Enzymatic preparation of ginsenosides Rg2, Rh1, and F1 from protopanaxatriol-type ginseng saponin mixture

Article abstract of DOI:10.1055/s-2003-38476

During investigations on the hydrolysis of a protopanaxatriol-type saponin mixture by various glycoside hydrolases, it was found that two minor saponins, ginsenosides Rg₂ and Rh₁, were formed in high yields by crude β-galactosidase from Aspergillus oryzae and crude lactase from Penicillium sp., respectively. Moreover, a crude preparation of naringinase from Penicillium decumbens readily hydrolyzed a protopanaxatriol-type saponin mixture to give an intestinal bacterial metabolite, ginsenoside F₁ as the main product. A crude preparation of hesperidinase from Penicillium sp. selectively hydrolyzed ginsenoside Re into ginsenoside Rg₁. This is the first report on the enzymatic preparation of minor saponins, ginsenosides Rg₂ and Rh₁, and of an intestinal bacterial metabolite, ginsenoside F₁, with a high efficiency from a protopanaxatriol-type saponin mixture.

Full text of DOI:10.1055/s-2003-38476

being similar in R values to Rh on thin layer chromatograms.
Similar incubation with EG afforded compound II (main prod-
f
1
Enzymatic Preparation of
Ginsenosides Rg , Rh , and F from
Protopanaxatriol-Type Ginseng
Saponin Mixture
uct) showing R values consistent with Rg , together with both
compound III and a small amount of compound IV having Rf
2
1
1
f
2
values similar to those of F . The complete disappearance of
1
the Ppt-type saponin mixture and a remarkab le accumulation
of compound III were observed in the incubated reaction mix-
ture with EL. EN converted the initial saponin mixture into a re-
markable amount of compound IV having a slightly higher Rf
1
1
2
2
Sung-Ryong Ko , Kang-Ju Choi , Kei Uchida , Yukio Suzuki
value than Rh , in addition to a small amount of compound III.
1
Abstract
The yields of compounds I, II, III, and IV measured by TLC densi-
tometry [10] were 70, 45, 90, and 70% based on total ginseno-
During investigations on the hydrolysis of a protopanaxatriol- sides in a Ppt-type saponin mixture, respectively. These com-
type saponin mixture by various glycoside hydrolases, it was pounds were isolated in crystalline form or as white powders
found that two minor saponins, ginsenosides Rg and Rh , were from the incubated reaction mixtures on a semipreparative
2
1
formed in high yields by crude b-galactosidase from Aspergillus scale by extraction with n-butanol and column chromatogra-
oryzae and crude lactase from Penicillium sp., respectively. More- phy, and identified by spectroscopy as follows: compound I,
over, a crude preparation of naringinase from Penicillium Rg ; compound II, Rg ; compound III, Rh , and compound IV, F
1
2
1
1
decumbens readily hydrolyzed a protopanaxatriol-type saponin (Fig.1).
mixture to give an intestinal bacterial metabolite, ginsenoside
F1 as the main product. A crude preparation of hesperidinase Crude preparations of four glycosidases were examined for the
from Penicillium sp. selectively hydrolyzed ginsenoside Re into hydrolytic activities of Re, Rf, Rg , and Rg . EH readily hydro-
1
2
ginsenoside Rg . This is the first report on the enzymatic prepara- lyzed Re into Rg , and both Rf and Rg into Rh , but had no activ-
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1
2
1
tion of minor saponins, ginsenosides Rg and Rh , and of an intes- ity against Rg . EG hydrolyzed Re into Rg , Rf into Rh , and also
2
1
1
2
1
tinal bacterial metabolite, ginsenoside F , with a high efficiency Rg into Rh as a main product and a small amount of F , but had
1
1
1
1
from a protopanaxatriol-type saponin mixture.
no activity against Rg . EL hydrolyzed all of Re, Rf, Rg , and Rg
2 1 2
into Rh . It was also already observed with EL that Re was hy-
1
drolyzed into Rh via Rg , not via Rg , during the investigation
1
1
2
Recently cytotoxic effects of minor saponins, such as ginsenoside on the time-course of Rh formation from Re [8]. EN completely
1
Rh (Rh ) and ginsenoside Rh (Rh ), on the growth of various hydrolyzed both Re and Rg into F , and also hydrolyzed both Rf
