Isolation, identification, and quantification of triterpene saponins in the fresh fruits of Panax notoginseng

Article abstract of DOI:10.1080/14786419.2021.1938038

This study is to develop a method for isolation, identification, and quantitative determination of dammarane-type triterpene saponins in the Panax notoginseng fruits (PNF). The saponins were isolated by a serious of chromatographic methods, and their structures were elucidated on the basis of spectroscopic evidence and comparison with those of literature reports. Quantitative assay was performed on an ultra-performance liquid chromatography-UV (UPLC-UV) method. As a result, 22 saponins were isolated from the extract of PNF, among them, compound 1 was a new saponin, named as malonylgypenoside IX, compounds 3–10, and 14–18 were isolated from the PNF for the first time. As to quantitative analysis, the calibration curves showed good linearity (r > 0.998) within the concentration range, and the method validation provided good reproducibility and sensitivity for the quantification of eight major saponins with precision and accuracy of less than 3.0%.

Full text of DOI:10.1080/14786419.2021.1938038

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Isolation, identification, and quantification of
triterpene saponins in the fresh fruits of Panax
notoginseng
Zheng-yi Qu, He-cheng Wang, Yin-ping Jin, Ya-li Li & Ying-ping Wang
To cite this article: Zheng-yi Qu, He-cheng Wang, Yin-ping Jin, Ya-li Li & Ying-ping Wang (2021):
Isolation, identification, and quantification of triterpene saponins in the fresh fruits of Panax
notoginseng, Natural Product Research, DOI: 10.1080/14786419.2021.1938038
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1938038
Isolation, identification, and quantification of triterpene
saponins in the fresh fruits of Panax notoginseng
Zheng-yi Qua, He-cheng Wangb, Yin-ping Jina, Ya-li Lia and Ying-ping Wangc
aInstitute of Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences,
b
Changchun, P.R. China; School of Life Science and Medicine, Dalian University of Technology,
c
Panjin, Liaoning, P.R. China; State Local Joint Engineering Research Center of Ginseng Breeding and
Application, Jilin Agriculture University, Changchun, P.R. China
ABSTRACT
ARTICLE HISTORY
Received 13 December 2020
Accepted 26 May 2021
This study is to develop a method for isolation, identification, and
quantitative determination of dammarane-type triterpene saponins in
the Panax notoginseng fruits (PNF). The saponins were isolated by a
serious of chromatographic methods, and their structures were eluci-
dated on the basis of spectroscopic evidence and comparison with
those of literature reports. Quantitative assay was performed on an
ultra-performance liquid chromatography-UV (UPLC-UV) method. As a
result, 22 saponins were isolated from the extract of PNF, among
them, compound 1 was a new saponin, named as malonylgypenoside
IX, compounds 3–10, and 14–18 were isolated from the PNF for the
first time. As to quantitative analysis, the calibration curves showed
good linearity (r> 0.998) within the concentration range, and the
method validation provided good reproducibility and sensitivity for
the quantification of eight major saponins with precision and accur-
acy of less than 3.0%.
KEYWORDS
Panax notoginseng; fruits;
saponins; NMR;
quantification
1. Introduction
Panax notoginseng (Burk.) F. H. Chen is a species of the genus Panax, family
Araliaceae, and has been widely used as a traditional Chinese medicine for the treat-
ment of cardiovascular diseases (Guo et al. 2010). Panax notoginseng saponins (PNS)
are the major bioactive constituents of P. notoginseng (Wang et al. 2006; Jin et al.
CONTACT Ying-ping Wang
yingpingw@yeah.net; He-cheng Wang
wanghc@dlut.edu.cn
Supplemental data for this article can be accessed online at https://doi.org/10.1080/14786419.2021.1938038.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
Z.-Y. QU ET AL.
