1
Fermentative transformation of ginsenoside Rb1 from Panax ginseng C.A. Meyer to Rg3
2
and Rh2 by Lactobacillus paracasei subsp. tolerans MJM60396
3
Sasikumar Arunachalam Palaniyandi1,2,†, Byeong Mo Son1,†, Karthiyaini Damodharan2,3, Joo-Won
4
Suh2,3,* and Seung Hwan Yang1,2,*
5
1
6
Pharmaceutical Materials, 3Division of Bioscience and Bioinformatics, College of Natural Science,
7
Myongji University, Cheoin-gu, Yongin, Gyeonggi-Do 449-728, Korea.
Graduate School of Interdisciplinary program of Biomodulation, 2Center for Nutraceutical and
8
9
10
*
Correspondence
SH Yang:
11
12
Phone: 82-31-330-6880; Fax: 82-31-321-7361
E-mail: [email protected]
JW Suh:
13
Phone: 82-31-330-6190; Fax: 82-31-321-7361
E-mail: [email protected]
14
15
†
These authors contributed equally to this work
16
17
18
Short Title: Ginsenoside transformation by Lactobacillus paracasei
19
20
21
22
23
24
25
26
1
27
Abstract
28
Lactic acid bacteria (LAB) were screened for ginsenoside transforming activity using crude ginseng
29
extract. TLC analysis of fermented ginseng extract showed that LAB strain MJM60396 possessed
30
higher transformation ability converting major ginsenosides into minor ginsenosides such as Rg3 and
31
Rh2. MJM60396 also showed high β-glucosidase activity. Strain MJM60396 was identified based on
32
16S rRNA gene sequence as Lactobacillus paracasei subsp. tolerans. Further, the strain MJM60396
33
was incubated with pure ginsenoside Rb1 to delineate the pathway of production of the minor
34
ginsenosides Rg3, and Rh2. HPLC analysis of the samples showed the appearance of Rg3 and Rh2 peak
35
from reaction mixture containing Rb1. Furthermore, β-glucosidase enzyme was prepared from strain
36
MJM60396 and pH and temperature were optimized for maximum activity. The cell-free enzyme
37
hydrolyzed the ginsenoside Rb1 along the following pathway: ginsenoside Rb1 → Rd → Rg3 → Rh2.
38
This is the first report of transformation of ginsenosides Rb1 to Rg3 and Rh2 by a Lac. paracasei
39
subsp. tolerans strain. Our results indicate that Lac. paracasei subsp. tolerans MJM60396 has the
40
potential to be applied for the preparation of ginsenosides Rg3 and Rh2 for nutraceutical preparations.
41
42
Keywords: Transformation, Ginsenoside Rg3, Ginsenoside Rh2, fermentation, Lactobacillus
43
paracasei subsp. tolerans
44
45
46
47
48
49
50
51
52
53
54
2
55
56
Introduction
Ginseng (Panax ginseng C.A.Meyer), a member of the Arialiaceae family of plants, is a
57
widely used medicinal plant in oriental medicine for thousands of years [1]. Ginseng roots are the
58
main part of the plant that contains high amount of the pharmacological components called
59
ginsenosides, which are triterpenoid glycosides [2]. These triterpenoid glycosides are grouped into
60
three major types such as the dammarane-type aglycones protopanaxadiol (PPD) and protopanaxatriol
61
(PPT), ocotillol-type and oleanane-type aglycones [3]. The number of naturally occurring
62
ginsenosides and their derivatives is debatable as the literature suggests variable numbers. However,
63
the most abundantly occurring ginsenosides are ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1, which
64
constitute more than 90% of the total ginsenosides in ginseng roots [4].
65
Of the various ginsenosides, ginsenoside Rg3, which is present in Korean red ginseng
66
(steamed and dried ginseng roots) and absent from Korean white ginseng (fresh ginseng roots), has
67
been shown to exert high anti-cancer activity compared to major ginsenosides like Rb1 and Rc [5].
68
Ginsenoside Rg3 was shown to exhibit anti-angiogenic activity towards lung cancer, when combined
69
with low-dose cyclophosphamide [6] and gemcitabine [7], anti-metastatic activity in intestinal
70
adenocarcinoma in rats and B16 melanoma in mouse [8,9]. In addition, Rg3 has been shown to
71
sensitize cancer cells to drugs such as docetaxel [10], doxorubicin [11] and cisplatin [12] and has
72
potential to treat drug-resistant cancer. Evaluation of ginsenoside Rg3 combined with gemcitabine and
73
cisplatin in patients with advanced oesophageal cancer showed that the combination treatment is
74
effective in inhibiting angiogenesis of esophageal cancer, reduced chemotherapy side effects, and
75
improved the patient’s life and survival rates [13]. More clinical trials in cancer patients have
76
validated the adjuvant effect of ginsenoside Rg3 in cancer treatment [14,15].
