Saponins from European Licorice Roots (Glycyrrhiza glabra)

Article abstract of DOI:10.1021/acs.jnatprod.8b00022

European licorice roots (Glycyrrhiza glabra), used in the food and beverage industry due to their distinctive sweet and typical licorice flavor, were fractionated, with the triterpenoid saponins isolated and their chemical structures determined by means o

Full text of DOI:10.1021/acs.jnatprod.8b00022

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Saponins from European Licorice Roots (Glycyrrhiza glabra)
Christian Schmid, Corinna Dawid, Verena Peters, and Thomas Hofmann*
̈
̈
Chair of Food Chemistry and Molecular Sensory Science, Technische Universitat Munchen, Lise-Meitner-Straße 34, D-85354
Freising, Germany
S
* Supporting Information
ABSTRACT: European licorice roots (Glycyrrhiza glabra),
used in the food and beverage industry due to their distinctive
sweet and typical licorice flavor, were fractionated, with the
triterpenoid saponins isolated and their chemical structures
determined by means of ESIMS, ESIMS/MS, HRESIMS, and
1D/2D NMR experiments. Next to the quantitatively
predominant saponin glycyrrhizin (11) and some previously
known saponins, the structures of 10 monodesmosidic
saponins were assigned unequivocally for the first time,
namely, 30-hydroxyglycyrrhizin (1), glycyrrhizin-20-metha-
noate (2), 24-hydroxyglucoglycyrrhizin (3), rhaoglycyrrhizin
(4), 11-deoxorhaoglycyrrhizin (5), rhaoglucoglycyrrhizin (6),
rhaogalactoglycyrrhizin (7), 11-deoxo-20α-glycyrrhizin (8), 20α-galacturonoylglycyrrhizin (9), and 20α-rhaoglycyrrhizin (10).
Although a total of 43 saponins have been reported in G.
uralensis,19,27,32−38 which is mostly used in oriental medicine
systems, mass-spectrometry-based metabolome profiling in-
dicated a series of not yet fully characterized saponins in
several Glycyrrhiza species.15,18,22,39−42 In particular, the
present knowledge on the saponin composition in European
licorice (Glycyrrhiza glabra L.), mainly used in the food and
beverage industry, is rather fragmentary. Therefore, the
objective of the present study was to isolate and identify the
chemical structure of oleanane-type triterpenoidic saponins in
European licorice roots by means of ESIMS, ESIMS/MS,
HRESIMS, and 1D/2D NMR experiments.
everal herbaceous perennial species from the genus
SGlycyrrhiza, also known as licorice, are well accepted for
their attractive flavor properties as well as other biological
activities that are due to oleanane-type triterpenoid sap-
onins.1−5 The legume genus Glycyrrhiza consists of about 30
species native to Europe, Asia, North and South America, and
Australia, including G. glabra, G. uralensis, G. inflata, G. aspera,
G. korshinskyi, and G. eurycarpa, respectively. Owing to the
distinctive sweet and licorice sensation of their roots,
Glycyrrhiza spp. are named after the Greek words “glycys”
(sweet) and “rhize” (roots) and are used for production of
food, confectionary, beverage, and cosmetic products, as well
as to flavor cigarettes.2 Besides uses as a low-caloric sweetener
and flavor additive, licorice extracts have a long-standing
history as a botanical drug in Europe and Asia, dating back to
at least 2100 B.C.6−10 For centuries, licorice extracts have been
used in China to treat respiratory (asthma), gastrointestinal
(chronic gastritis), urogenital (bladder infection), and skin
diseases (atopic dermatitis), respectively.2,9,11
Various polyphenols and saponins are reported to be
responsible for the pharmacological activity of licorice.2,9,11−23
Among the oleanane triterpenoid saponins, glycyrrhizin has
been reported as the major secondary metabolite in licorice;
this monodesmosidic saponin exhibits a 18β-glycyrrhetinic acid
skeletal structure, derived from β-amyrin, linked to a
disaccharide unit bearing two glucuronic acid moieties at
position C-3.7,8,24−28 In addition to its anti-inflammatory,
antimicrobial, antiviral, and antitumor activities,2,9,11−23
glycyrrhizin has been reported to inhibit 11β-hydroxysteroid
dehydrogenase (11βHSD2) and to induce mineralocorticoid
excess syndrome,29,30 leading to hypokalemia, hypertension,
and hypernatremia in humans.2,9,11,31
RESULTS AND DISCUSSION
■
In order to isolate and identify oleanane-type saponins from
the roots of G. glabra, a MeOH−H2O extract was fractionated
by means of an offline multidimensional preparative strategy
(Figure S1, Supporting Information). An initial preseparation
of different classes of phytochemicals in licorice roots was
achieved by taking up the MeOH−H2O extractables in H2O
and preseparating them into four fractions by means of MPLC.