2017; Huang et al. 2019a). The known PNS are divided into 3 major groups due to the
structure differentiation of sapogenins, protopanaxadiol (PPD), protopanaxatriol (PPT),
and C-17 side-chain varied type (Yang et al. 2014). The majority of previous isolation
and quantification studies have focused on the roots and leaves parts of P. notogin-
seng (Sakah et al. 2013; Liu et al. 2017; Gu et al. 2018; Xu et al. 2019), however, there
are very few research on the P. notoginseng fruits (PNF) (Shi et al. 2010). In the interest
of further exploitation and utilization of this precious medicinal plant, we recently
reported six malonyl-substituted ginsenosides, which were isolated from the H2O frac-
tion of PNF extraction (Qu et al. 2021). A continuation of phytochemical investigation
on PNF led to the isolation of another new malonyl-substituted ginsenoside, named as
malonylgypenoside IX (1), along with 21 known saponins. Meanwhile, an ultra-per-
formance liquid chromatography-UV (UPLC-UV) method was successfully applied to
the quantification of eight major saponins in PNF.
2. Results and discussion
2.1. Extraction, isolation, and identification of saponins
In MS1 spectra, the deprotonated molecular ion [M–H]ꢀ in negative ion mode provided
accurate molecular weight, and in MS2 spectra, ginsenosides can be rapidly identified
through the sapogenin ions at m/z 459 (PPD-type) and 475 (PPT-type), together with the loss
of 162 Da (glucose), 146 Da (rhamnose), and 132 Da (xylose or arabinose) sugar units. On this
basis, the saponins were purified from PNF with LC-MS guided, a scheme of the total proced-
ure was as follows: First of all, the PNS were extracted by 85% aqueous EtOH, and then
enriched by liquid-liquid extraction with n-butanol (n-BuOH), after that, the saponins were
further isolated by a serious of chromatographic methods, eventually, their structures were
established by MS and NMR spectroscopies, and comparison with those of literature date.
Following the approach described above, 22 saponins were separated from the n-
butanol fraction of PNF extraction (Figure 1). Among them, malonylgypenoside IX (1),
a new acidic saponin, which was belonged to a malonyl-substituted ginsenoside. It is
noteworthy that malonyl-substituted ginsenosides commonly acylation on the
appended sugar moieties and widely distributed in fresh or dried Panax genus (Wan
et al. 2015; Shi et al. 2018), and exhibit potential antidiabetic activity (Liu et al. 2009;
Liu et al. 2013; Qiu et al. 2017). However, malonylginsenosides are thermal instability
with hydrolysis or decarboxylation to their respective neutral ginsenosides or acety-
lated ginsenosides, therefore, they are prone to structure transformation during extrac-
tion and separation process, and the isolated malonylginsenosides were probably
much lower than actually occurs (Shi et al. 2018). Up to now, no more than 30 malo-
nylginsenosides were isolated from Panax species, thus, the isolation of this new malo-
nylginsenoside is quite significant for studying the diversity of their structures and
laying the foundation for further research on other malonylginsenosides.
For the rest of 21 known saponins, including 20(R)-ginsenoside Rg3 (2) (Li and
Gong 2019), ginsenoside Mc (3) (Han et al. 2007), notoginsenoside Fe (4) (Yang et al.
1983), gypenoside IX (5) (Takemoto et al. 1983), gypenoside XIII (6) (Takemoto et al.
1983), notoginsenoside ST-4 (7) (Pei et al. 2011), notoginsenoside Ft1 (8) (Chen et al.
2006), notoginsenoside K (9) (Ma et al. 1999), ginsenoside Rb2 (10) (Zhou et al. 2016),

NATURAL PRODUCT RESEARCH
3
Figure 1. The structures of compounds 1–22. Glc, b-D-glucopyranosyl; Xyl, b-D-xylopyranosyl. Ara
(p), a-L-arabinopyranosyl; Ara (f), a-L-arabinofuranosyl.