77
On the other hand, ginsenoside Rh2 is a minor ginsenoside not originally produced in the
78
plant but can be obtained by enzymatic conversion or microbial fermentation of major ginsenosides
79
through a pathway that include Rg3 as an intermediate [16]. Ginsenoside Rh2 has been shown to
80
possess 5 to 15-fold stronger anti-proliferative activity in various cancer cell lines compared to Rg3
81
[17]. Ginsenoside Rh2 has been shown to induce apoptosis and reversal of multi-drug resistance in
3
82
MCF7/ADM cell lines [18] and inhibition of invasiveness in glioblastoma [19]. However, there are no
83
human clinical trials to date that validate the anti-cancer effects of ginsenoside Rh2.
84
The concentrations of ginsenoside Rg3 and Rh2 are extremely low in fresh ginseng, and red
85
ginseng possess variable amounts of these ginsenosides and contain several artefactual compounds
86
[20]. Also, production of red ginseng is time consuming and labour intensive. Alternatively,
87
ginsenosides Rg3 and Rh2 can be produced through microbial fermentation of whole ginseng extract
88
or purified major ginsenosides such as Rb1, Rc, and Rb2. In this study several lactic acid bacteria
89
(LAB) isolated from fermented milk were screened for ginsenoside transforming activity. A candidate
90
strain Lactobacillus paracasei subsp. tolerans MJM60396 was further studied for its ability to
91
transform major ginsenoside Rb1 to Rg3 and Rh2. Whole ginseng extracts and purified Rb1 were
92
fermented with MJM60396 and transformed ginsenosides were analyzed and quantified by TLC and
93
HPLC, respectively. Furthermore, β-glucosidase from MJM60396 was studied for its ginsenoside
94
transforming ability, and pH and temperature for maximum conversion of Rb1 to Rg3 and Rh2 was
95
optimized.
96
97
Materials and methods
98
Microbial strains and growth conditions
99
Lactic acid bacteria (LAB) were isolated from fermented milk samples collected from
100
Tiruchirappalli district, Tamil Nadu, India. The samples were serially diluted and plates on de Man
101
Rogosa and Sharpe (MRS) agar medium and incubated at 37°C for 48 h. The colonies were picked
102
and used for screening ginsenoside-transforming activity.
103
104
105
Preparation of Ginseng saponin extract
One hundred gram of Panax ginseng roots were washed with tap water, chopped and dried
106
under shade to remove moisture. The dried roots were ground into fine powder and extracted with 30
107
volumes of 80% ethanol at 80°C for 1 h (Shaking water bath) and filtered through 3 mm filter paper
108
(Whatman). The remainder was then extracted with 20 volumes of 80% ethanol and filtered. After an
4
109
additional extraction, ginseng extract was evaporated under reduced pressure to remove the ethanol
110
using a vacuum evaporator and then dissolved in 10 volumes of water.
111
112
113
Screening for ginsenoside-transforming activity of LAB strains
The bacterial strains were cultured in 1 ml of MRS liquid medium for 16 h. The bacterial
114
suspension (500 µl) was added to 10 ml of MRS liquid medium with 0.5% Cysteine-HCl containing 1
115
ml of ginseng extract and incubated anaerobically at 37°C for 7 days. After 7 days, the microbial
116
culture was centrifuged at 5000 g to remove the microbial cells and the supernatant was extracted
117
with equal volumes of n-butanol. The n-butanol fraction was collected after overnight incubation at
118
room temperature and evaporated to dryness using a vacuum evaporator. The resultant extract was
119
dissolved in 1 ml of methanol and analyzed by thin-layer chromatography (TLC). TLC was performed
120
with silica gel plates (60 F254, Merck, Darmstadt, Germany) with a developing solvent of
121
chloroform: methanol: water (65:35:10, v/v/v, lower phase). Spots on the TLC plates were detected by
122
spraying the plates with 10 % H2SO4, followed by heating at 110°C for 10 min [21].
123
124
Screening of LAB strains for β-glucosidase activity
125
β-glucosidase activity of LAB strains was tested using Esculin-R2A agar according to [22].
126
The LAB strains were spot inoculated on Esculin-R2A agar in square dishes and incubated at 37°C
127
for 3 days. Simultaneously, the LAB strains were cultured in MRS broth for 48 h and the culture
128
supernatant was tested for β-glucosidase activity by esculin gel diffusion assay [23].
129
130
131
Phylogenetic analysis of candidate strain
Phylogenetic analysis of the candidate LAB strain was done using 16S rRNA gene sequence. The
132
genomic DNA of the strain was isolated using genomic DNA isolation kit (GeneALL, Seoul,
133
Republic of Korea), following manufacturer’s protocol. The16S rRNA gene sequence of the strain
134
was amplified using the forward primer 27F (5’-AGAGTTTGAT CCTGGCTCAG-3’) and the reverse
135
primer 1492R (5’-GGTTACCT TGTTACGACTT-3’) and the PCR products were sequenced by
136
SolGent sequencing company (SolGent, Republic of Korea) with the primers 27F and 785F (5’5
137
GGATTAGATACC CTGGTA-3’) to get a partial 16S rRNA gene sequence. The sequence was
138
BLAST searched for similar sequences in the Eztaxon database and a neighbour-joining phylogenetic
139
tree was constructed by MEGA 6 software [24] using sequences showing greater than 97% similarity.
