mLof sterilized liquid broth and 1.5 g ginseng total saponins for a period of 10 d in a shaking incubator (30°C, 150 rpm). Comparing the change of saponin content before and after fermentation by HPLC analysis, the content of protopanaxadiol-type major saponins (Rb1, Rb2, Rc, and Rd) and protopanaxatriol-type major saponins (Rg1 and Re) was generally decreased, and Rb1 was almost completely transformed after 10 d of incubation. At the same time, a significant increase in the content of minor ginsenosides F2, C-K, and Rh1 was observed (Fig. 5). To further analyze the source of minor ginsenosides F2, C-K, and Rh1, major ginsenosides Rb1, Rb2, Rc, Rd, and Rg1 were incubated with endophyte JG0; these standards of ginsenosides were then injected into HPLC. Q13
3.4. Bioconversion pathways of major ginsenosides
In order to investigate the biotransformation pathway of major ginsenosides into minor ginsenosides by endophyte JG09, the conversion of ginsenosides Rb1, Rb2, Rc, Rd, and Rg1 was carried out by inoculating the ginsenoside into precultured suspensions, followed by 7 d of incubation, during which different individual samples from the same batch at different times were extracted. As
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65 Q17
Luteibacter sp. V87 (DQ778039)
Luteibacter rhizovicinus strain TX1B2 (KJ589471) — Luteibactor rhizovicina isolate OUCZ70 (AY785744) Luteibacter rhizovicinus strain LJ96 (NR042197) ' Luteibactor rhizovicina strain LJ79 (AJ580497) " Luteibacter rhizovicinus strain IMER-B1-47 (FJ796463) Luteibacter sp. IMP04 (FR727720)
-Luteibacter sp. AJM040 (FR714940)
Luteibacter rhizovicinus strain HRT22 (JF778702)
Luteibacter sp. NORREL-LÍ2 (KC342052)
Luteibacter sp. b8(2) (GU975789)
_Г Luteibacter yeojuensis strain: NBRC 106387 (AB682403)
-Luteibacter yeojuensis strain R2A16-10 (NR 043618)
Fig. 2. A 16S rDNA sequenced-based phylogenetic tree constructed by the Neighbor joining method. The numbers at the nodes are bootstrap values based on 1,000 replications.
shown in Fig. 6, it is clear that endophyte JG09 could convert major ginsenosides Rbl, Rb2, Rc, and Rd into minor ginsenosides F2 and C-K and convert major ginsenoside Rgl into minor ginsenoside Rhl, based on the Rf values of standards.
The HPLC profile of the reaction mixture of ginsenoside Rbl and the endophyte JG09 after incubation is shown in Fig. 7. In the HPLC chromatogram, Rbl was hydrolyzed by endophyte JG09 to produce ginsenosides Rd, F2, and C-K, following 7 d of incubation. The transformation products of ginsenosides Rb2, Rc, Rd, and Rgl were also analyzed by HPLC, which yielded the same results to the findings of TLC (Fig. 6).
In the coculture of endophyte JG09 and ginsenoside Rbl, endophyte JG09 converted ginsenoside Rbl into C-K as well as Rd and F2; the concentration of Rbl, Rd, F2, and C-K exhibited regular change with reaction time. From 4 h to 9 h, the content of ginsenoside Rbl and Rd gradually decreased and that of F2 and C-K gradually increased. Ginsenoside Rd was completely converted after 9 h of incubation. It proved that the metabolite Rd was the
precursor of metabolite F2 and C-K. Fig. 7 shows that a peak area of ginsenoside F2 increased after 4 h with the generation of ginse-noside Rd, but decreased after 1 d of incubation. In the meantime, the content of ginsenoside C-K significantly increased after 1 d. The peak area of ginsenoside F2 reached the maximum value within 9 h, then the generation of ginsenoside C-K went into the rapidly developing period and this stage should take a longer time. At last, ginsenoside Rb1 was completely transformed and the corresponding peak disappeared (Figs. 6A, 7). This suggested that Rb1 was converted in the following sequence: Rb1 /Rd/F2/C-K, as shown in the pathway in Fig. 8, by the different b-glucosidase from endophyte JG09, consecutively hydrolyzing the terminal glucose and inner glucose at the C-3 position, and terminal glucose at the C-20 position.
