Journal of Pharmaceutical and Biomedical Analysis 125 (2016) 355–359

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Short communication

A new two-dimensional chromatographic method for separation of

saponins from steamed Panax notoginseng
Jimmy K. Lelu a , Qi Liu a , Raphael N. Alolga a , Yong Fan a , Wei-Lie Xiao b , Lian-Wen Qi a,∗ ,
Ping Li a,∗
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, No.24 Tongjia Lane, Nanjing 210009, China
b
Kunming Institute of Botany, CAS, China

a r t i c l e i n f o a b s t r a c t

Article history: The root and rhizome of Panax notoginseng (PNG) are used as folk medicine. Recent studies have reported
Received 19 February 2016 PNG to possess immunomodulatory, cardioprotective, hepatoprotective, anti-diabetic and anticancer
Received in revised form 12 April 2016 activities among a host of other pharmacological effects. The main active constituents responsible for
Accepted 14 April 2016
these pharmacological effects are saponins. It has also been proven that the chemical constituents of
steamed PNG differs from the raw form. Traditional methods of separating individual components in
Keywords:
crude extracts are usually tedious, almost irreproducible and time-consuming. In this study, an auto-
Panax notoginseng
mated multi-step preparative separation system, known as Sepbox afforded a quick, reproducible and
Saponins
Sepbox
fast separation of saponins from PNG. With Sepbox, a total of 11 saponins of high purity were obtained in a
Rare ginsenosides short period of time. The separated compounds were identified as notoginsenosides R1, T5, ginsenosides
Rb1, Rg1, Rg2, Rh1, Rh4, Rd, 20 (S) −Rg3 and a mixture of ginsenosides Rk1 and Rg5.
© 2016 Published by Elsevier B.V.

1. Introduction
Panax notoginseng (PNG), one of the Panax species is a common
used traditional Chinese herb. It is traditionally used for its hemo-
static and restorative properties. It has been cultivated for more
than 400 years in Wenshan prefecture, in the Yunnan Province,
China [1]. The major bioactive ingredients of PNG are saponins
often referred to as Panax notoginseng saponins (PNS). PNG has
been found to possess a wide range of pharmacological activi-
ties including, hepatoprotective properties [2,3], hypoglycemic [4]
or anti-diabetic [5–7] activities, immunomodulatory [8] proper-
ties, cardioprotective [9] effects, hypolipidemic [10–12] effects, and
anticancer [13–15] activities among others.
The traditional method of separating individual components
in crude extracts of natural products is usually tedious, time-
consuming and mostly irreproducible [16]. However, recent
advancement in isolation processes and technologies have broken
new grounds in ginseng analysis. In recent times, an increasing
number of investigations have focused on rare ginsenosides that
exhibit increased anticancer activities upon steaming [17–19]. To
∗ Corresponding author.
E-mail addresses: xlw@mail.kib.ac.cn (W.-L. Xiao), Qilw@cpu.edu.cn (L.-W. Qi),
liping2004@126.com (P. Li).
this end, several rare ginsenosides with potential bioactivities have
been discovered and identified [20–22].
To separate and isolate saponins of high purity from extracts
of PNG, several strategies and techniques have been reported. A
strategy of centrifugal partition chromatography (CPC), combined
with evaporative light scattering detection (ELSD), was estab-
lished and regarded as a fast and efficient tool for separation
of high-purity dammarane saponins [23]. High-speed counter-
current chromatography (HSCCC) coupled with evaporative light
scattering detector (ELSD) was also applied to separate saponins
from P. ginseng [24,25].
Two-dimensional liquid chromatography (2D-LC) systems are
proven to have superior resolution and separation in the analysis
of highly complex samples on the analytical scale [26–28]. Further-
more, on-line comprehensive preparative 2D NPLC × RPLC system
had been developed for the successful separation of components
with high purity [29]. However, efficient methods are still limited
for producing saponins, especially the rare types which do not nat-
urally exist in plants. Recently, a new automated chromatographic
separation − Sepbox® - is able to separate each ingredient of a com-
plex mixture in a single run.
Sepbox® 2D-5000 consists of 4 preparative HPLC pumps, 1 injec-
tion column, 1 main separation column, 3 s separation columns (6
with Polar Setup), 28 trap columns (for enrichment), 2 UV detec-
tors and 1 ELSD detector. The Sepbox fraction collector has six trays

