Purification of saponins from leaves of Panax notoginseng using ...

Anal Bioanal Chem (2013) 405:3413–3421 DOI 10.1007/s00216-013-6721-8

ORIGINAL PAPER

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography Xiujie Guo & Xiuli Zhang & Jiatao Feng & Zhimou Guo & Yuansheng Xiao & Xinmiao Liang

Received: 24 October 2012 / Revised: 28 November 2012 / Accepted: 10 January 2013 / Published online: 10 February 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C18 columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C18 column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C18 and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C18 column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-6721-8) contains supplementary material, which is available to authorized users. X. Guo : X. Zhang (*) : J. Feng : Z. Guo : Y. Xiao : X. Liang (*) Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China e-mail: [email protected] e-mail: [email protected]

effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of lowcontent and novel active saponins from natural products. Keywords Saponins . Purification . Reversed-phase liquid chromatography . Hydrophilic interaction chromatography . Panax notoginseng (Burk.) F.H.Chen

Introduction Saponins are the main ingredients in many medicinal herbs and are responsible for their biological activities [1, 2]. A high-quality chemical library of saponins is important for high-throughput screening and research into the structure– activity relationship of compounds of this chemical class. Saponins are complex molecules with complicated syntheses. Isolation from natural products is therefore an important source of saponins. Saponins are usually present in natural products as multicomponent mixtures of structurally related compounds with similar polarities, making the isolation process time-consuming and tedious. The isolation and purification of saponins from natural products are generally accomplished by low-pressure column chromatography [3], preparative high-speed countercurrent chromatography [4–9], and preparative highperformance liquid chromatography (HPLC) [10]. Lowpressure column chromatography is preferred owing to its simplicity and relatively low cost; however, tedious procedures involving several time- and solvent-consuming steps are generally required owing to the complex nature of the saponin mixtures. Recently, preparative high-speed countercurrent chromatography was shown to be a convenient and

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Fig. 1 Structures of dammarane-type triterpene saponins from Panax notoginseng. MW molecular weight, glu β-D-glucopyranosyl, xyl β-Dxylopyranosyl, araF α-L-arabinofuranosyl, araP α-L-arabinopyranosyl, rha α-L-rhamnopyranosyl

efficient technique for purification of target saponins from natural products. However, it is inefficient when purifying several saponins simultaneously owing to its long separation time and low resolution, especially for unknown saponins. Currently, preparative HPLC is widely used because of its higher column efficiency and better repeatability. Nevertheless, it is difficult to provide sufficient chromatographic resolution for

separation of saponins with one-dimensional (1-D) liquid chromatography (LC) [10]. Two-dimensional (2-D) LC has been successfully applied to improve the separation of complex samples [11–18]. Different separation modes can be combined to construct a 2-D LC method according to the sample properties. Hydrophilic interaction LC (HILIC) has comparative separation power and good orthogonality to reversed-phase LC (RPLC),

Purification of saponins using preparative 2-D RPLC/HILIC

making it a good choice for constructing a 2-D coupled system [19–21]. Saponins are complex molecules consisting of nonpolar aglycones coupled with one or more polar monosaccharide moieties, resulting in good retention in RPLC and HILIC modes in most cases [1, 2, 22–24]. The 2-D HILIC/RPLC approach has been applied to analyze various complex samples [12, 14, 17]. Liu et al. [25] developed a preparative 2-D RPLC/HILIC system for the preparative isolation of active bufadienolides from Bufo bufo gargarizans Cantor toad skin. A 2-D RPLC/HILIC method was used for comprehensive characterization of Stevia rebaudiana by Fu et al. [26]. Panax notoginseng (Burk.) F.H.Chen (P. notoginseng) is a well-known traditional Chinese medicine because of its wide range of therapeutic properties with regard to cardiovascular diseases, including atherosclerosis and coronary heart disease [27]. Dammarane-type triterpene saponins from leaves of P. notoginseng belong to two major groups: the protopanaxadiol type with sugar moieties attached to C-3 and/or C-20, and the protopanaxatriol type with sugar moieties at C-6 and/or at C-20 (Fig. 1). These compounds are responsible for the biological activities of the traditional Chinese medicine. Many of them are structurally similar, with 1-D HPLC not capable of resolving all saponins in this complex mixture [10, 28]. A 2-D RPLC/HILIC system with high separation power and good orthogonality could be an effective method for the purification of saponins. In this work, a preparative 2-D RPLC/HILIC orthogonal system was applied to isolate saponins from leaves of P. notoginseng. A C18 column was employed as the first dimension, and a XAmide column was used as the second dimension. Twenty-five fractions rich in saponins were prepared in the first dimension, and three representative fractions were chosen for further purification in the second dimension. The results show that this orthogonal purification system was efficient in isolating saponins from natural products.