4
Z.-Y. QU ET AL.
ginsenoside Rd (11) (Zhou et al. 2016), ginsenoside Rb3 (12) (Sanada and Shoji 1978),
notoginsenoside Fc (13) (Yang et al. 1983), notoginsenoside FP2 (14) (Wang et al.
2008), ginsenoside Rc (15) (Zhou et al. 2016), notoginsenoside Fa (16) (Huang et al.
2019b), notoginsenoside D (17) (Yoshikawa et al. 1997), notoginsenoside S (18)
(Yoshikawa et al. 2003), ginsenoside Rb1 (19) (Zhou et al. 2016), notoginsenoside R1
(20) (Zhou et al. 1981), ginsenoside Rg1 (21) (Nagai et al. 1971), ginsenoside Re (22)
(Sanada et al. 1974). As far as we know, compounds 3–10, and 14–18 were obtained
from PNF for the first time.
Compound 1 was obtained as a white amorphous powder, the molecular
formula of C50H82O20 was determined by the HRESIMS at m/z 1001.53448 [M–H]ꢀ
(calcd for C50H81O20, 1001.53267) with 10 degrees of unsaturation. The
characteristic
fragmentation
pattern
was
identified
42Da
162Da621 [M–H]ꢀ, and
162Da
ꢀ
as
1001
[M–H]ꢀ,
[M–H]ꢀ,
44Da957
[M–H]ꢀ,
42Da915
44Da
ꢀ
ꢀ
132Da783 [M–H]ꢀ,
132Da
162Da
ꢀ
ꢀ
ꢀ
162Da459 [M–H] , indicating the molecule was belonged to a PPD
sapogenin. Analysis of the 13C NMR and DEPT spectra showed the presence of 50 car-
bon resonances, including 3 carbon signals for a malonyl group, 17 carbons for 3
1
sugar moieties and 30 carbons for aglycone. The H NMR date of 1 included 8 methyls
[dH 0.81 (3H, s), 0.96 (6H, s), 0.98 (3H, s), 1.31 (3H, s), 1.62 (3H, s), 1.66 (6H, s)], one ole-
finic proton [dH 5.33 (1H, t-like)], and 3 anomeric protons [dH 4.92 (1H, d, J ¼ 7.5 Hz), dH
5.16 (1H, d, J ¼ 7.5 Hz), dH 5.01 (1H, d, J ¼ 7.3 Hz)]. The NMR data (Table S1) of 1 were
similar to those of gypenoside IX (Takemoto et al. 1983) except for an additional
malonyl substituent, including 2 carbonyl groups at dC 168.1 and dC 169.6 and 1
methylene [dC 42.8/dH 3.82 (2H, d, J ¼ 6.3 Hz)], further verified by the HMBC correla-
tions from dH 3.82 to dC 168.1. Malonylation were observed at 60-OH of the 3-Glc by
the long-range HMBC correlation from H-60 [dH 5.14 (1H, m)] to the carbonyl carbon
(dC 168.1) (Figure S1), representing the sigals of C-50 shifts to upfield with 3.3 ppm and
C-60 shifts to downfield with 2.2 ppm compared with those of gypenoside IX. The gly-
cosyl moieties of 1 were confirmed to be two b-D-glucopyranosyl and one b-D-xylo-
pyranosyl due to the coupling constant [(H-10, J ¼ 7.5 Hz), (H-100, J ¼ 7.5 Hz), (H-1000,
J ¼ 7.3 Hz)] and UPLC analysis of the acid hydrolysate. Furthermore, the linkages of
sugar units were proved by the long-range HMBC correlations from H-10 to C-3 (dC
89.2), H-100 to C-20 (dC 83.4), and H-1000 to C-600 (dC 70.1). The relative configuration of
3-OH and C-20 were deduced to be b and S according to the ROESY correlations from
H-3 [dH 3.38 (dd, J ¼ 11.6, 4.1 Hz)] to H-5 (dH 0.77, m) and H-17 (dH 2.59, m) to H-21 (dH
1.66, s), respectively. Based on the evidence above, the structure of compound 1 was
established as 3-O-[(6-O-malonyl)-b-D-glucopyranosyl]-(3b,12b,20S)-trihydroxydammar-
ane-24-ene-20-O-b-D-xylopyranosyl(1!6)-b-D-glucopyranoside, and named as malonyl-
gypenoside IX.