140
141
142
Transformation of ginsenoside Rb1 by LAB strain MJM60396
The LAB strain MJM60396 was cultured in 10 ml MRS liquid broth by inoculating a single
143
colony from a pure culture from a MRS agar medium. The inoculated MRS broth was incubated at
144
37°C for 24 h. After 24 h of incubation, the cells were harvested by centrifugation at 8000 g for 10
145
min. The cells were washed with sterile phosphate buffered saline (PBS) twice.
146
The LAB strain MJM60396 cells prepared as described above was used for the transformation
147
of ginsenoside Rb1. Cells from 10 ml of MRS medium was added to 10 ml of ginsenoside Rb1 in
148
sterile distilled water. The inoculated ginseng extract was incubated at 37°C anaerobically for 7 to 10
149
days. A 1 ml aliquot of the fermentate was collected each day and extracted with equal volume of n-
150
butanol by shaking the mixture for 1 h and incubating at room temperature for a further 12 h. The n-
151
butanol fraction was collected and evaporated to dryness in a speed vac (vacuum concentrator). The
152
dried extract was dissolved in methanol and analyzed by HPLC.
153
154
HPLC analysis of ginseng saponin
155
HPLC analysis done as described in our previous report [25]. Analysis were done in a Waters
156
HPLC system using a Sunfire C18 column (4.5 mm × 25 cm). HPLC-grade acetonitrile (A) and water
157
(B) were used as mobile phase. The analysis was performed with mobile phase flow rate of 1 ml per
158
min using a solvent gradient of 0 ~ 8 min, 20 ~ 30% A; 8 ~ 12 min, 30 ~ 40% A; 12 ~ 15 min, 40 ~
159
65% A; 15 ~ 20 min, 65 ~ 100% A, 20 ~ 30 min, 100% A; 30 ~ 35 min, 100 ~ 30% A; 35 ~ 40min, 30
160
~ 20% A, and column equilibration for 5min with 20% A as reported in [25]. The column was
161
injected with 20 µl of samples using an automated sample injector. The elution of various
162
ginsenosides was monitored at 203 nm. Throughout the run the column was maintained at a constant
163
temperature of 40°C using a column incubator. The ginsenoside transformation was monitored by the
164
comparison with the chromatogram of standard ginsenosides Re, Rg1, Rb1, Rf, Rc, Rb2, Rb3+Rg2, Rh1,
6
165
Rd, Rg3, compound K and Rh2 mixture. Ginsenosides Rb1, Rb2, Rc, Re, Rg2 and Rg3 were a kind gift
166
from Prof. Nam-In Baek (Natural Products Chemistry Lab, Kyung Hee University, Korea). Other
167
ginsenosides were obtained from Chromadex (USA). HPLC analysis of each sample was performed
168
in triplicates to obtain confirmatory results.
169
170
Preparation of β-glucosidase enzyme from strain MJM60396
Strain MJM60396 was cultured in 1 l of MRS broth for 36 h at 37°C. Bacterial cells were
171
172
removed by centrifugation at 6000 x g for 30 min at 4°C. The supernatant was collected and
173
precipitated with ammonium sulphate to 80% saturation and incubated overnight at 4°C. The
174
precipitate was collected by centrifugation at 13000 x g for 10 min. The pellet was resuspended in
175
NaOAc/HOAc buffer (pH 5.0) and dialyzed against 50 volumes of the same buffer at 4°C for 16 h,
176
using a cellulose acetate dialysis membrane (make). After dialysis, the enzyme solution was
177
concentrated 10 fold by freeze drying. This preparation was stored at 4°C until used for further
178
studies.
179
180
181
Assay of β-glucosidase
β-glucosidase assay was done following the method described in [26]. The enzyme assay was
182
performed in 96 well microtitre plates. Briefly, 20 μl of the enzyme was mixed with 20 μl of p-
183
nitrophenyl-β-D-glucoside (pNPG, 10 mmol/l), and 60 μl of 50 mM NaOAc-AcOH buffer (pH 5.0),
184
and incubated at 37°C for 10 min. The reaction was stopped by adding 100 μl of 0.5 M NaOH.
185
Absorbance of the reaction mixture was measured at 405 nm using a 96-well plate reader (infinite
186
M200 PRO, Tecan Austria Gmbh, Untersbergstr). One β-glucosidase unit is the amount of enzyme
187
that releases 1 μmol of p-nitrophenol (pNP) from pNPG per min under the described assay conditions.
188
189
190
191
Determination of optimum pH and temperature for β-glucosidase enzyme activity
To determine optimal pH for the β-glucosidase activity, pNP-β-D-glucosidase activity was
studied at 37°C in 0.2M sodium phosphate dibasic-citric acid monohydrate buffer from pH 3–8 [27].
7
192
The effect of temperature on enzyme activity was tested after incubation of the enzyme at various
193
temperature ranging from 20 to 70°C for 10 min in optimum pH in 0.2M Na2HPO4-Citric acid buffer.
