Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 400–408

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A simple and rapid method to identify and quantitatively analyze triterpenoid saponins in Ardisia crenata using ultrafast liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry Ling Ma a,b , Wei Li a,∗ , Hanqing Wang c , Xinzhu Kuang a , Qin Li d , Yinghua Wang b , Peng Xie b , Kazuo Koike a a

Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan Ningxia Institute for Drug Control, Yinchuan 750004, People’s Republic of China c College of Pharmacy, Ningxia Medical University, Yinchuan 750004, People’s Republic of China d Laboratory Center, The Fourth Affiliated Hospital, China Medical University, Shenyang 110032, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 4 August 2014 Received in revised form 10 October 2014 Accepted 11 October 2014 Available online 23 October 2014 Keywords: Ardisia crenata Saponin LC–MS Identification Quantitative determination

a b s t r a c t Ardisia plant species have been used in traditional medicines, and their bioactive constituents of 13,28epoxy triterpenoid saponins have excellent biological activities for new drug development. In this study, a fast and simple method based on ultrafast liquid chromatography coupled to electrospray ionization mass spectrometry (UFLC–MS) was developed to simultaneously identify and quantitatively analyze triterpenoid saponins in Ardisia crenata extracts. In total, 22 triterpenoid saponins, including two new compounds, were identified from A. crenata. The method exhibited good linearity, precision and recovery for the quantitative analysis of eight marker saponins. A relative quantitative method was also developed using one major saponin (ardisiacrispin B) as the standard to break through the choke-point of the lack of standards in phytochemical analysis. The method was successfully applied to quantitatively analyze saponins in commercially available plant samples. This study describes the first systematic analysis of 13,28-epoxy-oleanane-type triterpenoid saponins in the genus Ardisia using LC-ESI–MS. The results can provide the chemical support for further biological studies, phytochemotaxonomical studies and quality control of triterpenoid saponins in medicinal plants of the genus Ardisia. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Triterpenoid saponins, which are structurally defined as the glycosides of triterpenes, are among the most widely distributed plant component types with a large diversity of chemical structures and biological activities [1,2]. The triterpenoid saponins with a 13,28epoxy moiety in their sapogenin structure are distributed only in certain plant genera of the families of Myrsinaceae, Primulaceae, Aceraceae, Icacinaceae and Apiaceae, but numerous compounds of this group of triterpenoid saponins are reported to have excellent biological activities [3]. For example, saikosaponins from Bupleurum chinense (Apiaceae) have significant anti-inflammatory and hepatoprotective effects [4,5], maesabalides from Maesa balansae (Myrsinaceae) have potent and specific in vitro and in vivo

∗ Corresponding author. Tel.: +81 47 4721161; fax: +81 47 4721404. E-mail address: [email protected] (W. Li). http://dx.doi.org/10.1016/j.jpba.2014.10.013 0731-7085/© 2014 Elsevier B.V. All rights reserved.

antileishmanial activity [6], and sakuraso-saponins from Primula sieboldii (Primulaceae) have strong antifungal activities against Candida albicans [7]. Ardisia, which is the largest genus in the family Myrsinaceae, contains approximately 500 species. The plants that belong to the genus Ardisia are evergreen shrubs or trees, which are found throughout subtropical and tropical regions, and various species are used in traditional medicines. The plants that belong to the genus Ardisia are also important sources of 13,28-epoxy triterpenoid saponins [8,9]. Until now, approximately 50 triterpenoid saponins have been isolated from 10 Ardisia species, and these Ardisia saponins are reported to have biological activities such as cAMP phosphodiesterase inhibitory activity [10], a prostaglandin E2-like effect [11], and cytotoxicity against human cancer cells [12]. In addition, we recently reported that 13,28-epoxy triterpenoid saponins from Ardisia japonica selectively inhibited the proliferation of liver cancer cells without affecting normal liver cells [13]. The structural features and diverse biological

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Table 1 UFLC-ESI–MS analysis for triterpenoid saponins in Ardisia. Saponins

1b,d 2b,d 3b,d 4c 5b,d 6 7b,d 8b,d 9b,d 10c 11b,d 12b,d 13b,d 14 15 16b,d 17b,d 18b,d 19b,d 20b,d 21 22b,d 23d 24d 25d 26d 27d 28d 29d 30d 31d 32d 33d 34d a b c d e

Rt a (min)

