Conversion of ginsenoside RB1 into six types of highly bioactive ginsenoside Rg3 and its derivatives by FeCl3 catalysis

Article abstract of DOI:10.1248/cpb.c18-00426

Ginsenoside Rb1 is an important saponin of ginseng(s); however, Rb1, with 3-O- and 20-O-sugar moieties, has low bioavailability. Here, we report the derivatization of ginsenoside Rb1 to completely generate six types of highly bioactive minor ginsenoside R

Full text of DOI:10.1248/cpb.c18-00426

Vol. 66, No. 9
Chem. Pharm. Bull. 66, 901–906 (2018)
901
Note
Conversion of Ginsenoside Rb1 into Six Types of Highly Bioactive
Ginsenoside Rg3 and Its Derivatives by FeCl3 Catalysis
,a
Hongshan Yu,* Yu Wang,a Chunying Liu,b Jiamei Yang,a Longquan Xu,a Guanheng Li,a
,a
Jianguo Song,a and Fengxie Jin*
a College of Biotechnology, Dalian Polytechnic University; Qinggong-Yuan No. 1, Ganjingzi-qu, Dalian 116034, P.
b
R. China: and School of Life Science and Biotechnology, Liaoning Marine Microbial Engineering and Technology
Center, Dalian University; Xuefu-Dajie No. 10, Economic Technological Development Zone, Dalian 116622, P. R.
China.
Received June 6, 2018; accepted June 29, 2018
Ginsenoside Rb1 is an important saponin of ginseng(s); however, Rb1, with 3-O- and 20-O-sugar moi-
eties, has low bioavailability. Here, we report the derivatization of ginsenoside Rb1 to completely generate six
types of highly bioactive minor ginsenoside Rg3 and its derivatives by FeCl3 catalysis, the reaction conditions
are similar to enzymatic reaction conditions. In FeCl3 catalysis, the only 20-O-sugar-moiety of ginsenoside
Rb1 was decomposed into the minor ginsenosides Rk1 and Rg5 with newly produced C-20 ethylene bands;
but also hydrolyzed into 20(S)-Rg3 and 20(R)-Rg3; subsequently the C-24(25) ethylene bands of 20(S)-Rg3
and 20(R)-Rg3 were hydrated to 20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3. After separation of reaction
mixture from 34g ginsenoside-Rb1 by silica-gel-column, the 3.3g sample I of TLC top-band consisting of
Rg5 and Rk1, 8.7g sample II of TLC middle-band consisting of 20(S)-Rg3 and 20(R)-Rg3, 3.5g sample III
of TLC bottom-band consisting of unknown product-I and -II including 20(S)-25-OH-Rg3, were obtained.
The sample III consisting of unknown product-I and -II was purified by crystallization, and identified to
20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3 by HPLC-Evaporative Light Scattering Detector (ELSD) and
NMR. Therefore, six types of minor-ginsenosides Rk1, Rg5, 20(S)-Rg3, 20(R)-Rg3, 20(S)-25-OH-Rg3 and
20(R)-25-OH-Rg3 were successfully prepared from ginsenoside Rb1 by FeCl3 catalysis. FeCl3 has low toxic-
ity and is inexpensive, and the reaction conditions are similar to enzymatic reaction conditions; thus, this
method is applicable to the development of ginseng-based drugs.