http://dx.doi.org/10.1016/j.jpba.2016.04.019
0731-7085/© 2016 Published by Elsevier B.V.
356 J.K. Lelu et al. / Journal of Pharmaceutical and Biomedical Analysis 125 (2016) 355–359
Fig. 1. Chemical structures of saponins separated.
with 96 glass vials each. The fraction capacity is 572 fractions with
a volume of up to 45 mL each [30]. Literature investigation revealed
that Sepbox® coupled with high throughput bioassay-guided frac-
tionation was successfully applied for the rapid identification and
separation of bioactive compounds from two South African plants
[31], and Pimpinella anisum L. [32], Momordica charantia Vine [33],
Phlomis tuberosa [34].
In the present study, Sepbox chromatography led to the isolation
of notoginsenosides R1, T5, ginsenosides Rb1, Rg1, Rg2, Rh1, Rh4,
Rd, 20 (S)-Rg3 and a mixture of Rk1 and Rg5 from PNG. The purity
of each compound was assessed by HPLC to be higher than 85%,
and structures of all purified saponins were characterized by 1 H
and 13 C NMR spectroscopy and reference saponins. The chemical
structures of the saponins are given in Fig. 1.
2. Materials and methods
2.1. General experimental procedures
Separation was performed on Sepbox® 2D-5000 (Sepiatec,
Berlin, Germany). HPLC analysis was performed on an Agi-
lent 1200HPLC system (Agilent Technologies, Santa Clara, USA)
consisting of a G1311C QuatPump, a G1315D DAD detector,
a G1329B autosampler, and a G1316A thermostatted column
compartment equipped with an Agilent Zorbax SB-C18 column
(250 mm × 4.6 mm, i.d. 5 m). Semi-preparative HPLC was per-
formed on an Agilent 1200 liquid chromatograph with a Zorbax
SB-C18, 9.4 mm × 25 cm column. TLC Silica gel 60 F254 plates
(Merck, Darmstadt, Germany) were used for TLC analysis. Fractions
were monitored by TLC and spots were visualized by heating silica
gel plates sprayed with 10% H2 SO4 in EtOH.
All the solvents used for preparation of the crude extracts were
of analytical grade (Jinan Reagent Factory, Jinan, China). The HPLC
grade solvents for HPLC were obtained from Merck (Darmstadt,
Germany). Deionized water (18 M cm−1 ) was prepared by dis-
tilled water through a Milli-Q system (Millipore, Bedford, MA, USA).
Reference saponins, R1, Rb1, Rd, Rh1, Rh4, Rg1, 20(S)-Rg3, Rk1
and Rg5 were purchased from Jilin University (Changchun, China).
Their structures were further characterized by spectroscopic meth-
ods (1 H, 13 C NMR and MS) with a purity of higher than 95% for each
compound in our laboratory.
2.2. Plant material
4-year cultivated P. notoginseng (Burk.) F.H. Chen was bought
from Wenshan (Yunnan, China). The voucher samples were
deposited at the herbarium of the Department of Pharmacognosy,
China Pharmaceutical University.
2.3. Sample preparation
To obtain the steamed notoginseng, dried raw P. notoginseng
root powder was steamed at 120 ◦ C for 4 h [35], aired and cooled
to room temperature. Steamed notoginseng (50 g) was repeatedly
extracted four times with 500 mL of methanol, each by sonication
for 2 h at room temperature. The extraction each time was filtered
and the combined filtrates were concentrated in vacuo at 50 ◦ C. The
extract (7.39 g) was stored at −20 ◦ C as the steamed notoginseng
extract.
2.4. SepBox® fractionation
The Sepbox® system combines the advantages of HPLC and
SPE and was coupled to an HPLC/SPE/HPLC setup to allow for
two-dimensional separation. 4.9980 g of the steamed notoginseng
extract was diluted to 50 mL with methanol. The dilution was cen-
trifuged for ten minutes at 3000 rpm, membrane filtered and then
mixed with C4 silica (29.95 g), which yielding a free-flowing pow-
der. The sample was transferred to the injection column which was
flushed with water to remove any water soluble sugars, starch, etc.
Those fractions directly went into the fraction collector. The sample
remaining eluted to the main separation column and were sepa-
rated. The process of fractionation was monitored and detected by
UV (203 nm) and ELSD detectors. All the processes involved in the
automated 2D-chromatography are shown in Fig. 2.
2.4.1. First-dimension separation
The separation was performed on a C4 main separation column
with a high-pressure gradient solvent system of water-acetonitrile.
After first-dimension separation, 15 fractions, namely Fra.1–Fra.15
were obtained. These fractions were collected by 15 SPE trap
columns which were preconditioned with water prior to first-
dimension separation. All fractions were eluted with methanol
from trap columns leading to the next separation.
 J.K. Lelu et al. / Journal of Pharmaceutical and Biomedical Analysis 125 (2016) 355–359 357