Structure Aglycone

Sugar

I I III VI VI VII VII VIII VIII IX IX X X X XII XII X X X X XII XII II III IV V X XI XIII XIII XIII XIII XIII XIII

S4x S4r S4x S4x S4r S4x S4r S4x S4r S4x S4r S4x S4r S3g S4x S4r S3x S3r S2g1 S2g2 S3x S3r S4r S4r S4r S4r S5x S7x S7x S4r S6x S3g S3x S3r

4.46 4.62 6.62 7.05 7.23 7.98 8.28 8.39 8.73 9.35 9.53 11.73 12.04 12.46 12.83 13.14 14.49 14.56 15.47 15.58 16.60 16.76 6.27 6.65 6.97 7.35 11.39 12.53 18.19 18.23 19.10 19.98 20.81 20.95

Formula

C58 H94 O28 C59 H96 O28 C52 H84 O23 C52 H84 O22 C53 H86 O22 C52 H84 O23 C53 H86 O23 C52 H86 O22 C53 H88 O22 C52 H82 O23 C53 H84 O23 C52 H84 O22 C54 H88 O21 C47 H76 O18 C52 H82 O22 C53 H84 O22 C46 H74 O17 C47 H76 O17 C41 H66 O13 C41 H66 O13 C46 H72 O17 C46 H72 O17 C53 H88 O22 C53 H86 O23 C53 H86 O23 C52 H86 O22 C58 H94 O27 C70 H114 O37 C70 H116 O36 C53 H88 O21 C64 H106 O31 C47 H78 O18 C46 H76 O16 C47 H78 O16

MW

1239.35 1253.38 1077.21 1061.20 1075.24 1077.21 1091.24 1063.23 1077.25 1075.19 1089.22 1061.21 1075.24 929.00 1059.19 1072.55 899.10 913.10 766.45 766.45 897.10 911.20 1077.25 1091.24 1091.21 1063.24 1223.35 1547.60 1533.62 1061.25 1371.55 915.10 885.12 899.13

Positive ion (m/z)

Negative ion (m/z)

[M+Na]+

[Aglycone-H2 O+H]+

Others

[M−H]−

[M+HCOOH-H]−

n.d.e n.d.e 1100.10 1084.35 1097.90 1099.55 1113.80 1085.50 1101.10 1097.80 1111.05 1083.85 1097.95 951.15 1081.70 1096.10 921.55 936.15 789.10 789.20 921.75 934.20 1099.75 1114.10 1113.85 1086.15 1245.65 1570.10 1555.95 1084.10 1393.85 938.20 907.55 921.70

471.35 471.15 471.20 455.15 455.10 471.30 471.10 457.25 457.20 469.00 469.20 455.15 455.15 455.15 453.30 453.20 455.15 455.10 455.20 455.30 453.35 453.15 457.15 471.10 471.15 425.10 455.15 455.20 441.15 441.10 441.25 441.20 441.30 441.15

453.10, 435.25 453.10, 435.20 453.15, 435.10 437.20, 419.15 437.05, 419.20 453.21, 435.10 453.10, 435.20 439.10, 421.15 439.15, 421.10 453.15 453.15 437.10, 419.25 437.20, 419.10 437.35, 419.20 435.10, 417.35 435.00, 417.10 437.20, 419.25 437.30, 419.15 437.20, 407.05 437.30, 407.05 435.15, 417.20 435.2, 417.35 439.15, 421.10 453.10, 435.20 453.15, 435.15 407.15 437.20, 419.15 437.20 423.10, 405.15 423.10, 405.10 423.15, 405.15 423.10, 405.20 423.20, 405.15 423.10, 405.20

1237.80 1252.30 1075.65 1060.10 1073.75 1076.00 1090.00 1061.50 1076.10 1073.30 1087.20 1060.10 1073.60 927.20 1057.85 1071.75 898.10 912.10 765.30 765.50 896.45 910.10 1076.35 1090.20 1090.15 1062.25 1222.40 1545.95 1532.35 1060.25 1370.45 914.10 883.65 898.10

1283.65 1298.25 1121.30 1105.95 1120.30 1122.00 1136.00 1107.80 1121.65 1139.55 1133.10 1106.45 1119.60 973.30 1104.30 1117.90 944.30 957.65 811.20 811.45 942.10 956.55 1122.20 1136.10 1136.15 1108.10 1266.65 1591.50 1578.75 1106.10 1416.35 960.15 929.75 944.05

Rt , retention time. Structurally confirmed by comparison with reference compounds. New compounds. Reference. n.d., not detectable.