Key words ginsenosides Rb1; ginsenoside conversion; FeCl3 catalysis; ginsenoside Rg3; Rg3 derivative
Ginseng, a well-known traditional medicinal herb, com- (Rg3 and its derivatives) from high-abundance ginsenosides
prises 14 species within the genus Panax (Araliaceae fam- with low bioavailability is key to the development of natural
ily); however, the most widely used Panax species are Panax saponins and ginseng-based medicines. The highly active
ginseng (Korean or Asian ginseng), P. quinquefolium (Ameri- minor ginsenoside Rg3 is obtained from the less active gin-
can ginseng) and P. notoginseng (Notoginseng or Sanchi senoside Rb1 using acidic or basic conversion methods, but
ginseng).1–3) The ginsenosides, which are believed to be the this methodology usually results in the formation of many
main active components in ginseng, are a special group of tri- by-products and severe environmental pollution.17) Moreover,
terpenoid saponins that can be classified into dammarane- and enzymatic or microbiological methods have been widely used
oleanane-type ginsenosides. To date, over 150 ginsenosides in the conversion of ginsenoside into Rg3, Rg5, Rk1 and
have been identified from ginseng plants4); however, 80–90% 25-OH-Rg318–20) as these methods possess many advantages
of ginsenosides are dammarane-type ginsenosides (Rb1, Rb2, such as mild conditions, fewer by-product, and increased en-
Rc, Rd, Rg1, Re, and Rf in Korea ginseng; Rb1, Re, Rb2, Rc, vironmental friendliness; however, the enzymatic method has
Rd, and Rg1 in American ginseng; and Rg1, Rb1, R1, Rd, and many shortcomings such as high cost and ease of inactiva-
Re in Notoginseng), which shows that Rb1 is a representative tion.21,22) Metals or ionic liquids can be used in the production
ginsenoside of ginseng(s).5) Ginsenoside Rb1 cannot be di- of low-molecular-weight organic compounds such as in the
rectly absorbed by the human body due to the four glycosides conversion of sugar into hydroxymethyl furfural, and appli-
of 3-O-Glc-Glc- and 20-O-Glc-Glc-substituted on the Rb1 cations of metal catalysis in the conversion of biomass and
molecular core, so Rb1 has low bioavailability.6–8) After oral in the iron-catalyzed C–C bond-forming functionalization of
intake of ginseng, the major natural ginsenosides are hydro- heterocycles have revolutionized contemporary chemistry.23,24)
lyzed in the human intestinal tract, and the major ginsenosides Nevertheless, reports of metal catalysis for the production of
are converted into more active forms of the minor ginsen- high-molecular-weight natural products such as saponins are
osides, which are subsequently absorbed and can then exhibit rare. Herein, we report the inexpensive conversion of low
their physiological activity; however, the rates of conversion bioavailability ginsenoside Rb1 into highly bioactive minor
are very low.9–12) The minor ginsenosides Rg3, Rg5, Rk1 and ginsenoside Rg3 and its derivatives by FeCl3.
other derivatives have various important pharmacological
activities such as antitumor, antidiabetes, antithrombotic and
Results and Discussion
Minor Ginsenoside Products from Ginsenoside Rb1 by
anti-Alzheimer’s disease effects.13–16) Therefore, the production
of highly bioactive and highly bioavailable minor ginsenosides FeCl3 Ginsenoside Rb1 has two sugar-moieties, 3-O-Glc-
*To whom correspondence should be addressed. e-mail: hongshan@dlpu.edu.cn; fxjin@dlpu.edu.cn
© 2018 The Pharmaceutical Society of Japan

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Fig. 1. Products from the Reaction of Ginsenoside Rb1 with FeCl3
Ginsenoside Rb1 was reacted for 12h at 40°C with 0.65mol FeCl3. (A) TLC of the products from Rb1; solvent: chloroform–methanol–water=7:3:0.5 (v/v/v); 10% H2SO4
was used as the chromogenic agent. Rb1, Rg5, Rk1, 20(S)-Rg3, 20(R)-Rg3 and 20(S)-25-OH-Rg3 were used as standards. Lanes 1 and 2 are the products from the reaction
of ginsenoside Rb1 with catalytic FeCl3. (B) HPLC chromatogram of the reaction products from Rb1; the peaks of product-I and product-II are unknown ginsenosides.
(Right) Effect of reaction time (10min, 12, 48h) on ginsenoside Rb1 conversion by FeCl3 in HPLC. 1% Rb1 was reacted by 0.65mol FeCl3 at 40°C.