2.2. Quantification of saponins
2.2.1. Optimization of UPLC separation
A good UPLC conditions was required for obtaining chromatograms with high reso-
lution of adjacent peaks in a short time. The most common organic solvent of the

NATURAL PRODUCT RESEARCH
5
mobile phase for the separation of saponins was methanol or acetonitrile, however,
no satisfied separation was found when the organic solvent was methanol, so the
acetonitrile was chosen as organic solvent, and the resolution was improved.
Phosphoric acid, phosphorate buffer, and acetic acid were also added to the aqueous
phase for optimizing the separation, the results indicated that phosphoric acid could
show higher resolution and better peak shapes. The column temperature was main-
tained at 35 ꢁC for its good baseline resolution and suitable analysis duration. Under
the above optimization conditions, a good separation of the saponins in mixed stand-
ards and sample was shown in Figure S11.
2.2.2. Quantitation of eight major saponins in PNF
The developed UPLC-UV method was applied to quantitative determination of the
eight major saponins in PNF, and giving the dry weight contents of 2.04 0.07 mg/g
for notoginsenosides FP2, 1.57 0.11 mg/g for ginsenoside Rb1, 2.99 0.14 mg/g for
notoginsenoside Fc, 2.02 0.13 mg/g for ginsenoside Rc, 1.76 0.12 mg/g for ginseno-
side Rb2, 3.01 0.14 mg/g for ginsenoside Rb3, 0.88 0.15 mg/g for notoginsenoside
Fe, and 0.94 0.12 mg/g for gypenoside IX, respectively. The result showed that the
major saponins were PPD type in PNF, which were similar to those of P. notoginseng
leaves (Liu et al. 2018).
2.2.3. Validation of UPLC-UV method
As shown in Table S2. The calibration curves showed good linearity (r > 0.998) in a
relatively wide concentration range. The limit of detection (LOD) and limit of quantifi-
cation (LOQ) were in the range of 2.38–3.4 lg/mL and 5.13–8.5 lg/mL, respectively.
These results revealed that the method exhibited good sensitivity. The relative stand-
ard deviation (RSD) values of intra-day and inter-day precision were less than 3.0% for
all the analytes, which indicated that the precision of the analytical method was excel-
lent. The repeatability of the method was good with a RSD of < 3.0%. The recoveries
of the method were in the range of 93.9–106.8%, with RSD value less than 3.0% as
shown in Table S3, which suggested the good accuracy of this method.
3. Experimental
3.1. General experimental procedures
One-dimensional (1 D) and two-dimensional (2 D) nuclear magnetic resonance (NMR)
spectra were determined on Bruker Avance Neo 600 MHz NMR spectrometer (Bruker
€
Co., Fallanden, Switzerland) with tetramethylsilane as an internal standard. IR spectra
were run on Bruker Vertex 70 FT-IR spectrophotometer (Bruker Co., Ettlingen,
Germany). The electrospray ionization mass spectrometry (ESI-MS) and high resolution
electrospray ionization mass spectrometry (HRESIMS) spectra were obtained from an
Agilent 1200 HPLC with a 6300 Ion-trap liquid chromatography-mass spectrophotom-
etry and a Vanquish UHPLC instrument connected to the Orbitrap Fusion mass spec-
trometer. Medium pressure liquid chromatography (MPLC) system was composed of a
€
€
C-660 fraction collector (Buchi), two C-601 pumps (Buchi), and a RP-C18 column
(Merck, 40-63lm, 49 ꢂ 460 mm). Reversed-phase semi-preparative HPLC was performed

6
Z.-Y. QU ET AL.