Experimental Apparatus and reagents The dynamic axial compression column system (first dimension) consisted of two preparative HPLC pumps (DEAIC P280), a UV detector (DEAIC UV200 II), a sample injector (Rheodyne 7725i), and an HPLC workstation (DEAIC EC2000). The preparative HPLC system (second dimension) consisted of two HPLC pumps (Hanbon-NP7000C), a UV detector (Hanbon-NU3000C), a sample injector, and an HPLC workstation (Easychrom-1000). An Agilent 1100 HPLC system (fraction analysis) equipped with a quaternary pump, a degasser, an autosampler, a column thermostat, and

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a diode-array detector was used. A Waters Alliance HPLC system coupled with a Waters 2489 UV/visible detector was used for analysis of saponin standards, extracts, and isolated compounds. Data acquisition and processing were conducted using the Waters Empower software package. Identification of pure compounds was done by mass spectrometry and NMR spectroscopy. Mass spectrometry was performed with a system comprising a Waters ACQUITY™ UPLC chromatograph coupled to a Q-Tof Premier spectrometer (Waters, USA), a quadrupole and orthogonal acceleration time-of-flight tandem mass spectrometer, equipped with a LockSpray interface and an electrospray ionization interface. 1H and 13C NMR spectra were measured with a Bruker DRX-600 spectrometer (1H NMR at 600 MHz; 13C NMR at 150 MHz). The HPLC columns were as follows: XUnion C18 (150 mm×4.6 mm, 5-μm particle diameter, 100-Å pore size, and 220 mm×80 mm, 10-μm particle diameter from Acchrom, Beijing, China), XTerra MS C18 (150 mm×4.6 mm, 5-μm particle diameter, 100-Å pore size from Waters, Milford, MA, USA), SunFire C18 (150 mm×4.6 mm, 5-μm particle diameter, 100-Å pore size from Waters, Milford, MA, USA), Atlantis HILIC silica (150 mm×4.6 mm, 3-μm particle diameter, 100-Å pore size from Waters, Milford, MA, USA), ZIC-HILIC (150 mm× 4.6 mm, 5-μm particle diameter, 200-Å pore size from SeQuant, Sweden), and XAmide (150 mm×4.6 mm, 5-μm particle diameter, 100-Å pore size, and 250 mm×20 mm, 10-μm particle diameter from Acchrom, Beijing, China). Acetonitrile (ACN) for preparative HPLC and analytical HPLC was industrial grade and HPLC grade (Merck, Darmstadt, Germany), respectively. Water for the HPLC mobile phase was purified with a Milli-Q water purification system (Millipore, Billerica, MA, USA). Extracts from leaves of P. notoginseng were purchased from Guangxi Changzhou Natural Pharmaceutical (Guangxi, China). The saponin standards ginsenosides Rg1, Re, and Rb1, and notoginsenoside R1 were purchased from the National Institutes for Food and Drug Control (Beijing, China). The saponin standards ginsenosides Rb3, Rc, Rd, and F2 and notoginsenoside Fc were isolated and purified in our laboratory. Their structures were confirmed by UV spectroscopy, electrospray ionization mass spectrometry, and 1H and 13C NMR spectroscopy, and by comparison with literature values [29–32]. The structures of these reference saponins are shown in Fig. 1 (S1–S9). Chromatographic conditions For the analysis of saponin standards, the chromatographic conditions were as follows. Mobile phase A was water and mobile phase B was ACN. A linear gradient was used to increase the proportion of mobile phase B from 20 % to 30 % in 5 min, and it then reached 55 % at 20 min in RPLC mode. A