Glc- and 20-O-Glc-Glc-. We accidentally found that only the 20(S)-25-OH-Rg3 (5), which requires further structural clari-
20-O-sugar moiety of ginsenoside Rb1 can be completely re- fication.
acted into highly bioactive 20(S)-Rg3 (1), 20(R)-Rg3 (2), Rg5
The effects of the concentration of substrate Rb1, the etha-
(3), Rk1 (4), unknown product-I [20(S)-25-OH-Rg3] (5) and nol concentration in the reaction solvent, the temperature, and
unknown product-II [20(R)-25-OH-Rg3] (6) by FeCl3 catalysis the reaction time on the Rb1 conversion were examined. The
using conditions similar to the enzymatic reaction conditions good condition of ginsenoside Rb1 conversion by FeCl3 was
at a much lower cost than can be achieved with the enzymatic that 1% Rb1 in 35 to 40% ethanol solvent was reacted at 40°C
method. Minor ginsenoside products 20(S)-Rg3, 20(R)-Rg3, by 0.65mol FeCl3. To understand the effect of reaction time,
Rg5 and Rk1 were identified by HPLC using standards gin- a 1% solution of ginsenoside Rb1 in 35% ethanol was reacted
senosides 20(S)-Rg3, 20(R)-Rg3, Rg5 and Rk1. Unknown with 0.65mol FeCl3 at 40°C for 10, 30min, 1, 5, 9, 10, 12, 24
minor ginsenoside product-I [20(S)-25-OH-Rg3] and product- and 48h. After reacting for 10min, minor ginsenosides Rg5,
II [20(R)-25-OH-Rg3] were identified by HPLC-Evaporative Rk1, 20(S)-Rg3 and 20(R)-Rg3 began to be produced from
Light Scattering Detector (ELSD) and NMR methods.
ginsenoside Rb1; after reacting for 12h, the ginsenoside Rb1
A 2% solution of ginsenoside Rb1 in 40% ethanol was substrate was completely converted into Rk1, Rg5, 20(S)-Rg3,
mixed with the same volume of 1.3mol FeCl3 solution, and the 20(R)-Rg3, unknown product-I and product-II. After reacting
mixture was reacted at 40°C for 12h (the final Rb1 concentra- for 48h, the yields of product-I and product-II had increased,
tion was 1%, and the FeCl3 concentration was 0.65mol). After and the yields of Rk1, Rg5, 20(S)-Rg3 and 20(R)-Rg3 had
the reaction, the reaction mixture was diluted with water, decreased (Fig. 1 Right). Therefore, the best conditions for
extracted with water-saturated n-butanol, washed with water, producing the six types of minor ginsenosides from ginsen-
and dried by vacuum distillation to obtain the derivatized oside Rb1 were a 1% solution of ginsenoside Rb1 in 35–40%
samples. Each sample (4mg) was dissolved in 1mL methanol ethanol solvent reacting with 0.65mol FeCl3 at 40°C for 12h.
and analyzed by TLC and HPLC as shown in Figs. 1A, B. However, if unknown product-I and product-II are desired, the
Figure 1A, B shows that the products from the reaction reaction time can be extended to 48h.
of ginsenoside Rb1 with FeCl3 appear as three bands in the
According above results, when producing the six types of
TLC (Fig. 1A) and six peaks in the HPLC chromatogram minor ginsenosides Rk1, Rg5, 20(S)-Rg3, 20(R)-Rg3, product-
(Fig. 1B). Comparing the TLC and HPLC results, the product I and product-II from Rb1, the 1% ginsenoside Rb1 in 35 to
corresponding to the top band of the TLC was the mixture 40% ethanol solvent was reacted at 40°C for 12h by 0.65mol
of the isomers Rg5 (3) and Rk1 (4), the product correspond- FeCl3.
ing to the middle band of the TLC was the mixture of the
Preparation and Separation of Products from Rb1 by
isomers 20(S)-Rg3 (1) and 20(R)-Rg3 (2), and the product FeCl3 In the preparation of minor ginsenosides, the 22g of
corresponding to the bottom band of the TLC was the mix- ginsenoside Rb1 was dissolved in 1L of 35% ethanol–water,
ture of unknown product-I (5) and product-II (6) including and the solution was mixed with the same volume of 1.3mol

Vol. 66, No. 9 (2018)
Chem. Pharm. Bull.