on a Prostar/Dynamax system consisting of a Prostar 325 UV-Vis detector and two
Varian PS-218 pump, UV detection was carried out at 203 nm with a flow rate of 3 mL/
min. Column chromatographies (CC) were carried out on silica gel (200-300 mesh,
Qingdao Marine Chemical Group Co., Qingdao, China), RP-C18 (Merck Co., 40-63 lm,
Damstadt, Germany). Thin-layer chromatography (TLC) was carried out on silica gel
60 RP-18 F254s and silica gel 60 F254. Spots were detected under UV light or by spraying
with 10% sulfuric acid in ethanol (EtOH) followed by heating at 105 ꢁC.
3.2. Plant material
The fresh fruits of Panax notoginseng (Burk.) F. H. Chen were collected from Wenshan
prefecture, Yunnan province, China in December 2018, and authenticated by Prof.
Peihe Zheng. A voucher specimen (No. FN20181225) was deposited at Institute of
Special Wild Economic Animals and Plants, Chinese Academy of Agricultural Sciences.
3.3. Extraction and isolation
The fresh fruits of P. notoginseng (18 kg) were rubbed with appropriate amount H2O
and removed the seeds, after filtration, the remaining residue was extracted twice in
85% EtOH-H2O at 25 ꢁC. Combined the aqueous and EtOH solutions and evaporated
under vacuum at < 40 ꢁC, the residue (605 g) was dissolved in H2O and partitioned
successively with petroleum ether (PE, 60–90 ꢁC) and n-BuOH. The n-BuOH fraction
(165 g) was separated via silica gel CC with a gradient of CH2Cl2-MeOH-H2O (20:1:0.1-
1:1:0.1, v/v) to afford 17 fractions (Frs.1–17), Fraction 5 (26.5 g) was separated by MPLC
system (MeOH-H2O, 40:60 ꢀ 90:10, 6 mL/min) to yield 6 fractions (Frs.5.1 ꢀ 5.6). Fraction
5.5 (6.1 g) was purified via RP-C18 CC again with a constant gradient of MeOH-H2O
(60:40, v/v) to yield compounds 2 (14 mg), 3 (22 mg), and 8 (25 mg). Fraction 5.4
(4.3 g) was isolated by semi-preparative HPLC on a XBridge BEH C18 OBD column
(10 ꢂ 250 mm, 5 mm; Waters) with a constant gradient of CH3CN-H2O (45:55, v/v) to
afford compounds 4 (51 mg, tR ¼ 8.9 min), 5 (42 mg, tR ¼ 10.8 min), 11 (30 mg, tR ¼
6.3 min), and 12 (46 mg, tR ¼ 5.2 min). Fraction 5.2 (2.9 g) was isolated by semi-pre-
parative HPLC on a Gemini C18 Column (10 ꢂ 250 mm, 5 mm; Phenomenex) with a con-
stant gradient of CH3CN-H2O (45:55, v/v) to afford compounds 6 (56 mg, tR ¼ 8.9 min)
and 7 (32 mg, tR ¼ 10.8 min). Fraction 11 (23.4 g) was separated by MPLC system
(MeOH-H2O, 30:60 ꢀ 90:10, 6 mL/min) to yield 8 fractions (Frs.11.1 ꢀ 11.8). Fraction 11.4
(12.2 g) was isolated by semi-preparative HPLC on a Gemini C18 Column (10 ꢂ 250 mm,