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linear gradient was programmed to increase the proportion of mobile phase A from 13 % to 25 % in 20 min for the HILIC method. The flow rate was 1.0 mL/min, the injection volume was 10 μL, and the monitoring wavelength was 203 nm. For the analysis of extracts from leaves of P. notoginseng, the chromatographic conditions were as follows. Mobile phases A and B were water and ACN, respectively. A gradient program was used for RPLC according to the following profile: 0–5 min, 20–32 % mobile phase B; 5– 45 min, 32–68 % mobile phase B; 45–50 min, 68–95 % mobile phase B; 50–55 min, 95 % mobile phase B. A linear gradient was programmed to increase the proportion of mobile phase A from 5 % to 26 % in 30 min, and it then reached 40 % at 35 min for the HILIC method. The flow rate was 1.0 mL/min, the injection volume was 2 μL, and chromatograms were recorded at 203 nm. The separation in the first dimension was performed on a XUnion C18 column (220 mm×80-mm inner diameter, 10-μm particle diameter). Mobile phases A and B were water and ACN, respectively. A gradient program was used according to the following profile: 0–5 min, 20–32 % mobile phase B; 5– 45 min, 32–68 % mobile phase B; 45–50 min, 68–100 % mobile phase B; 50–55 min, 100 % mobile phase B. The flow rate was 300 mL/min, the injection volume was 10 mL, and chromatograms were recorded at 203 nm. The reanalyses of fractions collected from the first dimension were performed on a XAmide column (150 mm× 4.6-mm inner diameter, 5-μm particle diameter). Mobile phases A and B were water and ACN, respectively. The linear gradient for fractions 6–30 was from 5 % mobile phase A to 26 % mobile phase A in 30 min, and then reached 40 % mobile phase A at 35 min. The isocratic elution for analysis of fractions 9, 11, and 13 was done with 76 % mobile phase B, 80 % mobile phase B, and 80 % mobile phase B, respectively. The temperature of the column oven was set at 30 °C, the flow rate was 1.0 mL/min, and chromatograms were recorded at 203 nm. The second-dimension preparation was performed on a XAmide column (250 mm×20-mm inner diameter, 10-μm particle diameter). Mobile phase A was water and mobile phase B was ACN. Fractions collected from the firstdimension preparation were further separated on the XAmide column to isolate compounds. Fractions 9, 11, and 13 were taken as examples for further purification in the second dimension. The isocratic elution to isolate compounds from fractions 9, 11, and 13 was done with 76 % mobile phase B, 80 % mobile phase B, and 80 % mobile phase B, respectively. The flow rate was 20 mL/min, the injection volume was 1.0 mL, and chromatograms were recorded at 203 nm. The purity analyses of prepared compounds were performed on a XUnion C18 column (150 mm×4.6-mm inner diameter, 5-μm particle diameter). Mobile phases A and B

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were water and ACN, respectively. A gradient program was used according to the following profile: 0–5 min, 20–32 % mobile phase B; 5–25 min, 32–50 % mobile phase B. The flow rate was 1.0 mL/min, the injection volume was 2 μL, and chromatograms were recorded at 203 nm. Sample preparation A 45-g portion of the extracts from leaves of P. notoginseng was dissolved in 150 mL ACN/water (20:80, v/v) and filtered through 0.45-μm membranes to make the crude saponin sample with a concentration of about 300 mg/mL.