194
195
Enzymatic transformation of ginsenoside Rb1
196
The reaction mixture contained 1 ml of ginseng Rb1 (1 mg/ml) in 50 mM NaOAc-AcOH
197
buffer (pH 5.0) and 100 µl of β-glucosidase enzyme with an activity equivalent to 30 mU of pNP-β-
198
glucosidase activity was incubated at 45°C for 48 h. Samples were collected at 0, 3, 6, 12, 24, 36, and
199
48 h. The reaction mixture was extracted with equal volumes of n-butanol, and the n-butanol fraction
200
was analyzed by HPLC after removal of n-butanol by vacuum evaporation and dissolving the
201
resultant transformation products in methanol.
202
203
Results
204
Screening of LAB strains for ginsensoide-transforming and β-glucosidase activities
205
206
207
Of the several strains screened strain MJM60396 showed higher ginsenoside transforming
activity (Fig. 1A) and selected for further study.
The plate based screening for β-glucosidase activity showed three positive strains, among
208
which MJM60396 showed larger zone of black colour formation around the colony indicating higher
209
activity (Fig. 1B). Furthermore, screening of culture filtrate from the LAB for β-glucosidase using
210
esculin gel diffusion assay showed activity in those strains that did not show activity in esculin-R2A
211
agar (Fig. 1C). MJM60396 showed stronger activity in the gel diffusion assay (Fig. 1C).
212
213
214
Identification of MJM60396
The 16S rRNA gene sequence of strain MJM60396 (Genbank accession no. KT962976) was
215
aligned with other strains found to have the closest taxonomic relationship. Strain MJM60396 was
216
grouped with Lactobacillus species, and the highest degree of 16S rRNA gene sequence identity was
217
to Lactobacillus paracasei subsp. tolerans (99.8 %) (Fig. 2).
218
219
Transformation of ginsenoside Rb1 by L. paracasei subsp. tolerans strain MJM60396
8
220
The transformation of ginsenoside Rb1 by L. paracasei subsp. tolerans strain MJM60396 was
221
monitored by HPLC against standard ginsenosides as reference. The chromatogram of standard
222
ginsenosides is given in supplementary Fig. 1. Fermentation of ginsenoside Rb1 with strain
223
MJM60396 showed that it could transform ginsenoside Rb1 into ginsenosides Rd, Rg3 and Rh2 over
224
the incubation period of 10 days (Fig. 3). The concentration of Rb1 (Fig. 3A) decreased continuously
225
while Rg3 peak appeared on the 3rd day (Fig. 3b), gradually increased by 5th day (Fig. 3C) with
226
ginsenoside Rd as the intermediate compound. The highest amount of ginsenoside Rg3 was observed
227
on 7th day (Fig. 3D) and started to decrease. Whereas, ginsenoside Rh2 first appeared on the 7th day
228
(Fig. 3D) and gradually increased on the 10th day (Fig. 3E). The relative amount of ginsenoside Rb1
229
was 1.2% on 7th day compared to 76.1% for ginsenoside Rg3, 13.6% of Rd and 8.7% Rh2. And, the
230
relative amounts of ginsenoside Rb1 was 0.8% on 10th day compared to 48.1% for ginsenoside Rg3,
231
0.76% of Rd and 49.7% Rh2.
232
233
234
Optimization of β-glucosidase enzyme activity and transformation of Rb1
The effect of temperature and pH on the activity of β-glucosidase from MJM60396 is shown
235
in Fig. 4. Maximum β-glucosidase activities were observed at 45°C (Fig. 4A) and pH 5.0 (Fig. 4B).
236
The enzyme maintained ≥80% of activity between the temperature of 35 - 55°C (Fig. 4A) and pH of 4
237
to 6 (Fig. 4B).
238
Enzymatic transformation of ginsenoside Rb1was investigated over 48 h using the crude β-
239
glucosidase preparation from MJM60396. Transformation products over the time were monitored by
240
HPLC analysis and the chromatogram is given in Fig. 4C. The transformed products were identified
241
by comparing the retention times of standard ginsenosides given in supplementary Fig. 1. Enzymatic
242
transformation of Rb1 resulted in the conversion of Rb1 to Rd as early as 3h and peaking at 6 h (Fig.
243
4C i, ii & iii). However, the formation of Rg3 took longer as the maximum Rg3 concentration was
244
observed at 24 h and no Rh2 was detected at this time point with a decrease in Rd level (Fig. 4C iv).
245
Formation of Rh2 was observed at 36 h and gradually increased at 48 h, while a gradual decrease in
246
Rg3 level was observed in these time points (Fig. 4C v & vi).
247
9
248
249
Discussion
Transformation of major ginsenosides into minor ginsenoside by microbial fermentation and
250
enzymatic transformation has been proposed as a viable option for the production of non-natural
251
ginsenosides [28]. Biotransformation of ginsenosides and the mechanism of transformation has been
252
researched extensively (see review [16]). However, microbes isolated from soil or from other
253
environments were not suitable for food and pharmaceutical-grade preparation of ginsenosides.
254
Hence, screening of food grade microbes with GRAS (generally regarded as safe) status for
255
ginsenoside transforming activity is essential [16].