5 mm; Phenomenex) with a constant gradient of CH3CN-H2O (35:65, v/v) to afford com-
pounds 9 (15 mg, tR ¼ 23.0 min) and 14 (73 mg, tR ¼ 16.4 min). Fraction 11.6 (2.6 g)
was isolated by semi-preparative HPLC on a ZORBAX SB-C18 column (9.4 ꢂ 250 mm,
5 lm; Agilent) with a constant gradient of CH3CN-0.05% TFA (43:57, v/v) to afford com-
pounds 1 (21 mg, tR ¼ 12.2 min). Fraction 12 (6.7 g) was chromatographed over RP-C18,
eluted with MeOH-H2O (28:72–80:20, v/v) to yield compounds 10 (24 mg), 15 (39 mg),
and 19 (88 mg). Fraction 14 (17.4 g) was separated by MPLC system (MeOH-H2O,
25:75–70:30, 6 mL/min) to yield 7 fractions (Frs.14.1–14.7). Fraction 14.3 (9.6 g) was
purified by semi-preparative HPLC on a Gemini C18 Column (10 ꢂ 250 mm, 5 mm;

NATURAL PRODUCT RESEARCH
7
Phenomenex) with a constant gradient of CH3CN-H2O (30:70, v/v) to afford com-
pounds 13 (38 mg, tR ¼ 33.2 min) and 16 (8 mg, tR ¼ 25.0 min). Fraction 14.5 (1.6 g)
was then purified by semi-preparative HPLC on
a ZORBAX SB-C18 column
(9.4 ꢂ 250 mm, 5 lm; Agilent) with a constant gradient of CH3CN-H2O (30:70, v/v) to
afford compounds 17 (18 mg, tR ¼ 8.4 min) and 18 (12 mg, tR ¼ 11.5 min). Fraction 16
(30.8 g) was separated by was repeated separation on RP-C18, eluted with MeOH-H2O
(20:80–60:40, v/v) to yield compounds 20 (33 mg), 21 (18 mg), and 22 (12 mg).
3.3.1. Malonylgypenoside IX (1)
25
White amorphous powder; ½aꢃD þ6.3 (c 0.1 MeOH); IR (KBr) ꢀmax 3401, 2946, 1752,
1676, 1385, 1168, 1077, 1041 cmꢀ1; HRESIMS m/z 1001.53448 [M-H]- (calcd for
C50H81O20, 1001.53267). 1H NMR (600 MHz, pyridine-d5, d in ppm, J in Hz), dH: [1.74
(1H, m), 1.01 (1H, m), H-1], [2.33 (1H, m), 1.86 (1H, m), H-2], 3.38 (1H, dd, J ¼ 11.6,
4.1 Hz, H-3), 0.77 (1H, m, H-5), [1.49 (1H, m), 1.36 (1H, m), H-6], [1.49 (1H, m), 1.21 (1H,
m), H-7], 1.45 (1H, m, H-9), [2.04 (1H, m), 1.57 (1H, m), H-11], 4.21 (1H, m, H-12), 2.00
(1H, m, H-13), [1.57 (1H, m), 1.01 (1H, m), H-15], [1.86 (1H, m), 1.36 (1H, m), H-16], 2.59
(1H, m, H-17), 0.96 (3H, s, H-18), 0.81 (3H, s, H-19), 1.66 (3H, s, H-21), [2.42 (1H, m),
1.83 (1H, m), H-22], [2.63 (1H, m), 2.38 (1H, m), H-23], 5.33 (1H, t-like, H-24), 1.62 (3H, s,
H-26), 1.66 (3H, s, H-27), 1.31 (3H, s, H-28), 0.96 (3H, s, H-29), 0.98 (3H, s, H-30), 4.92
(1H, d, J ¼ 7.5 Hz, 3-Glc-10), 4.05 (1H, m, 3-Glc-20), 4.18 (1H, m, 3-Glc-30), 4.09 (1H, m, 3-
Glc-40), 4.11 (1H, m, 3-Glc-50), [5.14 (1H, m), 4.92 (1H, d, J ¼ 7.5 Hz), 3-Glc-60], 5.16 (1H,
d, J ¼ 7.5 Hz, 20-Glc-100), 3.94 (1H, m, 20-Glc-200), 4.22 (1H, m, 20-Glc-300), 4.09 (1H, m,