Results and discussion Construction of the orthogonal separation system Test of separation modes and columns RPLC is the commonest chromatographic mode for separation and isolation of compounds from a complex matrix. A saponin molecule consists of a nonpolar aglycone moiety and several glycosyl moieties. The aglycone moiety makes saponins suitable for RPLC. In this study, we investigated the selectivity differences of three C18 columns, namely, XUnion C18, XTerra MS C18, and SunFire C18 columns, with different matrix and bonding densities to separate nine saponin standards (S1–S9, Fig. 1). The results showed that the elution order of the standards was identical on the three columns and that their retention strongly depended on the position of the sugar moieties attached to the aglycone skeleton (Fig. 2), with the protopanaxadiol group (S1, S3, and S6–S9) retained more strongly than the protopanaxatriol group (S2, S4, and S5). The retention times of these standards on the XUnion C18 (Fig. 2, chromatogram a) and XTerra MS C18 (Fig. 2, chromatogram b) columns were similar, and slightly shorter than that on the SunFire C 18 column (Fig. 2, chromatogram c). As shown in Table 1, the resolution of these standards was similar for the three columns, with ginsenosides Rg1 (S2) and Re (S4), and ginsenoside Rc (S6) and notoginsenoside Fc (S9) being coeluted on all three columns. The coelution of ginsenosides Rg1 (S2) and Re (S4) could be attributed to their similar structures and properties, both bearing the same protopanaxatriol aglycone moiety, as shown in Fig. 1. The curves of these two ginsenosides nearly overlapped during an investigation on the effect of the concentration of ACN in the mobile phase on the retention values of ginsenosides in RPLC mode, which indicated a very


a 0.3

HILIC is a technique that offers a different retention mechanism compared with RPLC and is, therefore, a good candidate for orthogonal HPLC method development. The presence of polar glycosyl moieties in a saponin molecule makes possible retention on HILIC columns. Structural variations in HILIC-type stationary phases with chemically bonded amino, amido, diol, and zwitterionic sulfobetaine ligands provide different hydrophilicity and selectivity, which is important for the retention and separation of analytes [34]. In this study, three types of HILIC columns were investigated. The Atlantis HILIC silica column displayed the poorest retention of these standards (Fig. 3a), indicating that the hydrophilic interaction between saponins and the water-enriched layer is too weak. Three standards, S3–S5, were coeluted owing to their weak retention on the column. A ZIC-HILIC column and a XAmide column were subsequently selected to separate these standards using the same chromatographic conditions (Fig. 3, chromatograms b, c). The results indicated good retention on both columns, with the XAmide column showing better retention and selectivity of the standards. The elution order of the standards on these HILIC columns mainly corresponds to their molecular weight, which is consistent with literature reports [22, 24]. However, within a similar range of molecular weight, retention depended on the position of the sugar moieties attached to the aglycone skeleton. The elution order of S3–S5 on the ZIC-HILIC column was S4, S5, and S3, with S4 and S5 being coeluted, indicating that the protopanaxadiol saponin (S3) was retained more strongly on the column than the protopanaxatriol saponins (S4 and S5) (Fig. 3b). Interestingly, the elution order on the XAmide column was S3, S4, and S5 as shown in chromatogram c in Fig. 3, suggesting that the retention on the column of the protopanaxadiol saponin (S3) was weaker than that of the protopanaxatriol saponins (S4 and S5). Therefore, as the results show, different HILIC-type stationary phases can provide different selectivity for separation of saponins. Moreover, for saponins that were coeluted (S2 and S4, S6 and S9) on the XUnion C18 column, good resolution was obtained on the XAmide column, and the elution order of the standards was different on the two columns. The correlation coefficient between retention times of the standards on the XUnion C18 and XAmide HILIC

XUnion

S2,S4

0.2

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AU

S6,S9 S3 S7 S5

0.1

S8

S1

0.0

b

0.3

S2,S4

XTerra S6,S9 S3

AU

0.2 S7 S5

0.1

S1

S8

0.0

c

0.4

S2,S4

SunFire

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0.3 S6,S9 S3 S7

0.2 S5

S8

0.1

S1

0.0 0

5

10

15

20

Time (min) Fig. 2 High-performance liquid chromatography (HPLC) chromatograms of saponin standards obtained in reversed-phase liquid chromatography (RPLC) mode on a a XUnion C18 column (150 mm×4.6-mm inner diameter, 5-μm particle diameter), b a XTerra MS C18 column (150 mm×4.6-mm inner diameter, 5-μm particle diameter), and c a SunFire C18 column (150 mm×4.6-mm inner diameter, 5-μm particle diameter). Mobile phases A and B were water and acetonitrile (ACN), respectively. The mobile phase gradient was as follows: 0–5 min, 20– 30 % mobile phase B; 5–20 min, 30–55 % mobile phase B. The flow rate was 1 mL/min, the temperature was 30 °C, and UV detection was at 203 nm

small difference in their retention times [33]. Ginsenoside Rc (S6) and notoginsenoside Fc (S9) both have the same protopanaxadiol aglycone but with different glycosyl moieties (Fig. 1), resulting in different polarities. The polar selectivity of C18 columns is therefore insufficient, requiring an orthogonal separation mode to resolve these standards.