Fig. 2. Schematic presentation of all processes involved in automated 2D-chromatography.

Table 1 Table 2
Gradient elution program of HPLC analysis. The yield and purities of saponins separated.

Time (min) Water (A%) Acetonitrile (B%) Peak Fractions Identity Yield (mg) Purity (%)

0 82.5 17.5 1 SN 13–14 Notoginsenoside R1 19.9 96.9

20 79 21 2 SN 7–8 Ginsenoside Rg1 97 99.3
23 74 26 SN 15–17
42 74 26 SN 20–21
55 64 36 3 SN 25 Notoginsenoside R2 64.6 97.2
64 50 50 SN 31
73 32 68 4 SN 26 Ginsenoside Rh1 287 93.7
80 20 80 SN 32–34
SN 41–42
5 SN 43–44 Ginsenoside Rb1 43 99.5
2.4. 2 s-dimension separation SN 55
6 SN 59–60 Ginsenoside Rd 55 97.4
All the 15 fractions trapped in each SPE column were passed
SN 71
through a C18 s separation column for second-dimension sep- 7 SN 49 Notoginsenoside T5 31 89.7
aration. Subsequent separation was performed using different 8 SN 63–64 Ginsenoside Rh4 34.6 87.5
gradients of water, methanol and acetonitrile. A total of 116 individ- 9 SN 65–66 20(S)-ginsenoside Rg3 75.3 89.5
ual subfractions, namely SN.1–SN.116) were collected by 10 s trap SN 77–78
10 SN 82–86 Ginsenoside Rk1 18.6 99.2
columns. Acetone was introduced to replace much of the water 11 SN 96–98 Ginsenoside Rg5 16.5 99.2
in the subfractions before they were collected so as to simplify
subsequent sample handling.
3. Results and discussion
2.5. Analytical HPLC & TLC analyses
From both TLC spots and HPLC data, it was realized that the
In order to enrich the isolated compounds, all the subfractions obtained 116 subfractions could be further merged. SN 7–8, SN
were examined by TLC and HPLC with reference ginsenosides. 13–14, SN 15–17, SN 20–21, SN 25, SN 31, SN 32–34, SN 49, SN 55,
Under the guidance of two systems, same subfractions were gath- SN 59–60, SN 64, SN 65–66, SN 71 and SN 77–78 contained only one
ered together. Subfractions which were determined to contain two major compound and some other subfractions (SN 26, SN 41–42,
or above compounds in HPLC analysis were further purified by SN 43–44, SN 63, SN 82–86 and SN 96–98) contained less than
Semi-preparative HPLC. The isolated pure compounds were sepa- two major compounds. The subfractions were identified by com-
rately analyzed by an Agilent 1200HPLC system. The mobile phase paring their chromatograms with that from a previous study [35].
was a mixture of water (A) and acetonitrile (B). The detailed gradi- as shown in Fig. 3. After individually collected, concentrated and
ent elution program is given in Table 1. The flow rate was 1 mL/min further purified by semi-preparative HPLC, 11 compounds were
and the effluents were monitored by a DAD detector at 203 nm. obtained including notoginsenoside R1 (19.9 mg), R2 (64.6 mg), T5
(29.8 mg), ginsenosides Rb1 (9 mg), Rg1 (97 mg), Rh1 (287 mg),
2.6. Identification Rh4 (34.6 mg), Rd (55 mg), 20(S)-Rg3 (75.3 mg) and a mixture of
Rk1 (18.6 mg) and Rg5 (16.5 mg). HPLC analyses demonstrated that