20-Glc-400), 4.09 (1H, m, 20-Glc-500), [4.76 (1H, d, J ¼ 10.9 Hz), 4.33 (1H, m), 20-Glc-600],
5.01 (1H, d, J ¼ 7.3 Hz, Xyl-1000), 4.05 (1H, m, Xyl-2000), 4.16 (1H, m, Xyl-3000), 4.22 (1H, m,
Xyl-4000), [4.35 (1H, m), 3.70 (1H, m), Xyl-5000], 3.82 (2H, d, J ¼ 6.3 Hz, 60-Mal). 13C NMR
(150 MHz, pyridine-d5, d in ppm), dC: 39.2 (C-1), 26.7 (C-2), 88.2 (C-3), 39.7 (C-4), 56.5
(C-5), 18.5 (C-6), 35.1 (C-7), 40.1 (C-8), 50.2 (C-9), 37.0 (C-10), 30.8 (C-11), 70.1 (C-12),
49.5 (C-13), 51.7 (C-14), 31.0 (C-15), 26.8 (C-16), 51.4 (C-17), 16.0 (C-18), 16.3 (C-19),
83.4 (C-20), 22.3 (C-21), 36.2 (C-22), 23.2 (C-23), 126.1 (C-24), 131.0 (C-25), 25.8 (C-26),
18.0 (C-27), 28.2 (C-28), 16.8 (C-29), 17.4 (C-30), 107.0 (3-O-Glc-10), 75.6 (3-O-Glc-20),
79.4 (3-O-Glc-30), 71.6 (3-O-Glc-40), 74.9 (3-O-Glc-50), 65.5 (3-O-Glc-60), 98.2 (20-O-Glc-100),
74.9 (20-O-Glc-200), 78.6 (20-O-Glc-300), 71.7 (20-O-Glc-400), 77.0 (20-O-Glc-500), 70.1 (20-O-
Glc-600), 105.9 (Xyl-1000), 74.9 (Xyl-2000), 78.0 (Xyl-3000), 71.1 (Xyl-4000), 67.0 (Xyl-5000), 168.1
(-O-CO), 42.8 (-CH2), 169.6 (-COOH).
a 25
½ ꢃD
3.3.2. Acid hydrolysis and UPLC analysis
Acid hydrolysis was carried out as reported previously (Li et al. 2012; Qu et al. 2021),
in brief, compound 1 (2 mg) were hydrolyzed in 2 M HCl (3 mL) and stirred at 90 ꢁC for
3 h. The resultant of reaction was diluted with water and extracted by EtOAc (3 mL),
the aqueous solution was neutralized with Ag2CO3 and subsequently analyzed by
UPLC equipped with evaporative light scattering detector (ELSD), and an Acquity BEH
Amide column (2.1 ꢂ 50 mm, 1.7 lm; Waters). The configurations of D-xylose (2.41 min)

8
Z.-Y. QU ET AL.
and D-glucose (3.33 min) were determined by comparison of the retention time of
authentic standard.
3.4. Quantification of saponins
3.4.1. Calibration curves
The mixed standards solution were prepared by dissolving the accurately weighed of
eight standard reference compounds (i.e., notoginsenosides FP2, Fc, Fe, and ginseno-
sides Rb1, Rc, Rb2, Rb3, and gypenoside IX. All standards were isolated from this study,
and the purity of all standards > 95%) with methanol, and giving a appropriate con-
centration ranges for the establishment of calibration curves. Five concentrations of
the mixed standards solution were determined under the same chromatographic con-
ditions, and their calibration curves were obtained by plotting the peak area versus
the corresponding concentration of saponin concerned. The LOD and LOQ were deter-
mined based on signal-to-noise ratios of 3 and 10, respectively.