256
The present study was aimed at identifying a LAB strain from fermented milk samples that
257
were able to transform ginsenosides particularly Rb1 to produce Rg3 and Rh2. Screening of fermented
258
ginseng extract by TLC analysis showed L. paracasei subsp. tolerans has higher ginsenoside
259
transforming activity. This strain has also been observed to possess high β-glucosidase activity among
260
other strains tested. Fermentative transformation of ginsenosides involves transformation by organic
261
acid production and production of specific enzymes [29]. Mild organic acid treatment was reported to
262
transform major ginsenosides into minor compounds [30,31], however, this method is not selective
263
and produce side reactions, which leads to artefactual compounds [16]. Hence, in our study whole cell
264
biotransformation of ginsenoside Rb1 was studied in the absence of any nutrients to avoid
265
transformation by production of organic acids.
266
Glycosidase enzymes produced by microbes have been reported to transform several
267
ginsenosides (see review [16]), among which β-glucosidase has been reported to convert glucose-
268
containing major PPD ginsenosides such as Rb1, Rb2, Rc, and Rd into minor compounds such as Rg3,
269
F2, Compound K, and Rh2 [4,32,34] and major PPT ginsenosides Re, Rg1 and Rf into Rh1 [35,36].
270
Further, to confirm that β-glucosidase is involved in the transformation of ginsenoside Rb1, we
271
incubated Rb1 with the enzyme preparation and observed that the enzyme could convert it into Rg3
272
and Rh2.
273
Lactic acid bacteria such as Lactobacillus delbrueckii [37], Leuconostoc paramesenteroides
274
[37], Lactobacillus ginsenosidimutans [38], Lactobacillus plantarum [39], Lactobacillus pentosus
275
[4,40] and Lactobacillus paralimentarius were previously reported to transform ginsenosides.
10
276
However, to the best of our knowledge L. paracasei subsp. tolerans has not been reported to
277
transform ginsensides.
278
β-glucosidase from L. pentosus reported by [4] showed an optimum temperature of 37°C, pH
279
of 7 and could transform ginsenoside Rb1 to compound K via RdF2. Whereas β-glucosidase from
280
L. paralimentarius reported by [41] showed an optimum temperature of 30°C and pH of 6 was able to
281
convert Rbgypenoside XVII and RdF2compound K. Crude cell extract of Lactobacillus
282
delbrueckii was shown to transform Rb1 via RdF2Rh2 [37]. Compared to the above reports β-
283
glucosidase from MJM60396 was able to transform ginsenoside Rb1 via RdRg3Rh2 (Fig. 5) and
284
shows an optimum pH of 5 with more than 80% activity between pH 4 to 6 and optimum temperature
285
of 45°C with about 80% activity between 35 to 55°C.
286
In conclusion, this study demonstrated that ginsenoside Rg3 and Rh2 could be produced from
287
the major ginsenoside Rb1 via ginsenoside Rd by L. paracasei subsp. tolerans strain MJM60396
288
isolated from fermented milk. This study also demonstrated the transformation ability of β-
289
glucosidase enzyme preparation from strain MJM60396 to convert Rb1 into the anticancer compound
290
Rg3 and Rh2. Therefore, L. paracasei subsp. tolerans strain MJM60396 has the potential to be applied
291
for the preparation of Rg3 and Rh2 in the nutraceutical industry.
292
293
294
295
296
297
298
299
300
301
302
303
11
304
Acknowledgement
305
This work was carried out with the support of “Cooperative Research Program for Agriculture
306
Science & Technology Development (Project No. PJ009517), Rural Development Administration,
307
Republic of Korea.
308
309
References
310
1.Qi, L. W., C. Z. Wang, and C. S. Yuan (2011) Ginsenosides from American ginseng: chemical and
311
312
pharmacological diversity. Phytochemistry. 72: 689-699.
2.Ye, L., C. Q. Zhou, W. Zhou, P. Zhou, D. F. Chen, X. H. Liu, X. L. Shi, and M. Q. Feng (2010)
313
Biotransformation of ginsenoside Rb1 to ginsenoside Rd by highly substrate-tolerant Paecilomyces
314
bainier 229-7. Bioresour. Technol. 101 7872-7876.
315
316
317
3.Kim, D.-H. (2012) Chemical Diversity of Panax ginseng, Panax quinquifolium, and Panax
notoginseng. J. Ginseng Res. 36: 1-15.
4.Kim, S.-H., J.-W. Min, L.-H. Quan, S.-Y. Lee, D.-U. Yang, and D.-C. Yang (2012) Enzymatic
318
transformation of ginsenoside Rb1 by Lactobacillus pentosus strain 6105 from kimchi. J. Ginseng
319
Res. 36: 291-297.
320
5.Nag, S. A., J. J. Qin, W. Wang, M. H. Wang, H. Wang, and R. Zhang (2012) Ginsenosides as
321
anticancer agents: In vitro and in vivo activities, structure-activity relationships, and molecular
322
mechanisms of action. Front. Pharmacol. 3: 25.