Combining TLC with HPLC results, we also confirmed the conversion pathways of major ginsenosides Rb2, Rc, Rd, and Rg1 by endophyte JG09, the results are as follows: Rb2/C-O/C-Y/C-K;
Fig. 3. TLC analysis of metabolites of ginseng total saponins by endophyte JG09 cultured in different media: H2O (1), R2A (2), PDB (3), LB (4), NB (5), and LL (6). LB, ; LL, ; NB, ; PDB, ; R2A, ; S1 and S2, saponin standards.
Fig. 4. TLC analysis of metabolites of ginseng total saponins by endophyte JG09 cultured at different pH's of 3.0 (1), 4.0 (2), 5.0 (3), 6.0 (4), 7.0 (5), 8.0 (6), 9.0 (7), and 10.0 (8). S1 and S2, saponin standards.
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Fig. 5. HPLC analysis of (A) ginseng total saponins and (B) fermented ginseng total saponins. The peaks correspond to ginsenoside Rg1, Re, Rh1, Rb1, Rc, Rb2, Rd, F2, and C-K, respectively.
Fig. 6. Time-course TLC analyses of ginsenoside (A) Rb1, (B) Rb2, (C) Rc, (D) Rd, and (E) Rg1 by endophyte JG09. C, blank sample; S1—S5, standard.
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Fig. 7. (A) HPLC profile of the metabolites of ginsenoside Rbl converted by endophyte JG09. (B) The content change of ginsenoside Rbl, Rd, F2, and C-K in the fermentation broth.
Rc/C-Mcl/C-Mc/C-K; Rd/F2/C-K; Rgl/Rhl, as shown in the pathways in Figs. 8, 9.
3.5. Standard curve preparation and the maximum production rate calculation for minor ginsenoside F2 and C-K
Fig.l0 shows the standard curve of ginsenosides F2 and C-K by plotting the area against the concentrations of ginsenosides F2 and C-K. Under these conditions, the full linear range of the assay was extended from 0.003l25 mg/mL to 0.75 mg/mL, as indicated in
Fig. 10. The linear correlation coefficients R2 are 0.9985 and 0.9991, respectively, so the standard curve can be used to calculate the maximum yield of ginsenosides F2 and C-K during the transformation. By using the molecular weight and the initial concentration of Rb1, the theoretical yield of ginsenosides F2 and C-K were calculated. Under optimum conditions, when Rb1 was added to the culture broth of endophyte JG09, the content of ginsenosides F2 and C-K could reach a maximum value in 7 d. In order to determine the maximum production rate of minor ginsenosides F2 and C-K and assess the transformation effectiveness of endophyte JG09 to
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Fig. 8. Biotransformation pathway of ginsenosides Rb1, Rb2, Rc, and Rd by endophyte JG09.
ginsenoside Rb1, calculations were carried out using peak area and the standard curve of ginsenosides. The results show that the maximum production yield of ginsenoside F2 at 9 h reaches 94.53%, the maximum production yield of ginsenoside C-K at 7 d reaches 66.34% (Table 2).
It is well known that P. ginseng has various ingredients including ginsenosides and their derivatives. To date, ginsenosides have been isolated and identified structurally from approximately 60 species of the Panax genus [23]. Major ginsenosides such as Rb1, Rb2, Rc, Rd, and Rg1 are present in greater abundance than any other gin-senosides, however minor ginsenosides Rh1, F2, and C-K have greater pharmacological effects such as tumor-suppressing, anti-metastatic, anticarcinogenic, hepatoprotective, neuroprotective,
immune-stimulating and vasodilating effects, which are of great significance for human health, though their content is relatively low in ginseng. The anticancer activity of ginsenoside F2 was already applied and noticed [24,25]. Ginsenoside C-K is the main pharmacologically active metabolite detected in blood after the oral administration of major ginsenosides [26,27]. It has been shown that ginsenoside C-K possesses various biological activities in vivo or in vitro, such as antigenotoxic activity, antiallergic effect, and antitumor activity [27]. In addition, ginsenoside C-K shows potential hepatoprotective and anti-inflammatory activities and the pharmacological effects of ginsenoside C-K appear to be stronger than those of known minor ginsenosides and some pharmaceutical drugs including diphenyl dimethyl bicarboxylate and indomethacin
Fig. 9. Biotransformation pathway of ginsenosides Rg1 by endophyte JG09.
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Fig. lO. Standard curve of ginsenoside F2 and C-K.