activities of Ardisia saponins make them attractive targets for new drug discovery. However, no comprehensive profile of the saponins in Ardisia species is available, and analytical studies were not reported. Therefore, a simple and rapid analytical method to identify and quantify triterpenoid saponins in the genus Ardisia should be developed. HPLC-coupled MS detection is a powerful tool to both identify and quantitatively analyze triterpenoid saponins because it has a much better detection sensitivity and produces better structural information than classical analytical methods such as TLC, HPLC-UV, and HPLC-ELSD [14,15]. Ultrafast liquid chromatography (UFLC) results in short analysis time and increased peak resolution, capacity, and sensitivity using columns that contain particles with a diameter of CHO > CH2 OH > COOH > COOGlc. Various C-16 moieties extend the retention time in the following order: O > OH > 15,16epoxy moiety. An increased number of sugar moieties shortened the retention times. Among saponins with the same number of sugars, a saponin with a xylopyranosyl moiety exhibited a shorter retention time than that with a rhamnosyl moiety. The TIC chromatograms of the A. crenata extract in the negative mode are presented in Fig. 4.

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Fig. 4. TIC chromatograms of the Ardisia crenata extract in the negative-ionization mode.

3.3. Optimization of UFLC–MS conditions The MS conditions were: interface voltage (4.5, 4.0, and 3.5 kV in the positive-ion mode and −4.5, −4.0, and −3.5 kV in the negativeion mode), CDL voltage (25, 50, 75, 150 and 200 V in the positive-ion mode and −25, −50, −75, and −150 V in the negative-ion mode), nebulizing gas (1.5, 1.6 L/min), and in-source collision-induced dissociation (CID) to generate useful fragment ions by varying the Q-array DC voltages (from 10 to 80 V in the positive-ion mode and −10 to −80 V in the negative-ion mode). The MS conditions were optimized to acquire the highest signal-to-noise ratio, higher responses, and better detection. As a result, the optimum conditions were determined as described in Section 2.5. The total-ion chromatogram (TIC) of the mixed standard solution was analyzed by collecting its MS spectrum in the full-scan mode. The optimized UFLC–MS conditions showed that the MS scan in both positive- and negative-ion modes provided valuable information to confirm the molecular weights of both the saponins and their aglycones and were selected for the qualitative analyses. The negative-ion mode showed better sensitivity than the positiveion mode; therefore, the negative ionization was selected for the quantitative analysis. Then, the formate adducts [M+HCOOH-H]− ion was chosen because it was the highest or second highest peak in the MS spectra of the individual compounds that were used for the quantitative analysis. Comparing the signal-to-noise ratio value of the target peaks in HPLC-ESI-TIC and HPLC-ESI-SIM-TIC indicated that the SIM technique was more sensitive than full scanning.

The typical chromatograms of eight saponins in the SIM mode are presented in Fig. 5. The MS data of the 28 reference triterpenoid saponins were collected in both positive and negative modes for the ionization (Table 1 and Fig. 6), and their fragmentation behaviors were elucidated as follows. In the ESI positive-ion mode, all triterpenoid saponins showed a notably high tendency to form dehydrated protonated ions [aglycone-H2 O+H]+ and/or [aglycone-2H2 O+H]+ from the aglycone base peak with the less abundant ions [aglycone3H2 O+H]+ and [aglycone-4H2 O+H]+ . In addition, the ion peak for [M+Na]+ was notably weak or not observed in the ESI-MS spectra of these saponins. In the ESI negative-ion mode, the triterpenoid saponins showed a notably high tendency to form a dehydrogenated molecular ion [M−H]− and/or a formic acid adduct dehydrogenated molecular ion [M+HCOOH-H]− . However, the saponins with a C-30 carbonyl moiety in the sapogenin showed only a dehydrogenated molecular ion [M−H]− as the base peak, and the formic acid adduct dehydrogenated molecular ion [M+HCOOHH]− peak was notably weak. 3.4. Method validation of the quantitative analysis The proposed UFLC–MS method for the quantitation of saponins in A. crenata was validated to determine the linearity, LOD, LOQ, intra-day and inter-day precisions, stability and accuracy. The results are summarized in Tables 2 and 3. The correlation coefficient values (r2 ) were higher than 0.9920 for all analytes. These

Fig. 5. UFLC-ESI-SIM chromatograms of the Ardisia crenata extract.