903
ponents by TLC were pooled and dried by vacuum distilla-
tion to obtain three samples each showing one band in TLC;
3.3 0.8g of sample I (top band in TLC), 8.7 0.9g of sample
II (middle band in TLC), and 3.5 0.8g of sample III (bottom
band in TLC) as shown in Fig. 2A. The experiment was re-
peated 3 times.
When separated samples I, II and III, each showing one
band in TLC, from Rb1 were analyzed by HPLC with a pho-
todiode array detector; sample I was shown to contain the iso-
mers Rg5 and Rk1; and sample II was the isomers 20(S)-Rg3
and 20(R)-Rg3 based on comparison to standards of Rg5, Rk1,
20(S)-Rg3 and 20(R)-Rg3 (Fig. 2B); however, separated sam-
ple III containing unknown product-I and -II did not show an
HPLC-active peak. Sample III, containing unknown product-I
and -II, was examined using HPLC-ELSD and showed two
peaks as shown in Fig. 2C.
Purification and Identification of Unknown Product-I
and II from Sample III To further clarify the contents of
sample III (one band in TLC), containing unknown product-I
and -II, sample III was further separated and purified by crys-
tallization to obtain pure compounds. A 3g portion of sample
III was dissolved in 60mL of 60% methanol and stored at
room temperature to obtain 0.25g of a white precipitate
(product-II) in 98% purity. After the precipitation of product-
II, the remaining solution was dried by vacuum distillation,
dissolved in hot methanol, and cooled in an ice bath to gener-
ate a precipitate. This procedure was repeated several times to
generate 0.18g of pure product-I; the HPLC-ELSD retention
time of product-I (98% purity) was the same as that of the
20(S)-25-OH-Rg3 standard, which proved that product-I was
20(S)-25-OH-Rg3.
Fig. 2. Three Bands of Products from Rb1 Separated by Silica Gel Col-
umn Chromatography
An NMR method was used to determine the structures of
product-I and -II. Although product-I was identified by com-
parison to the 20(S)-25-OH-Rg3 standard by HPLC-ELSD,
(A) The separated three products, samples I, II and III, in TLC. Rg5, Rk1,
20(S)-Rg3, 20(R)-Rg3 and 20(S)-25-OH-Rg3 are standards. 1, Separated product
sample III. 2, Separated product sample II. 3, Separated product sample-I. (B)
HPLC chromatograms of separated product samples I and II. (C) HPLC-ELSD
chromatogram of separated product sample III.
13C-NMR
to more carefully confirm their structures, the
spectra of product-I and -II were collected. The NMR data
in Table 1 correspond with previous reports,19,20,25) which
confirms the structure of product-I is 20(S)-25-OH-Rg3:
FeCl3 in 35% ethanol–water, and then the mixture was reacted [12β,20(S),25-trihydroxy
dammar-3-O-β-D-glucopyranosyl-
at 40°C for 12h. After the reaction, water was added to the (1→2)-β-D-glucopyranoside], and the structure of product-II is
solution, and the mixture was treaded with AB-8 macropo- 20(R)-25-OH-Rg3: [12β,20(R),25-trihydroxy dammar-3-O-β-D-
rous-resin to remove sugars and other impurities and then glucopyranosyl-β-(1→2)-β-D-glucopyranoside].
subjected to a D-280 anion exchange resin column to decolor-
Pathways of Ginsenoside Rb1 Conversion by FeCl3 into
ize the material; the products were concentrated and dried by Rg3 and Its Derivates It is shown from above results that
vacuum distillation to afford 13g of the product showing three a 1% solution of ginsenoside Rb1 was completely converted
bands [top band: Rk1 and Rg5, middle band: 20(S)-Rg3 and into six types of minor ginsenosides Rg3 and its derivatives
20(R)-Rg3, and bottom band: unknown product-I and product- by 0.65mol FeCl3 at 40°C in 12h. According to Fig. 1 Right,
II including standard 20(S)-25-OH-Rg3] in TLC (Fig. 2A) and the minor ginsenosides 20(S)-Rg3 (1) and 20(R)-Rg3 (2),
six peaks in the HPLC chromatogram. This experiment was Rg5 (3) and Rk1 (4) began to be produced from ginsenoside
repeated 5 times.