To obtain pure compounds, fractions II/III and IV were
separated by HPLC into 23 and 18 subfractions, respectively.
While compounds 11, 12, and 19 could be obtained as pure
substances from fractions II/II-19, IV-11, and II/III-22,
respectively, additional semipreparative and preparative
HPLC separation was performed to afford compounds 1−10,
13−18, 20, and 21 from fractions II/III-15−II/III-18, II/III-
20, II/III-21, II/III-23, IV-6, IV-7, IV-9, IV-14, IV-15, and IV-
16, respectively (Figure S1, Supporting Information, colored in
Received: January 7, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00022
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Figure 1. Structures of new triterpenoid saponins 1−10 isolated from G. glabra.
black). Preparative fractionation, followed by HRESIMS, LC-
MS/MS, and 1D and 2D NMR experiments, led to the
identification of 21 triterpenoid saponins in European licorice
roots (Figure 1). To the best of our knowledge, the chemical
structures of 10 triterpenoid saponins (1−10) were deter-
mined unequivocally for the first time (Figure 1). In addition,
the presence of saponins 12, 13, 15, 16, 17, 18, and 21,
previously isolated from G. uralensis and G. inflata, is reported
for the first time in G. glabra. Furthermore, the NMR data of
compounds 12, 15, and 20 could be complemented.22,37,38,43,44
Mass spectrometric and NMR spectroscopic data (Tables S1−
S11) of several previously identified saponins corresponded
well with published data: glycyrrhizin (11),45 LS-B2 (11-
deoxoglycyrrhizin, 12),22,37,38 LS-G2 (24-hydroxyglycyrrhizin,
13),38,44 LS-J2 (11-deoxo-24-hydroxyglycyrrhizin, 14),33 glu-
coglycyrrhizin (15),43 araboglycyrrhizin (16),32,38 apioglycyr-
rhizin (17),32,38 glycyrrhetinic acid-monoglucuronide
(18),46,47 LS-H2 (20α-glycyrrhizin, 19),38,44 24-hydroxy-20α-
glycyrrhizin (20),38,44 and LS-C2 (11-deoxo-11,13-glycyrrhizin
diene, 21).22,37,38 A comparison of selected key NMR data,
shown in Figure 2, were used to support the structure
elucidation of unknown saponins and might be useful in the
future to determine new saponins.
HRESIMS analysis of 1 revealed a molecular formula of
C42H64O15, based on the deprotonated molecule [M − H]− at
m/z 807.4166, calcd for C42H63O15 (m/z 807.4167).