Table 1 Resolution of saponin standards on C18 columns

Columns XUnion C18 XTerra MS C18 SunFire C18

RS5,S2

RS2,S4

RS4,S8

RS8,S6

RS6,S9

RS9,S7

RS7,S3

RS3,S1

3.5 3.8 4.4

0 0 0

21.9 25.4 28.9

2.0 2.4 2.5

0 0 0

2.6 3.3 3.4

5.4 5.6 7.0

17.3 19.1 22.8

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a

0.24

S3,4,5

Atlantis HILIC Silica

AU

0.16 S2

0.08

S6

S1

S7 S9 S8

better in the initial RPLC mobile phase than in the HILIC mobile phase, we chose the XUnion C18 column as the firstdimension column to divide the sample into fractions and the XAmide column as the second-dimension column for further purification of saponins in the preparative 2-D HPLC system. Isolation of saponins by preparative 2-D HPLC

0.00 0.24

b

ZIC-HILIC

First-dimension preparation

AU

0.16 S3 S4,5 S6

0.08

S7

S9 S8

0.00 0.24

c

XAmide

0.16

AU

S3 S2

0.08

S7

S4

S9

S6

S1 S5

S8

0.00 0

5

10

15

20

Time (min) Fig. 3 HPLC chromatograms of saponin standards obtained in hydrophobic interaction liquid chromatography (HILIC) mode on a an Atlantis HILIC silica column (150 mm×4.6-mm inner diameter, 3μm particle diameter), b a SeQuant ZIC-HILIC column (150 mm×4.6mm inner diameter, 5-μm particle diameter, 200-Å pore size), and c a XAmide column (150 mm ×4.6-mm inner diameter, 5-μm particle diameter). Mobile phases A and B were water and ACN, respectively. The mobile phase gradient was as follows: 0–20 min, 13–25 % mobile phase A. The flow rate was 1 mL/min, the temperature was 30 °C, and UV detection was at 203 nm

The first-dimension preparation was conducted on a XUnion C18 column. The sample loading was 3.0 g and a single fractionation took 90 min, including 20 min for column equilibration, 15 min for column washing, and 55 min for fractionation. Fractionation of 36 g of extracts was completed in 12 runs in 18 h. Fractions were collected manually from 1 to 55 min with 1-min intervals and they were denoted as fractions 2–55 in order. In total, 54 fractions were collected in the firstdimension fractionation, with the saponins mainly distributed in fractions 6–30 according to the first-dimension chromatographic profile (Fig. 5a). The 25 fractions rich in saponins were reanalyzed on the XAmide HILIC column to compare the separation selectivity with that of the XUnion C 18 column. A threedimensional chromatogram was constructed to illustrate the 2-D RPLC/HILIC separation of the sample (Fig. 6). The presence of additional peaks, when the fractions eluted from the C18 column were reanalyzed, indicated that the 2-D RPLC/HILIC system was sufficiently orthogonal. The firstdimension fractionation efficiently simplified the sample and greatly improved the separation in the second dimension, and multiple saponins with high purity could be isolated during the second-dimension fractionation.

17.5

The first-dimension LC method selection As discussed previously, the separation selectivity of RPLC and HILIC is different for saponins. XUnion C18 and XAmide HILIC columns exhibited good retention and separation for P. notoginseng leaf extracts (Fig. 5a, b). However, the chromatographic patterns of the extracts on the two columns were entirely different (Fig. 5). Therefore, a high orthogonality 2-D system can be constructed using the XUnion C18 column and the XAmide HILIC column. Taking into account that the solubility of the extracts was

2

R =0.0126 15.0

Rt on XAmide Column (min)

columns was 0.0126 (Fig. 4), indicating good orthogonality between the two columns. Therefore, the XUnion C18 and XAmide HILIC columns were selected to separate saponins in leaves of P. notoginseng.