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L. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 400–408

Fig. 6. MS spectrum of ardisiacrispin B.

results indicated good correlations between the concentrations of the investigated compounds and their peak areas in the test ranges. The LODs and LOQs were in the ranges of 4.14–56.23 ng mL−1 and 14.20–172.69 ng mL−1 , respectively. The overall intra-day and inter-day variations (RSDs) of the eight analytes were in the ranges of 0.92–2.35% and 1.70–3.56%, respectively. The repeatability and stability, which were presented as the RSD, were in the ranges of 0.97–3.27% and 1.58–3.56%, respectively. The overall recoveries were 96.8–103.4% with an RSD of 3.14–7.80%. These results indicated that the developed UFLC–MS method was sensitive, repeatable, and accurate for the quantification analysis of these triterpenoid saponins.

3.5. Identification of saponins in A. crenata Samples of A. crenata were analyzed using the optimized UFLC–MS method. As shown in the TIC chromatograph in the positive and negative ESI scan modes, the saponin peaks are efficiently separated. In total, 22 triterpenoid saponins were consequently

identified based on their chromatographic behaviors and MS fragment data and by comparisons with the reference substances. Sixteen compounds were unambiguously identified as ardisiacrenoside D (1), ardisiacrenoside C (2), ardisicrenoside H (3), ardisianoside K (5), ardisiamamilloside F (7), ardisiacrenoside B (8), ardisiacrenoside A (9), ardisianoside J (11), ardisiacrispin A (12), ardisiacrispin B (13), ardisimamilloside B (16), primulanin (17), cyclamiretin (18), dexyloprimulanin (19), davuricoside C (20) and ardisimamilloside H (22) by comparing their retention times with those of the reference saponins and using the observed fragmentation information in the MS spectra. Among these compounds, 1 and 2, 8 and 9, 12 and 13, and 17 and 18 are four pairs of saponins, the structures of which differ from each other only by the terminal sugar, which is a xylopyranosyl or a rhamnopyranosyl moiety. All pairs of compounds have similar retention times, and for each pair, the compound with the xylopyranosyl moiety exhibited a shorter retention time. In the positive-ion ESI mode, each pair of compounds showed superimposable fragment ion characteristic of the aglycone portion, but in the negative-ion mode, they yielded [M−H]− ions that varied by 14 mass units.

Table 2 Quantitation ion, regression equation, correlation coefficients, linearity ranges and limit of detection (LOD) and quantitation (LOQ) of eight marker saponins. Analyte

Quantitation ion (m/z)

Calibration curvea

R2

8 9 12 13 15 16 17 18

1107.30 1121.35 1105.20 1119.25 1103.35 1117.25 943.35 957.15

y = 21354x − 474.28 y = 13131x + 2923.5 y = 24869x + 81,535 y = 19635x + 26,998 y = 43129x − 15,567 y = 27453x − 6310.5 y = 44721x − 3275.2 y = 70790x − 3845.1

0.9985 0.9988 0.9920 0.9979 0.9991 0.9993 0.9992 0.9989

a

Linear range (␮g/mL) 0.25–50.14 0.42–84.87 2.65–105.87 1.92–96.18 0.11–34.05 0.13–39.90 0.04–39.57 0.03–32.08

LOD (ng mL−1 )

LOQ (ng mL−1 )

50.01 56.23 21.70 22.20 21.76 39.90 4.14 4.61

149.03 172.69 64.71 77.70 67.48 79.80 23.80 14.20

y and x stand for the peak area and the concentration of each analyte, respectively.

Table 3 Precision, repeatability, stability and recovery of the eight marker saponins. Analytes

8 9 12 13 15 16 17 18

Precision (RSD, %)

Repeatability (RSD, %, n = 6)

Intraday (n = 6)

Interday (n = 6)

0.92 1.71 2.25 1.51 2.10 2.30 1.97 2.35

1.80 2.33 2.81 3.56 3.50 3.25 2.19 1.70

1.92 2.04 0.97 1.16 2.18 1.79 2.64 3.27

Stability (RSD, %, n = 6)

1.80 2.33 2.81 3.56 2.01 1.58 2.19 1.71

Recovery (%, n = 3) Mean

RSD (%)

100.5 97.6 93.2 96.9 97.7 103.4 101.9 96.8

4.72 3.14 5.80 7.84 7.80 5.20 5.64 4.46

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Table 4 The accuracies (%) of RQD method with eight triterpenoid saponins, respectively. Internal standard