3.4.2. Sample preparation
The fresh sample (drying rate, 27.1%) was accurately weighed (40.0 g), and extracted
with 500 mL ethanol in an ultrasonic bath (KQ-500DE, Kunshan, China) for 30 min and
filtered. This extraction was repeated two additional times, the combined filtrate was
evaporated to dryness in vacuo. The residue was dissolved with water, and then sub-
jected to a HP-20 macroporous resin (30.0 g, Mitsubishi Chemical Co., Tokyo, Japan)
CC, eluting with H2O (100 mL) and 90% ethanol solution (100 mL) at a flow rate of 2
BV/h, the 90% ethanol fraction was collected and evaporated to dryness, and then dis-
solved and diluted to the volume scale by methanol in a 25 mL volumetric flask. This
solution was filtered through 0.22 lm nylon filter membrane prior to UPLC analysis.
3.4.3. Chromatographic conditions
UPLC analysis was performed on an ACQUITY UPLC system (Waters Corp., Milford,
USA), equipped with a PDA detector and a binary pump solvent management system.
Chromatographic separation was carried out on a Waters ACQUITY UPLC BEH C18
(2.1 ꢂ 50 mm, 1.7 lm; Waters) at a flow rate of 0.5 mL/min. A gradient elution system
consisted of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v)
was as follows: 0–3 min, 10% A; 3–4 min, 10–28% A; 4–7 min, 28% A; 7–9 min, 28–32%
A; 9–10 min, 32% A; 10–11 min, 32–40% A; 11–12 min, 40–50% A; 12 ꢀ 14 min, 50% A;
14 ꢀ 14.5 min, 50 ꢀ 65% A; 14.5 ꢀ 16.5 min, 65% A; 16.5 ꢀ 17 min, 65 ꢀ 95% A;
17 ꢀ 19 min, 95% A; 19 ꢀ 19.5 min, 95 ꢀ 10% A; 19.5 ꢀ 22 min, 10% A. The detection
wavelength and column temperature were set at 203 nm and 35 ꢁC.
3.4.4. Method validations
The method precision was evaluated by intra-day and inter-day tests. Intra-day tests
were performed by analysis of six times (0 h, 5 h, 10 h, 15 h, 20 h, and 24 h) of the
same sample within one day. Inter-day experiments were carried out on three con-
secutive days in the same method as intra-day experiments. The RSD was taken as a
measure of precision. The method repeatability was evaluated with parallelly divided

NATURAL PRODUCT RESEARCH
9
the sample into six parts, and extracted and analyzed by the mentioned above.
Recovery test was used to evaluate the accuracy of this method. Accurate weights of
standard compounds were added to the sample of known amounts of these com-
pounds, the resultant samples were extracted and analysed with the described
method. The average recovery and RSD were taken as a measure of recovery.
4. Conclusion
In conclusion, a continuation of phytochemical study on PNF led to yield a new
malonyl-substituted ginsenoside, named as malonylgypenoside IX (1). Meanwhile,
other known saponins (2–22) were also isolated, among these known isolates, thirteen
(3–10, and 14–18) saponins were obtained from PNF for the first time. In addition,
this is the first report on the simultaneous determination of eight major saponins
[notoginsenoside FP2 (14), ginsenoside Rb1 (19), notoginsenoside Fc (13), ginsenoside
Rc (15), ginsenoside Rb2 (10), ginsenoside Rb3 (12), notoginsenoside Fe (4), and gype-
noside IX (5)] in PNF with UPLC-UV method, which was proved to be simple, rapid
and accurate via method validation. This study would provide scientific foundation for
the comprehensive utilization of PNF.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1ꢀS11 and
Tables S1ꢀS3.
Funding
The authors thank the National Key Research and Development Program of China
(2017YFC1702102). The General Research Subject of Higher Education in Jilin Province
(JGJX2020D547), and the Project of the Jilin Provincial Department of Science and Technology,
China (20170203004YY).
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