323
6.Zhang, Q., X. Kang, and W. Zhao (2006) Antiangiogenic effect of low-dose cyclophosphamide
324
combined with ginsenoside Rg3 on Lewis lung carcinoma. Biochem. Biophys. Res. Commun. 342:
325
824-828.
326
7.Liu, T. G., Y. Huang, D. D. Cui, X. B. Huang, S. H. Mao, L. L. Ji, H. B. Song, and C. Yi (2009)
327
Inhibitory effect of ginsenoside Rg3 combined with gemcitabine on angiogenesis and growth of
328
lung cancer in mice. BMC Cancer. 9: 250.
329
330
8.Iishi, H., M. Tatsuta, M. Baba, H. Uehara, A. Nakaizumi, K. Shinkai, H. Akedo, H. Funai, S.
Ishiguro, and I. Kitagawa (1997) Inhibition by ginsenoside Rg3 of bombesin-enhanced peritoneal
12
331
metastasis of intestinal adenocarcinomas induced by azoxymethane in Wistar rats. Clin. Exp.
332
Metastasis. 15: 603-611.
333
334
335
9.Chen, J., H. Peng, X. Ou-Yang, and X. He (2008 ) Research on the antitumor effect of ginsenoside
Rg3 in B16 melanoma cells. Melanoma Res. 18: 322-329.
10.Kim, S. M., S. Y. Lee, J. S. Cho, S. M. Son, S. S. Choi, Y. P. Yun, H. S. Yoo, Y. Yoon do, K. W.
336
Oh, S. B. Han, and J. T. Hong (2010) Combination of ginsenoside Rg3 with docetaxel enhances
337
the susceptibility of prostate cancer cells via inhibition of NF-kappaB. Eur. J. Pharmacol. 631: 1-
338
9.
339
11.Kim, S. W., H. Y. Kwon, D. W. Chi, J. H. Shim, J. D. Park, Y. H. Lee, S. Pyo, and D. K. Rhee
340
(2003) Reversal of P-glycoprotein-mediated multidrug resistance by ginsenoside Rg3. Biochem.
341
Pharmacol. 65: 75-82.
342
12.Lee, C. K., K. K. Park, A. S. Chung, and W. Y. Chung (2012) Ginsenoside Rg3 enhances the
343
chemosensitivity of tumors to cisplatin by reducing the basal level of nuclear factor erythroid 2-
344
related factor 2-mediated heme oxygenase-1/NAD(P)H quinone oxidoreductase-1 and prevents
345
normal tissue damage by scavenging cisplatin-induced intracellular reactive oxygen species. Food
346
Chem. Toxicol. 50: 2565-2574.
347
13.Huang, J.-Y., Y. Sun, and Q.-X. Fan (2009) Efficacy of Shenyi capsule combined with
348
gemcitabine plus cisplatin in treatment of advanced esophageal cancer-a randomized controlled
349
trial. J. Chin. Integr. Med. 7: 1047-1051.
350
14.Lu, P., W. Su, Z. Miao, H. Niu, J. Liu, and Q. Hua (2008) Effect and mechanism of ginsenoside
351
Rg3 on postoperative life span of patients with non-small cell lung cancer. Chin. J. Integr. Med.
352
14: 33-36.
353
15.Chen, Z., J. Cheng, Y. Huang, S. Han, N. Liu, G. Zhu, and J. Yao (2007) Effect of adjuvant
354
chemotherapy of ginsenoside Rg3 combined with mitomycin C and tegafur in advanced gastric
355
cancer. Chin J. Gastrointes. Surg. 10: 64-66.
356
16.Park, C. S., M. H. Yoo, K. H. Noh, and D. K. Oh (2010) Biotransformation of ginsenosides by
357
hydrolyzing the sugar moieties of ginsenosides using microbial glycosidases. Appl. Microbiol.
358
Biotechnol. 87: 9-19.
13
359
17.Wang, W., Y. Zhao, E. R. Rayburn, D. L. Hill, H. Wang, and R. Zhang (2007) In vitro anti-cancer
360
activity and structure-activity relationships of natural products isolated from fruits of Panax
361
ginseng. Cancer Chemother. Pharmacol. 59: 589-601.
362
18.Zhang, H., J. Gong, H. Zhang, and D. Kong (2015) Induction of apoptosis and reversal of
363
permeability glycoprotein-mediated multidrug resistance of MCF-7-ADM by ginsenoside Rh2. Int.
364
J. Clin. Exp. Pathol. 8: 4444-4456.
365
366
19.Li, S., Y. Gao, W. Ma, T. Cheng, and Y. Liu (2015) Ginsenoside Rh2 inhibits invasiveness of
glioblastoma through modulation of VEGF-A. Tumour Biol.
367
20.Her, Y., Y.-C. Lee, J.-H. Oh, Y.-E. Choi, C.-W. Lee, J.-S. Kim, H. M. Kim, and J.-W. Yang
368
(2012) An application of β-glycosidase to transformation of ginsenosides for the effective
369
production of specific ginsenosides with biological efficacy. Biotechnol. Bioproc. Eng. 17: 538-
370
546.