Rb1 after the same reaction time; subsequently, the C-24(25)
Then, 20g of reaction mixture from approximately 34g of double bonds of 20(S)-Rg3 and 20(R)-Rg3 were hydrated to
ginsenoside Rb1 was separated using a silica gel column con- 20(S)-25-OH-Rg3 (5) and 20(R)-25-OH-Rg3 (6). However, the
taining 400g of 300–400 mesh silica gel (ϕ35×700). The col- 3-O-sugar-moiety of ginsenoside Rb1 did not react.
umn was eluted with chloroform and methanol [8.8:1.2 (v/v)].
The reaction pathway for the conversion of ginsenoside Rb1
In the elution, the mixture of Rg5 and Rk1 (top band of the into high bioactive ginsenoside Rg3 and its derivatives by
reaction mixture in TLC) eluted first; the second band to elute FeCl3 should be that the only 20-O-sugar-moiety of ginsen-
was the mixture of the isomers 20(S)-Rg3 and 20(R)-Rg3 oside Rb1 was decomposed into the minor ginsenosides Rk1
(middle band of the reaction mixture in TLC); the last band and Rg5 with newly produced C-20 ethylene bands; but also
to elute was the mixture of unknown product-I and product-II hydrolyzed into 20(S)-Rg3 and 20(R)-Rg3; subsequently, the
(bottom band of the reaction mixture in TLC). After complete C-24(25) ethylene bands of 20(S)-Rg3 and 20(R)-Rg3 were
elution of product-I and -II, the fractions with the same com- hydrated to 20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3 as shown

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Vol. 66, No. 9 (2018)
Table 1. 13C-NMR Data of Product-I [20(S)-25-OH-Rg3] and Product-II [20(R)-25-OH-Rg3] (δ in ppm) (C-No., Carbon Number) and These Structures
C-No.
Product-I
Product-II
C-No.
Product-I
Product-II
C-1
39.34
26.91
89.12
39.88
56.58
18.64
35.38
40.21
50.59
37.12
32.24
71.20
48.77
51.92
31.61
27.07
54.88
16.03
16.57
73.55
27.44
36.73
39.27
26.80
89.03
39.84
56.50
18.58
35.32
40.15
50.51
37.05
32.30
70.98
49.42
51.89
31.54
26.88
50.87
15.97
16.52
73.43
23.01
44.21
C-23
19.33
45.89
69.92
30.07
30.37
28.30
16.79
17.23
18.86
45.74
69.83
30.06
30.35
28.25
16.73
17.46
C-2
C-24
C-3
C-25
C-4
C-26
C-5
C-27
C-6
C-28
C-7
C-29
C-8
C-30
C-9
3-O-Glc-
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
1′
2′
3′
4′
5′
6′
105.26
83.54
78.40
71.79
78.08
63.00
105.27
83.66
78.38
71.79
78.10
62.99
3-O-Glc-(1→2)-Glc
1″
2″
3″
4″
5″
6″
106.15
77.26
78.46
71.83
78.23
62.86
106.24
77.31
78.47
71.79
78.25
62.84
in Fig. 3.
analysis. The AB-8 macroporous resin and D-280 anion ex-
change resin were obtained from Nankai University, Tianjin,
P. R. China.
Conclusion
Six types of minor-ginsenosides Rk1, Rg5, 20(S)-Rg3,
General Experimental Procedures The ginsenoside Rb1
20(R)-Rg3, 20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3 are suc- and standard ginsenosides 20(S)-Rg3, 20(R)-Rg3, Rg5, Rk1
cessfully prepared from ginsenoside Rb1 by FeCl3 catalysis and 20(S)-25-OH-Rg3 were obtained from GreenBio Co.,
using conditions similar to the enzymatic reaction conditions Ltd. and Tianle Co., Ltd. The 60-F254 silica gel plates were
at a much lower cost than can be achieved with the enzymatic Merck (Darmstadt, Germany) to use for the TLC analysis;
method.