Compared to the spectrum of commercially available
glycyrrhizin (11) with a determined empirical formula of
C42H62O16, the HRESIMS data of 1 indicated a lack of an
oxygen atom and two protons. By means of additional ESIMS/
MS experiments of the protonated molecule [M + H]+, this
resulted in fragments of m/z 633 and m/z 439, indicating the
presence of two uronic acid moieties. The 13C NMR
spectroscopic data of 1, isolated from fraction II/III-21-4-2,
showed a total of 42 signals resonating between 17.1 and 199.8
ppm, among which 12 signals between 73.4 and 172.9 ppm
were almost identical with the disaccharide moiety of
glycyrrhizin (11), consisting of two β-D-glucuronic acids, as
reported earlier.43 Acid hydrolysis, followed by the determi-
nation of the monosaccharides, proved the presence of two D-
glucuronic acids. For the characterization of the aglycone
structure, carbon atoms with typical stand-alone 13C NMR
chemical shifts were used, such as 199.8 ppm for the carbonyl
atom C-11, 170.2 ppm for the quaternary olefinic carbon atom
C-13, and 89.5 ppm for the O-glycoside-bound carbon atom
C-3, respectively (Table 1). Using C-3, C-11, and C-13 as
starting points for NMR signal assignment and their
corresponding HMBC correlations with H-1 (δH 3.05, δH
1.03), H-2 (δH 2.10, δH 2.33), Me-23 (δH 1.43), Me-24 (δH
1.27)/C-3 (δC 89.5); H-9 (δH 2.44), H-12 (δH 5.75)/C-11 (δC
199.8); and H-12 (δH 5.75), Me-27 (δH 1.42), H-18 (δH
2.26)/C-13 (δC 170.2), the adjacent functional groups at
carbons 1, 2, 3, 9, 11, 12, 13, 18, 23, 24, and 27 were assigned.
Compared to glycyrrhizin (11), compound 1 showed a high-
field shift of H-12 with δH 5.75 (1H, s, 1) and H-18 with δH
2.26 (1H, dd, J = 13.5, 4.5 Hz, 1) with respect to H-12 with δH
5.98 (1H, s) and H-18 with δH 2.54 (1H, dd, J = 13.1, 3.7 Hz),
indicating a less electronegative functional group at the
triterpenoid ring E. With the absence of any signal at δC
179.5 (C-30), which was observed for glycyrrhizin (11), and
the presence of the two signals δH 3.80 (1H, d, J = 10.8 Hz)
and 3.72 (1H, d, J = 10.8 Hz) correlating with C-18 (δC 47.6)
this revealed a CH2OH group at position C-30 (Figure 2).
Further HMBC correlations of Me-29 (δH 5.16)/C-30 (δC
65.7), H2-30 (δH 3.72, δH 3.80)/C-29 (δC 28.6), and H-18 (δH
2.26)/C-29 (δC 28.6) also supported the presence of a
CH2OH group at position C-30. Moreover, ROE correlations
were observed between the proton pairs H-3/Me-23, H-3/H-5,
Me-23/H-5, H-5/H-9, and H-9/Me-27 of 1, which indicated
that the H-5 and H-9 protons as well as the methyl groups Me-
B
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1
Figure 2. H NMR chemical shifts of prominent positions in the aglycone and the carbohydrate moiety of saponins 1−21.
23 and Me-27 are α-oriented. In addition, ROE correlations of
protons Me-24/Me-25, Me-25/Me-26, Me-26/Me-28, H-18/
Me-28, and H-18/H2-30 established that H-18 and H2-30 are
β-oriented (Figure 3). Thus, compound 1 was determined as
(3β,20β)-20-hydroxymethyl-11-oxo-30-norolean-12-en-3-yl 2-
O-β-D-glucopyranuronosyl-β-D-glucopyranosiduronic acid (30-
hydroxyglycyrrhizin).
downfield shift of the C-30 methylene group protons of δH
4.29 (1H, m, 2) and 4.11 (1H, d, J = 11.0 Hz, 2), in
comparison to δH 3.80 (1H, d, J = 10.8 Hz, 1) and 3.72 (1H, d,
J = 10.8 Hz, 1), was detected. By means of the HMBC
correlation of H-30 (δH 4.29, δH 4.11)/C-31 (δC 162.1) the
position of an aldehyde group could be determined at position
C-30, and, consequently, compound 2 was assigned as
(3β,20β)-20-methylformate-11-oxo-30-norolean-12-en-3-yl 2-
O-β-D-glucopyranuronosyl-β-D-glucopyranosiduronic acid (gly-
cyrrhizin-20-methanoate).