[4,5,28—30]. There is a study showing that ginsenoside Rbl is hy-drolyzed by intestinal microorganism in the body and is changed to minor ginsenoside F2 [3l]. Some studies have looked for suitable microbes or enzymes that can transform major ginsenosides into minor ginsenosides [3,l2,32,33]. However, most methods suffer from the fact that they are small scale, difficult, expensive, and low yielding, and there are only a few reports of transforming major ginsenosides into minor ginsenosides by potential resource endophytes.
In this study, we isolated 32 ß-glucosidase-producing endophytes from P. grandiflorum and identified endophyte JG09 as Luc-teibacter sp., which shows a strong ability to bioconvert major ginsenosides into minor ginsenosides, as confirmed by TLC and HPLC analysis. This is the first report of conversion of major gin-senosides to minor ginsenosides by fermentation with P. grandiflorum endophytes. We found that endophyte JG09 can hydrolyze protopanaxadiol ginsenosides Rbl, Rb2, Rc, and Rd into minor ginsenosides F2 and C-K, and hydrolyze protopanaxatriol ginsenoside Rgl into minor ginsenoside Rhl simultaneously. The method was simple, low cost, and high yielding after optimizing the fermentation conditions. This new finding could have great significance in the research of ginsenosides, and this biotransformation has great potential to be applied in the preparation of minor ginsenosides F2 and C-K in the pharmaceutical industry.
Table 2
Actual yield and production rate of ginsenoside F2 and C-K
Conflicts of interest
All contributing authors declare no conflicts of interest.
Acknowledgments
This work was supported by the Natural Science Foundation of Jilin Province of China (No. 20150101184JC) and the National Natural Science Foundation of China (No. 20862017).
References
Strain Ginsenoside F2 Ginsenoside C-K
Peak area (9 h) Actual mass (mg) Yield (%) Peak area Actual mass (7 d) (mg) Yield (%)
JG09 872.3 0.06692 94.53 599.8 0.03323 66.34
Qi LW, Wang CZ, Yuan CS. Isolation and analysis of ginseng: advances and challenges. Nat Prod Rep 2011;28:467-95.
Jia L, Zhao Y. Current evaluation of the millennium phytomedicine-ginseng (I): etymology, pharmacognosy, phytochemistry, market and regulations. Curr Med Chem 2009;16:2475-84.
Wang L, Liu QM, Sung BH, An DS, Lee HG. Bioconversion of ginsenosides Rb1, Rb2, Rc and Rd by novel ß-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: Cloning, expression, and enzyme characterization. J Biotechnol 2011;156:125-33.
Li W, Zhang M, Zheng YN, Li J, Wang YP, Wang YJ, Gu J, Jin Y. Snailase preparation of ginsenoside M1 from protopanaxadiol-type ginsenoside and their protective effects against CCl4-induced chronic hepatotoxicity in mice. Molecules 2011;16:10093-103.
Chen Y, Xu Y, Zhu Y, Li X. Anti-cancer effects of ginsenoside compound k on pediatric acute myeloid leukemia cells. Cancer Cell Int 2013;13:24. Xu QF, Fang XL, Chen DF. Pharmacokinetics and bioavailability of ginsenoside Rb1 and Rg1 from Panax notoginseng in rats. J Ethnopharmacol 2003;84: 187-92.
Shin KC, Oh DK. Characterization of a novel recombinant ß-glucosidase from Sphingopyxis alaskensis that specifically hydrolyzes the outer glucoseat the C-3 position in protopanaxadiol-type ginsenosides. J Biotechnol 2014;172: 30-7.
Kim WY, Kim JM, Han SB, Lee SK, Kim ND, Park MK, Kim CK, Park JH. Steaming of ginseng at high temperature enhances biological activity. J Nat Prod 2000;63:1702-4.
Bae EA, Han MJ, Kim EJ, Kim DH. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch Pharm Res 2004;27:61-7. Q15 Noh KH, Son JW, Kim HJ, Oh DK. Ginsenoside compound K production from ginseng root extract by a thermostable beta-glycosidase from Sulfolobus solfataricus. Biosci Biotechnol Biochem 2009;73:316-21. Chi H, Ji GE. Transformation of ginsenosides Rb1 and Re from Panax ginseng by food microorganisms. Biotechnol Lett 2005;27:765-71. Wu LP, Jin Y, Yin CR, Bai LL. Co-transformation of Panax major ginsenosides Rb1 and Rg1 to minor ginsenosides C-K and F1 by Cladosporium cladospor-ioides. J Ind Microbiol Biotechnol 2012;39:521-7.