and the content ratio of ginsenosides in the silica gel plate
FeCl3 has low toxicity, and the reaction conditions are simi- was determined by scanning the TLC spots using a Shimadzu
lar to enzymatic reaction conditions, and FeCl3 is much less CS-930 TLC scanner (Shimadzu, Kyoto, Japan). The products
expensive than the enzyme, making this method suitable for 20(S)-Rg3, 20(R)-Rg3, Rk1 and Rg5 from ginsenoside Rb1
the development of ginseng-based drugs.
by FeCl3 were examined by HPLC (Waters 2695 Separations
The problems associated with the separation of Module with Waters 2996 Photodiode Array Detector, Wa-
20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3 involving the lack ters Corp., Milford, U.S.A.) according to previously reported
of peaks observed with HPLC with a photodiode array method. A Zhonghuida C-18 chromatographic column (5µm,
detector, but the presence of peaks from the mixture of ϕ4.6mm×250mm, Zhonghuida Corp., Dalian, China) was
20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3 with the Rk1, Rg5, used for separation using mobile phase A (acetonitrile) and
20(S)-Rg3, and 20(R)-Rg3 requires further study.
B (water) as follows: 0–20min, 20% A; 20–31min, A from
20 to 32%; 31–40min, A from 32 to 43%; and 40–70min, A
from 43 to 100%. The unknown product-I and -II from gin-
Experimental
Materials The ginsenoside Rb1 and standard ginsenosides senoside Rb1 conversion by FeCl3 were examined by HPLC-
20(S)-Rg3, 20(R)-Rg3, Rg5, Rk1 and 20(S)-25-OH-Rg3 were ELSD (Waters 2695 Separations Module with Waters 2424
obtained from GreenBio Co., Ltd. (Dalian, P. R. China) and Evaporative Light Scattering Detector Waters Corp., Milford,
Tianle Co., Ltd. (Shenyang, P. R. China). The 60-F254 silica gel U.S.A.). A Zhonghuida C-18 chromatographic column (5µm,
plates were Merck (Darmstadt, Germany) to use for the TLC ϕ4.6mm×250mm, Zhonghuida Corp.). The mobile phase A

Vol. 66, No. 9 (2018)
Chem. Pharm. Bull.
905
Fig. 3. Pathways for the Conversion of Ginsenoside Rb1 by FeCl3
(acetonitrile) and B (water) was as follows: 0–20min, 20% A;
The effects of substrate Rb1 concentration, temperature,
20–31min, A from 20 to 32%; 31–40min, A from 32 to 43%; solvent ethanol concentration, FeCl3 concentration and re-
and 40–70min, A from 43 to 100%. The injection volume was action time on the Rb1 conversion by FeCl3 catalysis were
20µL, the flow rate was 3.2mL/min, the column temperature examined. The substrate Rb1 concentration was fixed to 1.0,
was 35°C, the carrier gas was nitrogen under 0.1MPa pres- 2.0, 2.5, 3, 3.5, 4, 4.5, 5 and 5.5% (finally 0.5, 1, 1.15, 1.5,
sure, the ELSD drift tube temperature was 110°C, and the Im- 1.75, 2, 2.25, 2.5 and 2.75%); the temperature was fixed to
pact device was Off. All reagents were chromatographic grade 30, 35, 40, 45, 50, 55, 60°C; solvent of ethanol concentration
and were obtained from Merck, U.S.A.
was fixed to 10, 20, 30, 35, 40, 50, 60, 70, 80 and 95%, and
The product mixture from ginsenoside Rb1 conversion by the reaction time was fixed to 10, 30min, 1, 3, 5, 7, 9, 12, 24
FeCl3 was separated using AB-8 macroporous resin and D-280 and 48h; FeCl3, 0.50, 0.65, 0.70, 0.75 and 0.80M, respectively.