Accuracy (%) 8

8 9 12 13 15 16 17 18

100.50 98.12 99.86 100.50 105.29 95.23 100.72

9

12

13

15

16

17

18

107.29

89.20 106.79

97.99 132.73 93.92

87.74 89.59 86.83 90.54

93.41 95.35 92.82 94.88 95.35

102.97 90.04 87.63 104.04 90.04 94.60

113.76 97.35 94.90 109.35 97.35 100.07 92.05

105.08 108.55 109.58 107.59 103.51 107.54

94.51 106.79 84.63 90.08 88.82

132.73 89.06 109.92 93.72

From the observation of these similar behaviors, the structures of five compounds, 4, 6, 10, 15 and 21, were tentatively determined according to their corresponding compounds: 5, 7, 11, 16, and 22, respectively. Because 4 and 10 were new compounds, they were given the trivial names of ardisianoside L (4) and ardisianoside M (10), respectively. The known compounds were hederifolioside A (6), 3-O-[␤-d-xylopyranosyl-(1 → 2)-␤-d-glucopyranosyl(1 → 4)-[␤-d-glucopyranosyl-(1 → 2)]-␣-l-arabinopyranosyl]3␤-hydroxy-13␤,28-epoxy-16-oxooleanan-30-al (15) and 3-O-␤-d-xylopyranosyl-(1 → 2)-␤-d-glucopyranosyl-(1 → 4)-␣l-arabinopyranos-yl-3␤-hydroxy-13␤,28-epoxy-16-oxooleanan30-al (21). The remaining compound, 14, showed similar fragment ions to those of compounds 12, 13, 17, 18, 19, 20 in the positive-ion ESI mode, which suggests that they have a similar sapogenin structure. In the negative-ion ESI mode, the deprotonated molecule ion [M−H]− at m/z 927.20 suggests the presence of a trisaccharidic moiety. Further comparison of the two closely structurally related saponins 17 and 18 determined that 14 was cyclaminorin. Among these saponins, 4 and 10 are new compounds, and 5, 11, 15, 16, 19, 20, 21 and 22 were identified from A. crenata for the first time.

3.6. Quantitative analysis of the samples 3.6.1. Absolute quantitative determination The optimized UFLC-ESI–MS method was subsequently applied to simultaneously qualitatively and quantitatively analyze saponins in five A. crenata samples. Twenty-two triterpenoid saponins were characterized based on their retention behaviors, molecular weight and MS fragment data, or by comparison with the reference substances. Saponins in samples of A. crenata from various regions of China showed similar profiles; however, there were several differences. Compounds 19, 20, 21, and 22 were not found in samples 2 and 5, compounds 19, 20 were not detectable in samples 3 and 4, compound 16 was not

94.16 88.32 88.97

90.68 94.38

89.78

detectable in sample 2, and compound 18 was not detectable in sample 5. The developed UFLC-ESI–MS method was also applied to quantify the contents of eight saponins in A. crenata samples 1–5. All contents were calculated using the external-standard method, and the mean values and SDs from three parallel determinations of each sample are summarized in Table 4. In all A. crenata samples, ardisiacrispins A and B were the major constituents, and their contents were approximately 100 times higher than those of the other saponins. 3.6.2. Relative quantitative determination The relative quantitative determination (RQD) of triterpenoid saponins was performed using the relative correction factor (RCF) method [19,20]. In our paper, each triterpenoid saponin was used as the internal standard, and the RCF method was adopted to calculate the relative content of the other triterpenoid saponins. The RQD was determined as the relative content divided by the absolute content. The highest accuracy was subsequently selected, which made the triterpenoid saponin best suited as the internal standard. Ardisiacrispin B (13) was chosen as the internal standard to determine another seven triterpenoid saponins (Table 4) because it showed the best accuracy of the RQD, and its content was approximately 100 times higher than that of the other saponins. Table 5 showed the results of the relative quantitative determinations of triterpenoid saponins with the corresponding internal standard (No. 1 samples, for example). Compared with the result of absolute quantitative determinations, the accuracies of the RQD were 90.54–109.35% (average 100.25%). The RSD values of the relative correction factor in different concentrations were 2.24–5.72% as listed in Table 5. The result shows that the relative quantitative method for triterpenoid saponin determinations is feasible within acceptable errors. With the RQD method, the analytes can be quantified even if only a few reference substances are available. Hence, the RQD method may be a new method to fill up the standard shortage to phytochemically analyze triterpenoid saponins.