12.5

10.0

7.5

5.0

2.5 2.5

5.0

7.5

10.0

12.5

15.0

17.5

Rt on XUnion Column (min)

Fig. 4 Correlation coefficient between retention times (Rt) for the nine standards on the XUnion C18 and XAmide HILIC columns


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a

The samples should have good retention and a wide elution window in the first-dimension column to reduce the complexity of the fractions as much as possible. The first-dimension fractionation simplifies the sample and improves the separation in the second, orthogonal dimension. Also, good solubility of the sample in the initial mobile phase of the first-dimension preparation is needed.

XUnion

0.6

0.5

AU

0.4

0.3

0.2

Purification of saponins on the second-dimension column

0.1

0.0 0

5

10

15

20

25

30

35

40

45

50

55

Time (min)

b 0.6

XAmide

0.5

AU

0.4

0.3

0.2

0.1

0.0 0

5

10

15

20

25

30

35

Time (min)

Fig. 5 HPLC chromatograms of the extracts from leaves of P. notoginseng. a In RPLC mode on a XUnion C18 column (150 mm×4.6-mm inner diameter, 5-μm particle diameter). Mobile phases A and B were water and ACN, respectively. The mobile phase gradient was as follows: 0–5 min, 20–32 % mobile phase B; 5–45 min, 32–68 % mobile phase B; 45–50 min, 68–95 % mobile phase B; 50–55 min, 95 % mobile phase B. b In HILIC mode on a XAmide column (150 mm×4.6-mm inner diameter, 5-μm particle diameter). The mobile phase gradient was as follows: 0–30 min, 5–26 % mobile phase A; 30–35 min, 26–40 % mobile phase A. The flow rate was 1 mL/min, the temperature was 30 °C, and UV detection was at 203 nm Fig. 6 Reanalyses of fractions 6–30 on the XAmide HILIC column (150 mm×4.6-mm inner diameter, 5-μm particle diameter). Mobile phases A and B were water and ACN, respectively. The mobile phase gradient was as follows: 0– 30 min, 5–26 % mobile phase A; 30–35 min, 26–40 % mobile phase A. The flow rate was 1 mL/min, the temperature was 30 °C, and UV detection was at 203 nm

The second-dimension purification was performed on a XAmide HILIC column. As shown in Fig. 5a, peaks distributing from 8–13 min, corresponding to fractions 9–13, were insufficiently separated owing to the limited polar selectivity of the C18 column, requiring an orthogonal separation mode to resolve these relatively complex fractions. Therefore, to investigate the capability, feasibility, and validity of the method, fractions 9 (about 1.5 g), 11 (about 2.0 g), and 13 (about 2.0 g) were selected for further purification in the second dimension. Figure 7a shows the analyses of fraction 11 under optimized conditions in the second dimension. Taking into account the solubility of the sample in the mobile phase and the separation time, we selected isocratic elution in order to improve the solubility of the sample in the mobile phase and minimize the column equilibration time. The preparative isolation chromatogram of fraction 11 under optimized conditions is shown in Fig. 7b. For fraction 11, the overloading of the sample to improve preparative efficiency causes broadening of bands and peak fronting and results in decreased resolution (Fig. 7b). To obtain high-purity compounds, all the fractions were collected at a rate of 1 min per tube. Ultimately, five pure compounds (F11-1 to F11-5) and dozens of fractions were obtained. For fraction 9, the fractions were also collected at a rate of 1 min per tube, and then two pure compounds (F91, F9-2) and dozens of fractions were obtained from this fraction. Two pure compounds (F13-1, F13-4) and dozens of fractions were obtained from fraction 13. The minor compounds can be enriched for structure identification by

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a

800

F11-3

sufficiently separated on the XAmide column (Fig. 7). Therefore, more compounds could be purified in each fraction, including the unresolved and coeluted compounds in the 1-D preparation. Good orthogonality is necessary to improve separation in the 2-D preparation system, especially for minor compounds.