Yang XB, Hou J, Liu D, Deng S, Wang ZB, Kuang HX. Biotransformation of isoimperatorin by Cunninghamella blakesleana AS 3.970. J Mol Catal B: Enzym 2013;88:1-6.
Lv X, Liu D, Hou J, Dong PP, Zhan LB, Wang L. Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis activities of its metabolites. Food Chem 2013;138:2260-6.
Wilson D. Endophyte: the evolution of a term, and clarification of its use and definition. Oikos 1995;73:274-6.
Wang Y, Dai CC. Endophytes: a potential resource for biosynthesis, biotransformation and biodegradation. Ann Microbiol 2011;61:207-15. Agusta A, Maehara S, Ohashi K, Simanjuntak P, Shibuya H. Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant. Chem Pharm Bull 2005;53:1565-9.
Verza M, Arakawa NS, Lopes NP, Kato MJ, Pupo MT, Said S. Biotransformation of a tetrahydrofuran lignin by the endophytic fungus Phomopsis sp. J Braz Chem Soc 2009;20:195-200.
Yao ML, Liu JZ, Jin S, Jiao J, Gai QY, Wei ZF, Fu YJ, Zhao JD. A novel biotransformation of astragalosides to astragaloside IV with the deacetylation of fungal endophyte Penicillium canescens. Process Biochem 2014;49:807-12. Porebski S, Bailey LG, Baum BR. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 1997;15:8-15.
Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, et al. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 2012;62:716-21.
Tamura K, Dudley J, Nei M, Kumar S. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24: 1596-9.
Yang JL, Gao LL, Zhu P. Advances in the biosynthesis research of ginsenosides. Acta Pharmaceutica Sinica 2013;48:170-8.
80 81 82
99 100 101 102
110 111 112
120 121 122
L. Cui et al / Ginsenoside conversion by Platycodon grandiflorum endophytes
10 11 12 13
[24] Mai TT, Moon J, Song Y, Viet PQ, Phuc PV, Lee JM, Yi TH, Cho M, Cho SK. Ginsenoside F2 induces apoptosis accompanied by protective autophagy in breast cancer stem cells. Cancer Lett 2012;321:144-53.
[25] Shin JY, Lee JM, Shin HS, Park SY, Yang JE, Cho SK, Yi TH. Anti-cancer effect of ginsenoside F2 against glioblastoma in xenocraft model in SD rats. J Ginseng Res 2012;36:86-92.
[26] Hasegawa H. Proof of the mysterious efficacy of ginseng: basic and clinical trials: metabolic activation of ginsenoside: deglycosylation by intestinal bacteria and esterification with fatty acid. J Pharmacol Sci 2004;95:153-7.
[27] Bae EA, Choo MK, Park EK, Park SY, Shin HY, Kim DH. Metabolism of ginse-noside Rc by human intestinal bacteria and its related antiallergic activity. Biol Pharm Bull 2002;25:743-7.
[28] Lee ES, Choi JS, Kim MS, You HJ, Ji GE, Kang YH. Ginsenoside metabolite compound K differentially antagonizing tumor necrosis factor-a induced monocyte-endothelial trafficking. Chem Biol Interact 2011;194:13-22.
[29] Lee HU, Bae EA, Han MJ, Kim DH. Hepatoprotective effect of ginsenoside Rb1 and compound K on tert-butyl hydroperoxide-induced liver injury. Liver Int 2005;25:1069-73.
[30] Park EK, Shin YW, Lee HU, Kim SS, Lee YC, Lee BY, Kim DH. Inhibitory effect of ginsenoside Rb1 and compound K on NO and prostaglandin E2 biosyntheses of RAW264.7 cells induced by lipopolysaccharide. Biol Pharm Bull 2005;28: 652-6.
[31] Ko SR, Suzuki Y, Suzuki K, Choi KJ, Cho BG. Marked production of ginsenosides Rd, F2, Rg3, and compound K by enzymatic method. Chem Pharm Bull 2007;55:1522-7.
[32] Cheng LQ, Kim M, Lee JW, Lee YJ, Yang DC. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia. Biotechnol Lett 2006;28:1121-7.
[33] Fu Y, Yin ZH, Wu LP, Yin CR. Diversity of cultivable ß-glycosidase-producing microorganisms isolated from the soil of a ginseng field and their ginsenosides-hydrolysing activity. Lett Appl Microbiol 2013;58:138-44.
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