Saponin 3 showed the molecular formula C42H64O16 based
on the ion [M − H]− at m/z 823.4122, calcd for C42H63O16
(m/z 823.4116), in its HRESIMS. When compared to the
empirical formula of glycyrrhizin (11) (C42H62O16), com-
pound 3 showed two additional protons. An ESIMS/MS
experiment of compound 3 revealed, based on the protonated
molecular ion peak [M + H]+ at m/z 825, fragments at m/z
663 and m/z 469, indicating a hexose as terminal sugar moiety
and an uronic acid as the aglycone-bound sugar moiety.
Further, the aglycone fragment m/z 469 of 3 showed in
comparison with the corresponding fragment of glycyrrhizin
(11) at m/z 453 an aglycone with a 16 Da increased mass, thus
suggesting an additional hydroxy group linked to the
sapogenin. The 13C NMR spectrum displayed 42 resonances
like glycyrrhizin (11), with two signals shifted to δC 63.6 and
δC 61.9 (Table 1 and Table S1, Supporting Information).
These signals gave cross-peaks with proton resonances at δH
Compound 2 gave a molecular formula of C43H64O16, based
on the [M − COHH]− ion with m/z 807.4195, calcd for
C42H63O15 (m/z 807.4167), in the HRESIMS spectrum. In
contrast to the HRESIMS, the ESIMS data revealed a
protonated peak of m/z 837 [M + H]+, indicating an
additional carbonyl group when compared to 1. Further
ESIMS/MS fragmentation of compound 2 showed patterns of
m/z 809, 661, 457, and 442 with neutral mass losses of 176 +
219 Da, indicating the presence of two uronic acid moieties.
1
The H NMR spectrum of 2 displayed in contrast to 1 an
additional resonance at δH 8.39 (1H, s) with a correlation to
the carbon resonance at δC 162.1 in the HSQC spectrum,
which could be attributed to an aldehyde group (Table 1).
Comparison of 1H and 13C NMR resonances of compounds 1
and 2 revealed for both similar shifts for H-1 to H-28 and C-1
to C-28, including H-1′ to H-6″ and C-1′ to C-6″. With similar
results of the monosaccharides after acidic hydrolysis, 2 was
identical with 1 except for positions 29 and 30. For the methyl
group at C-29, a high-field shift of δH 0.96 (3H, s, 2), in
contrast to δH 1.16 (3H, s, 1), could be observed. In contrary, a
C
DOI: 10.1021/acs.jnatprod.8b00022
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1
Table 1. NMR Spectroscopic Data for Compounds 1−5 in Pyridine-d5 (500 MHz for H and 125 MHz for 13C, δ Values in
ppm, J Values in Hz)
1
2
3
4
5
position δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
2
3
39.9, CH2 3.05, dd (13.6, 39.8, CH2 3.05, dt (13.2, 39.4, CH2 2.97, dd (9.3,
39.8, CH2 3.04, dd (9.5,
3.5)