F11-1

600

mAU

F11-2 400

Structural identification of the isolated compounds 200

F11-4 F11-5

0 0

5

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15

20

Compounds P1–P8 (Fig. 1), obtained from fractions 9, 11, and 13, were identified by high-resolution mass spectrometry and 1 H and 13C NMR spectroscopy (data are given in Online Resource). Seven of them were identified as notoginsenoside FP2 (P1) [3], notoginsenoside Fa (P2) [32], ginsenoside Rc

Time (min)

b

F9-1

0.02

F11-3

F11-1

AU

100

0.01 0.00 0.30

80

AU

F11-2 mV

60

F9-2

0.15 0.00

40

0.10

AU

F11-4

F11-1

0.05

20

0.00

F11-5

0.12 6

9

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15

18

21

24

27

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AU

0

F11-2 0.06 0.00

Time (min)

AU

0.08

F11-3 0.04 0.00 0.16

AU

Fig. 7 Chromatograms of fraction 11 under optimized conditions: a HPLC analysis of fraction 11 with a XAmide column (150 mm×4.6mm inner diameter, 5-μm particle diameter); isocratic elution with ACN/water (80:20, v/v); flow rate 1.0 mL/min; UV detection at 203 nm. b Preparative HPLC chromatogram of fraction 11 obtained with a XAmide column (250 mm×20-mm inner diameter, 10-μm particle diameter); isocratic elution with ACN/water (80:20, v/v); flow rate 20 mL/min; UV detection at 203 nm

F11-5

0.08 0.00

F13-1

0.01 0.00 0.16

AU

repeated injection and collection. The purity of these compounds was determined by HPLC (Fig. 8), and all compounds were obtained with more than 95 % purity. The retention times of compounds F11-1 and F11-3 were almost identical (Fig. 8); therefore, these compounds were prone to coelution on C18 columns. However, they were sufficiently separated on the XAmide HILIC column (Fig. 7). A similar situation was encountered for compounds F11-2 and F11-5. Their retention times were similar on the C18 column (Fig. 8). Moreover, as the results in Fig. 7 show, the peak area of compound F11-2 is much larger than that of compound F11-5, so it would be more difficult to isolate compound F11-5 on C18 columns. Fortunately, they were also

AU

0.02

F13-4

0.08 0.00 6

8

10

12

14

16

18

20

Time (min)

Fig. 8 Purity evaluation of prepared compounds was performed on a XUnion C18 column (150 mm×4.6-mm inner diameter, 5-μm particle diameter). Mobile phases A and B were water and ACN, respectively. The mobile phase gradient was as follows: 0–5 min, 20–32 % mobile phase B; 5–25 min, 32–50 % mobile phase B. The flow rate was 1 mL/ min, the temperature was 30 °C, and UV detection was at 203 nm


(P3) [30], ginsenoside Rb1 (P4) [35], notoginsenoside Fc (P5) [32], ginsenoside Rd (P7) [29], and ginsenoside Rb3 (P8) [29] (Fig. 1). Notoginsenoside FP2 was isolated from leaves of P. notoginseng for the first time. By comparing the 1H NMR and 13 C NMR data with those of ginsenoside Ra1 (Fig. 1, R1) in the literature [30], we identified compound P6 as an isomer of notoginsenoside Q (Fig. 1, R2) [36]. To our knowledge, this is the first report of this isomer.

Conclusions A 2-D RPLC/HILIC purification method was successfully applied to realize an orthogonal separation at the preparative level for isolation of saponins from leaves of P. notoginseng. Nine saponins were used as reference standards to select columns. The correlation coefficient between retention times of the standards on the XUnion C18 column and the XAmide HILIC column was 0.0126, suggesting good orthogonality of the two columns. Therefore, a C18 column was employed as the first dimension, and a XAmide column was employed as the second dimension. Three representative fractions from the first dimension were selected for discussion. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from these three fractions in the second dimension. More compounds can be purified in each fraction, including the unresolved and coeluted compounds in the 1-D preparation. Therefore, this method will be efficient for the purification of saponins from natural products, especially for low-content and novel active saponins. Acknowledgments This work was supported by the Key Projects in the National Science & Technology Pillar Program in the 12th FiveYear Plan (2012BAI29B08 and 2008BAI51B01) and the Natural Science Foundation of China (21005077).

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