371
372
373
374
375
21.Chi, H., B. H. Lee, H. J. You, M. S. Park, and G. E. Ji (2006) Differential transformation of
ginsenosides from Panax ginseng by lactic acid bacteria. J. Microbiol. Biotechnol. 16: 1629-1633.
22.Kim, M. K., J. W. Lee, K. Y. Lee, and D.-C. Yang (2005) Microbial conversion of major
ginsenoside Rb1 to pharmaceutically active minor ginsenoside Rd. J. Microbiol. 43: 456-462.
23.Saqib, A. A. N., and P. J. Whitney (2006) Esculin gel diffusion assay (EGDA): A simple and
376
sensitive method for screening β-glucosidases. Enzyme Microb. Technol. 39: 182-184.
377
24.Tamura, K., G. Stecher, D. peterson, A. Filipski, and S. Kumar (2013) MEGA6: Molecular
378
379
Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729.
25.Palaniyandi, S. A., K. Damodharan, K. W. Lee, S. H. Yang, and J.-W. Suh (2015) Enrichment of
380
ginsenoside Rd in Panax ginseng extract with combination of enzyme treatment and high
381
hydrostatic pressure. Biotechnol. Bioproc. Eng. 20: 608-613.
382
26.Liu, L., X. M. Zhu, Q. J. Wang, D. L. Zhang, Z. M. Fang, C. Y. Wang, Z. Wang, B. S. Sun, H.
383
Wu, and C. K. Sung (2010) Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation
384
of 20(S, R)-Rg3 from black ginseng. Phytochemistry. 71: 1514-1520.
14
385
27.Desai, A. S., V. M. Chauhan, A. P. Johnston, T. Esler, and J. W. Aylott (2013) Fluorescent
386
nanosensors for intracellular measurements: synthesis, characterization, calibration, and
387
measurement. Front. Physiol. 4: 401.
388
28.Lee, B. H., H. J. You, M. S. Park, B. Kwon, and G. E. Ji (2006) Transformation of the Glycosides
389
from food materials by probiotics and food microorganisms. J. Microbiol. Biotechnol. 16: 497-
390
504.
391
29.Lee, Y., J. Oh, H. Lee, N. K. Lee, D.-Y. Jeong, and Y.-S. Jeong (2015) Lactic acid bacteria-
392
mediated fermentation of Cudrania tricuspidata leaf extract improves its antioxidative activity,
393
osteogenic effects, and anti-adipogenic effects. Biotechnol. Bioproc. Eng. 20: 861-870.
394
30.Kim, S. H., S. Y. Kim, H. Lee, K. S. Ra, H. J. Suh, S. Y. Kim, and K.-S. Shin (2011)
395
Transformation of ginsenoside-rich fraction isolated from ginseng (Panax ginseng) leaves induces
396
compound K. Food Sci. Biotechnol. 20: 1179-1186.
397
31.Quan, K., Q. Liu, J. Y. Wan, Y. J. Zhao, R. Z. Guo, R. N. Alolga, P. Li, and L. W. Qi (2015) Rapid
398
preparation of rare ginsenosides by acid transformation and their structure-activity relationships
399
against cancer cells. Sci. Rep. 5: 8598.
400
32.Su, J.-H., J.-H. Xu, H.-L. Yu, Y.-C. He, W.-Y. Lu, and G.-Q. Lin (2009) Properties of a novel β-
401
glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2. J.
402
Mol. Catal. B: Enzym. 57: 278-283.
403
33.Zhao, X., L. Gao, J. Wang, H. Bi, J. Gao, X. Du, Y. Zhou, and G. Tai (2009) A novel ginsenoside
404
Rb1-hydrolyzing β-D-glucosidase from Cladosporium fulvum. Process Biochem. 44: 612-618.
405
34.Gao, J., X. Zhao, H. Liu, Y. Fan, H. Cheng, F. Liang, X. Chen, N. Wang, Y. Zhou, and G. Tai
406
(2010) A highly selective ginsenoside Rb1-hydrolyzing β-D-glucosidase from Cladosporium
407
fulvum. Process Biochem. 45: 897-903.
408
35.Ruan, C. C., H. Zhang, L. X. Zhang, Z. Liu, G. Z. Sun, J. Lei, Y. X. Qin, Y. N. Zheng, X. Li, and
409
H. Y. Pan (2009) Biotransformation of ginsenoside Rf to Rh1 by recombinant beta-glucosidase.
410
Molecules. 14: 2043-2048.
15
411
36.Quan, L. H., J. W. Min, S. Sathiyamoorthy, D. U. Yang, Y. J. Kim, and D. C. Yang (2012)
412
Biotransformation of ginsenosides Re and Rg1 into ginsenosides Rg2 and Rh1 by recombinant
413
beta-glucosidase. Biotechnol. Lett. 34: 913-917.
414
415
37.Chi, H., and G. E. Ji (2005) Transformation of ginsenosides Rb1 and Re from Panax ginseng by
food microorganisms. Biotechnol. Lett. 27: 765-771.
416
38.Jung, H. M., Q. M. Liu, J. K. Kim, S. T. Lee, S. C. Kim, and W. T. Im (2012) Lactobacillus
417
ginsenosidimutans sp. nov., isolated from kimchi with the ability to transform ginsenosides.