Table 5 Contents (␮g/g) of eight marker saponins in Ardisia crenata by relative and absolute quantitative analyses. Analytes

8 9 12 13 15 16 17 18 a b c d

Relativea

Absoluteb

f¯c

RSD (%, n = 6)

1.01 0.63 1.35 1.00 1.54 1.05 2.10 3.32

3.58 2.70 5.72 0.00 4.90 2.24 2.69 2.82

c

Sample 1 827.02 1331.35 23,963.06 33,583.48 390.11 722.38 676.90 448.23

Sample1 828.18 1226.47 25,354.06 34,920.61 430.85 761.4 605.61 409.91

Sample2 ± ± ± ± ± ± ± ±

15.49 25.27 248.47 380.63 8.32 13.71 15.32 12.34

523.06 199.84 16,162.30 13,289.31 431.62 589.16 126.36 36.31

The content of sample 1 determined by ardisiacrispin B after corrected with. The content of sample using absolute quantitative determination. Relative standard deviation (%) = (SD/mean) × 100. Under the limit of detection.

Sample3 ± ± ± ± ± ± ± ±

10.04 3.84 164.86 127.58 9.58 13.61 3.45 1.95

617.25 ± 304.28 ± 20,062.51 ± 14,594.23 ± 79.76 ± –d 271.70 ± 106.99 ±

Sample4 17.16 7.21 369.15 274.37 1.87 4.56 3.17

798.53 913.76 22,033.3 26,178.87 760.11 947.95 471.19 309.96

Sample5 ± ± ± ± ± ± ± ±

18.29 18.46 182.88 465.98 12.69 23.60 12.68 7.19

337.2 ± 118.91 ± 12,463.66 ± 9198.97 ± 414.92 ± 289.22 ± 54.47 ± –d

5.80 3.59 183.22 146.26 12.95 6.10 2.49

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4. Conclusions In this study, a rapid, simple and sensitive UFLC-ESI–MS method was developed to simultaneously identify and quantitatively analyze triterpenoid saponins in A. crenata extracts, which exhibited good linearity, precision and recovery for the quantitative analysis of eight marker saponins. To the best of our knowledge, this study describes the first systematic analysis of 13,28-epoxy-oleananetype triterpenoid saponins in the genus Ardisia by LC-ESI–MS. Our study can provide the chemical support for further biological studies, phytochemotaxonomical studies and quality control of triterpenoid saponins in medicinal plants of the genus Ardisia species.

[7]

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[11]

[12]

Acknowledgment This work was partially supported by a Grant-in-Aid for Scientific Research (C) (No. 24590027) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] J.M. Augustin, V. Kuzina, S.B. Andersen, S. Bak, Molecular activities, biosynthesis and evolution of triterpenoid saponins, Phytochemistry 72 (2011) 435–457. [2] B. Dinda, S. Debnath, B.C. Mohanta, Y. Harigaya, Naturally occurring triterpenoid saponins, Chem. Biodivers. 7 (2010) 2327–2580. [3] K. Foubert, M. Theunis, S. Apers, A.J. Vlietinck, L. Pieters, Chemistry, distribution and biological activities of 13,28-epoxy-oleanane saponins from the plant families Myrsinaceae and Primulaceae, Curr. Org. Chem. 12 (2008) 629–642. [4] C.N. Lu, Z.G. Yuan, X.L. Zhang, R. Yan, Y.Q. Zhao, M. Liao, J.X. Chen, Saikosaponin a and its epimer saikosaponin d exhibit anti-inflammatory activity by suppressing activation of NF-␬B signaling pathway, Int. Immunopharmacol. 14 (2012) 121–126. [5] J. Fan, X. Li, P. Li, N. Li, T. Wang, H. Shen, Y. Siow, P. Choy, Y. Gong, Saikosaponind attenuates the development of liver fibrosis by preventing hepatocyte injury, Biochem. Cell Biol. 85 (2007) 189–195. [6] N. Germonprez, L. Maes, L. van Puyvelde, M. van Tri, D.A. Tuan, N. de Kimpe, In vitro and in vivo anti-leishmanial activity of triterpenoid saponins isolated

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A simple and rapid method to identify and quantitatively analyze triterpenoid saponins in Ardisia crenata using ultrafast liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry.

Ardisia plant species have been used in traditional medicines, and their bioactive constituents of 13,28-epoxy triterpenoid saponins have excellent bi...
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