anion exchange resin (Nankai University, Tianjin, P. R. China) The 0.2mL of reaction mixtures were diluted with 3 times the
column. The single band in TLC from product mixture from volume of water and extracted by water-saturated n-butanol;
ginsenoside Rb1 conversion by FeCl3 were separated using a the water-saturated n-butanol layer was washed with water (3
silica gel (Qingdao Haiyang Chemical Co., Ltd., P. R. China) times) and dried by vacuum distillation to obtain the reacted
column. The structures of the unknown product-I and II were ginsenosides, respectively. The reaction samples were dis-
analyzed using NMR. Each of these was dissolved inpyridine- solved in pure methanol, respectively, and analyzed by the
d5 and the NMR (Nuclear Magnetic Resonance) spectra were methods of TLC and HPLC.
recorded using the BrukeAVANCE 600 (1H: 600MHz;13C:
150MHz) NMR spectrometer (Switzerland).
Preparation and Separation of Products from Rb1
by FeCl3 Using 35–40% ethanol as the solvent system,
1.3–1.4 M FeCl3 solution was mixed with the same volume of
Conversion Condition of Ginsenoside Rb1 by FeCl3
A
2% Rb1 was dissolved in 35–40% ethanol–water, and mixed 2–2.2% ginsenoside Rb1 solution, and the mixture was re-
with the same volume of 1.3mol FeCl3 solution in 35–40% acted at 40°C for 12h. The reaction mixture was diluted with
ethanol–water, and reacted at 40°C for different time. The 3-6 times the volume of water, isolated using the previously
reaction mixture was diluted with 3 times the volume of water reported methods of the AB-8 macroporous resin column
and extracted by water-saturated n-butanol; the water-saturat- and the anion exchange resin D280 column.21,22) The reaction
ed n-butanol layer was washed with water (3 times) and dried mixture was eluted on the AB-8 macroporous resin column
by vacuum distillation to obtain the reacted ginsenosides.
(the column volume was 20 times of dried ginsenoside weight)

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Chem. Pharm. Bull.
Vol. 66, No. 9 (2018)
to adsorb ginsenoside; then the FeCl3 and sugar and other
impurities were rinsed with water (about 5–8 times of the col-
umn volume) elution; then the column was eluted by 80–84%
alcohol (about 4–5 times of the column volume) to wash the
ginsenosides; the alcohol eluant was eluted on the anion-
exchange resin D280 (volume was same with AB-8 column)
to decolorize; then the decolorized solution was concentrated
and dried by vacuum distillation to obtain the product mixture
(with three bands in TLC) containing six types of minor gin-
senosides Rg3 and derivatives.
Projects of China National Science and Technology No.
2012ZX09503001-003 and by the National High-end Foreign
Expert Project of China, No. GDT20152100019.
Conflict of Interest The authors declare no conflict of
interest.
Supplementary Materials The online version of this ar-
ticle contains supplementary materials.
The reaction product mixture from Rb1 (with three bands
in TLC) was separated using silica gel column.21,22) The mix-
ture products were dissolved in methanol and chloroform,
mixed with 80–100 mesh silica gels (2.3 times of the sample
weight), constantly stirred, and dried to form powder; then the
mixture powder was put in a column (diameter–height=1:15
to 20) containing 300–400 mesh samplesilica-gel (20 times
of sample weight); the top of column was put with 2cm cot-
ton. The column was first dredged by 100% chloroform, then
eluted with a solvent consisting of chloroform and methanol
[8.8:1.2 (v/v)], the fractions were about 250mL. In the elution,
the mixture of Rg5 and Rk1 (top band of reaction mixture
in TLC) was firstly eluted; second, the isomer mixture of
20(S)-Rg3 and 20(R)-Rg3 (middle band of reaction mixture
in TLC); the last, the mixture of unknown product-I and -II
(bottom band of reaction mixture in TLC) was eluted. After
completely eluted the product-I and -II, the fractions with the
same component by TLC were then collected and dried by
vacuum distillation to obtain sample I containing Rg5 and
Rk1 (top band in TLC), sample II containing 20(S)-Rg3 and
20(R)-Rg3 (middle band in TLC), and sample III containing
unknown product-I and -II (bottom band in TLC).
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Purification and Identification of Sampe III Containing
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Acknowledgments This work was supported by Major