418
Antonie Van Leeuwenhoek. 103: 867-876.
419
39.Kim, B.-G., S.-Y. Choi, M.-R. Kim, H. J. Suh, and H. J. Park (2010) Changes of ginsenosides in
420
Korean red ginseng (Panax ginseng) fermented by Lactobacillus plantarum M1. Process Biochem.
421
45: 1319-1324.
422
40.Quan, L.-H., L.-Q. Cheng, H.-B. Kim, J.-H. Kim, N.-R. Son, S.-Y. Kim, H.-O. Jin, and D.-C. Yang
423
(2010) Bioconversion of ginsenoside Rd into Compound K by Lactobacillus pentosus DC101
424
isolated from Kimchi. J. Ginseng Res. 34: 288-295.
425
41.Quan, L. H., Y. J. Kim, G. H. Li, K. T. Choi, and D. C. Yang (2013) Microbial transformation of
426
ginsenoside Rb1 to compound K by Lactobacillus paralimentarius. World J. Microbiol.
427
Biotechnol. 29: 1001-1007.
428
429
430
431
432
433
434
435
436
437
438
16
439
Figure Legends
440
Fig. 1 Screening of ginsenoside transforming and β-glucosidase activity of lactic acid bacteria. (A)
441
TLC analysis of the fermented ginseng extract. Control, non-fermented ginseng extract; 1, strain
442
MJM60450; 2, strain MJM60312; 3, strain MJM60396; 4, strain MJM12311; 5, MJM60460; 6,
443
MJM60406; 7, MJM60462; 8, MJM60463; 9, MJM60410; standard, mixture of pure ginsenosides
444
Rb1, Rb2, Rb3, Rc, Re, Rd, Rg1, Rg3, Rh1, Rh2 and Ck. (B) Screening of β-glucosidase activity in
445
lactic acid bacteria using Esculin-R2A agar, (C) β-glucosidase activity of culture broth of lactic acid
446
bacteria. Control 1, sterile distilled water; control 2, sterile MRS broth; control 3, 0.02 U of
447
Aspergillus niger β-glucosidase.
448
449
Fig. 2 Identification of strain MJM60396. Phylogenetic tree constructed based on 16S rDNA
450
sequence of the strain. The strain possesses 99% similarity with Lactobacillus paracasei subsp.
451
paracasei. GenBank accession numbers are indicated in parentheses after the strain name.
452
453
Fig. 3 Time course study of transformation of Rb1 by strain MJM60396. Samples were collected at
454
regular intervals and extracted with n-butanol, evaporated and the resultant extract was dissolved in
455
HPLC grade methanol and used for HPLC analysis. (A) 0 day, (B) 3 days, (C) 5 days, (D) 7 days, (E)
456
10 days.
457
458
Fig. 4 Optimization of reaction conditions for β-glucoside activity of strain MJM60396; (A)
459
temperature; (B) pH. (C) HPLC analysis of ginsenoside Rb1 transformation by β-glucoside from
460
MJM60396. (i) 0 h, (ii) 3 h, (iii) 6 h, (iv) 24h, (v) 36 h, and (vi) 48 h.
461
462
Fig. 5 Schematic representation of the hydrolytic pathway of ginsenoside Rg3 and Rh2 production
463
from Rb1 by strain MJM60396 (highlighted pathway) in comparison with previously reported
464
pathways.
465
466
17
467
468
469
470
Fig. 1
471
472
473
474
475
476
477
478
479
480
481
482
483
484
18
485
486
100
92
100
87
100
Lactobacillus paracasei subsp. tolerans JCM 1171(T) (D16550)
MJM60396 (KT962976)
Lactobacillus casei BL23(T) (FM177140)
Lactobacillus paracasei subsp. paracasei ATCC 25302(T) (ACGY01000162)
Lactobacillus zeae ATCC 15820(T) (D86516)
72
Lactobacillus rhamnosus JCM 1136(T) (BALT01000058)
42
Lactobacillus sakei subsp. sakei JCM 1157(T) (BALW01000030)
Lactobacillus oligofermentans AMKR18(T) (AY733084)
Lactobacillus brevis ATCC 14869(T) (KI271266)
Lactobacillus paraplantarum DSM 10667(T) (AJ306297)
97
100
73
83
Lactobacillus plantarum subsp. plantarum ATCC 14917(T) (ACGZ01000098)
Lactobacillus pentosus JCM 1558(T) (D79211)
Lactobacillus plantarum subsp. argentoratensis DKO 22(T) (AJ640078)
Lactobacillus fermentum NBRC 3956 (AP008937)
100
487
488
Lactobacillus reuteri JCM 1112(T) (AP007281)
0.01
Fig. 2
489
490
491
492
493
494
495
496
497
498
499
500
501
19
502
503
504
505
Fig. 3
506
507
20
508
509
510
511
Fig. 4
512
513
514
515
21
516
517
518
519
Fig. 5
520
521
522
22
523
524
Supplementary data
525
526
527
Supplementary Fig. 1 HPLC chromatogram of standard ginsenosides.
528
23