1
1
2
2
1
1
cancer cells [1], [2], [3], [4], and also inhibitory effects of human and Rg into Rh . The hydrolytic pathways of Ppt-type saponins
2
1
intestinal bacterial saponin metabolites, such as compound K by crude preparations of four glycosidases are shown in Fig. 2.
and protopanaxatriol, on the growth, invasion, and migration of These results reveal that EG, EL, and EN are extremely useful in
tumor cells have been reported [5], [6], [7]. In the previous paper the selective formation of minor saponins, Rg and Rh and an
285
2
1
[
8], we first reported that a minor saponin, Rh , was formed in intestinal bacterial metabolite, F , respectively, from a Ppt-
1
1
high yields (more than 90%) from major saponins such as ginse- type saponin mixture. The isolation and characterization of
noside Re (Re) and ginsenoside Rg (Rg ) by a crude lactase pre- purified enzymes, which specifically hydrolyze glycosidic lin-
1
1
paration from Penicillium sp. In this paper, we report that crude kages such as -O-b-
preparations of four glycosidases, Penicillium sp. hesperidinase cose(2¬1)-b-
-glucose, C-6-O-b-
EH), Aspergillus oryzae b-galactosidase (EG), Penicillium sp. lac- glucose, from crude preparations of four glycosidases will be
D
-glucose (2¬1)-a-
L
-rhamnose, -O-b- -glu-
D
D
D
-glucose, and C-20-O-b- -
D
(
tase (EL), and P. decumbens naringinase (EN) have high activities the subject of a future study.
in the formation of Rg , ginsenoside Rg (Rg ) (another minor sa-
1
2
2
ponin), Rh , and ginsenoside F (F ) (bacterial metabolite of Rg in
1
1
1
1
rat large intestine [9]), respectively, from a protopanaxatriol- Materials and Methods
type (Ppt-type) saponin mixture which was readily obtained
from ginseng extract.
A protopanaxatriol-type (Ppt-type) saponin mixture containing
Re, Rf, Rg , and Rg , and each of these ginsenoside were obtained
1
2
The incubation of a Ppt-type saponin mixture with EH for 48 h from standardized Ginseng extract containing 13±14% of total
gave a large quantity of compound I having R values similar to saponin (KT & G Central Research Institute) by the reported
f
those of Rg , together with a small quantity of compound III methods [11], [12]. F was isolated from leaves of the same plant
1
1
(Panax ginseng C. A. Meyer, Araliaceae) by the reported proce-
dures [13]. Rh1 was formed from Re by a crude lactase from
Penicillium sp. as described previously [8]. The purity of these
ginsenosides was assessed by TLC and HPLC. Crude preparations
of four glycosidases, i.e., a b-galactosidase from A. oryzae (EG)
(Kohjin Co. Ltd., Tokyo, Japan), a lactase from Penicillium sp. (EL)
Affiliation: 1 KT & G Central Research Institute, Taejon, Korea ´ 2 Research
Institute for Bioresources, Okayama University, Kurashiki, Japan
Correspondence: Dr. Sung-Ryong Ko ´ KT & G Central Research Institute ´ 302
Shinsong-dong ´ Yusong-ku ´ Taejon, 305-345, Korea
(KI Chemical Ind. CO., Ltd., Iwata, Shizuoka-ken, Japan), a hesper-
Received: July 17, 2002 ´ Accepted: October 20, 2002
idinase from Penicillium sp. (EH), and a naringinase from
P. decumbens (EN) (both from Sigma Chemical Co. St. Louis, MO,
USA) were purchased commercially.
Bibliography: Planta Med 2003; 69: 285±286 ´ ꢀ Georg Thieme Verlag Stuttgart ´
New York ´ ISSN 0032-0943

was extracted with n-butanol, and the butanol layer was evapo-
rated to dryness in vacuo. The residues obtained with EH, EG,
EL, and EN were purified by the reported methods [8], [11],
[
12], [13], using the combined column chromatography to af-
ford compounds I (163 mg), II (93 mg), III (182 mg), and IV
130 mg), respectively. These compounds I, II, III, and IV were
(
identified as Rg , Rg , Rh , and F , respectively, by the compari-
1
2
1
1
son of FAB-MS, IR, 1H- and C-NMR spectra with authentic
13
samples [14], [15].
Acknowledgements
This work was supported in part by a Korea Science and Techno-
logy Foundation brain pool project research fellowship to one of
the authors, Yukio Suzuki.
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Letter¼ Planta Med 2003; 69: 285±286