The 11 isolated compounds were identified by 1 H and 13 C NMR most of the compounds could be separated by Sepbox® with a good
analyses and compared with published data. The 1 H and 13 C NMR purity. The yield and purity of each compound is shown in Table 2.
data and spectra of these compounds are available as Supplemen- Sepbox 2D-5000 is a fully automated two-dimensional separa-
tary data. tion system. The high-performance liquid chromatography (HPLC)
358 J.K. Lelu et al. / Journal of Pharmaceutical and Biomedical Analysis 125 (2016) 355–359
Fig. 3. Identified subfractions obtained by comparison with published literature.
and solid phase extraction (SPE) series are particularly suitable for
the separation and purification of components in herbal medicines
within 24 h. The UV and ELSD detectors can effectively detect dif-
ferent kinds of samples which may have no UV absorption.
The common separation methods such as column chromatogra-
phy by using normal phase, reverse phase, gel and other materials,
through interaction of enrichment and separation, ultimately can
obtain pure compounds. But long and complex column separation
processes may lead to more losses. Besides, large amounts of sol-
vents and materials, as well as differences in the skills of researchers
constitute the disadvantages of column chromatography. The com-
mon separation techniques for ginsenoside separation include;
high-speed countercurrent chromatography (HSCCC) coupled with
evaporative light scattering detector (ELSD), centrifugal partition
chromatography (CPC) as well as other emerging technologies. In
summary, the use of Sepbox for the separation of ginsenosides ben-
efits from the following advantages over conventional methods;
separation time of less than a day (i.e. 24 h), higher sample loading
of about 5 g (i.e. 4.998 g), higher isolate number (11 isolates), higher
isolate yield and purity.
In this study, Sepbox was successfully applied to separate
saponins, including rare ginsenosides reported to possess strong
bioactivities. The results showed that, after the separation with
Sepbox, a monomer may appear in different subfractions. It sug-
gests that sample preparation and analyses should be paid more
attention.
4. Conclusion
This work is the first to report the simultaneous separation of
11 saponins from steamed PNG. Among the 11 saponins, 6 rare gin-
senosides (Rg2, Rh1, Rh4, 20(S)-Rg3, Rk1 and Rg5) which are only
found in steamed PNG were obtained. Five common saponins, i.e.
notoginsenosides R1, T5 and ginsenosides Rg1, Rb1, Rd were also
separated and identified. Using Sepbox® 2D-5000, a crude extract
of steamed PNG was eventually separated into a maximum of 116
subfractions by coupling of HPLC/SPE/HPLC system. Developing a
time-saving and efficient sample preparation method would fur-
ther enhance the speed of separation, analysis and identification.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jpba.2016.04.019.
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