39.1, CH2 1.39, m
3.5)
3.5)
4.1)
1.03, m
1.05, m
1.06, m
1.12, m
0.91, m
27.1, CH2 2.33, m
27.1, CH2 2.34, dd (13.9, 26.8, CH2 2.60, m
4.2)
26.5, CH2 2.26, dd (12.1, 26.8, CH2 2.18, m
4.4)
2.10, m
2.09, m
2.09, m
2.05, m
1.84, m
89.5, CH 3.36, dd (11.7, 89.5, CH 3.37, dd (11.8, 90.8, CH 3.54, dd (12.3, 89.8, CH 3.38, dd (11.7, 90.4, CH 3.34, dd (11.8,
4.5) 4.3) 5.4) 4.3) 4.5)
4
5
40.3, C
40.3, C
44.5, C
40.5, C
40.0, C
56.2, CH 0.83, m
55.8, CH 0.74, d (11.8) 55.7, CH 0.73, d (11.6) 56.1, CH 0.87, d (11.9) 55.4, CH 0.83, dd (12,
2.6)
6
7
8
18.0, CH2 1.29, 1.08, m
33.3, CH2 1.53, 1.27,m
45.9, C
17.9, CH2 1.50, 1.32, m
33.2, CH2 1.55, 1.23, m
45.9, C
18.0, CH2 1.58, 1.28, m
33.2, CH2 1.55, 1.24, m
45.7, C
18.0, CH2 1.59, 1.36, m
33.3, CH2 1.65, 1.29, m
45.9, C
18.9, CH2 1.56, 1.37, m
33.3, CH2 1.54, 1.30, m
40.4, C
9
62.4, CH 2.44, s
37.6, C
199.8, C
128.9, CH 5.75, s
170.2, C
44.0, C
62.4, CH 2.42, s
37.6, C
199.8, C
129.1, CH 5.73, s
169.3, C
43.8, C
62.1, CH 2.40, s
37.0, C
199.8, C
128.8, CH 5.95, s
170.3, C
43.7, C
62.5, CH 2.48, s
37.6, C
199.9, C
129.0, CH 5.94, s
169.9, C
43.8, C
48.2, CH 1.64, t (8.9)
37.2, C
24.1, CH2 1.81, m
123.2, CH 5.46, t (4.0)
145.5, C
42.1, C
27.0, CH2 1.81, 1.02, m
27.6, CH2 2.10, dt (13.8,
4.3)
10
11
12
13
14
15
16
27.1, CH2 1.68, 1.06, m
27.3, CH2 2.09, m
27.0, CH2 1.68, 1.04, m
27.0, CH2 2.01, m
27.1, CH2 1.68, 1.09, m
26.9, CH2 2.09, m
27.2, CH2 1.73, 1.12, m
27.0, CH2 2.05, m
0.90, m
32.9, C
47.6, CH 2.26, dd (13.5, 47.2, CH 2.17, m
4.5)
41.2, CH2 1.69, 1.49, m
36.4, C
30.3, CH2 1.69, 1.38, m
36.8, CH2 1.54, 1.28, m
28.5, CH3 1.43, s
17.2, CH3 1.27, s
0.85, m
32.8, C
0.94, m
34.4, C
0.95, m
32.5, C
0.92, m
32.7, C
17
18
49.0, CH 2.51, dd (13.3, 49.1, CH 2.53, dd (16.9, 49.0, CH 2.41, dt (13.7,
3.3)
42.0, CH2 2.13, 1.72, m
44.3, C
31.9, CH2 2.26, 1.46, m
38.6, CH2 1.68, 1.42, m
22.9, CH3 1.37, s
63.6, CH2 4.33, m
3.39, d (12.3)
3.3)
42.0, CH2 2.12, 1.74, m
44.5, C
31.9, CH2 2.29, 1.49, m
38.8, CH2 1.69, 1.44, m
28.8, CH3 1.48, s
17.2, CH3 1.27, s
3.3)
43.8, CH2 2.29, 1.85, m
44.7, C
32.2, CH2 2.30, 1.51, m
39.4, CH2 1.81, 1.47, m
28.8, CH3 1.47, s
17.3, CH3 1.25, s
19
20
21
22
23
24
40.6, CH2 1.62, 1.22, m
34.8, C
30.6, CH2 1.35, 1.36, m
36.4, CH2 1.41, 1.24, m
28.5, CH3 1.42, s
17.2, CH3 1.26, s
25
26
27
28
29
30
17.1, CH3 1.21, s
19.2, CH3 1.05, s
23.9, CH3 1.42, s
29.0, CH3 0.83, s
28.6, CH3 1.16, s
17.1, CH3 1.21, s
19.1, CH3 1.04, s
23.9, CH3 1.38, s
28.8, CH3 0.79, s
28.1, CH3 0.96, s
16.9, CH3 1.06, s
18.9, CH3 1.00, s
23.8, CH3 1.39, s
28.3, CH3 0.76, s
29.1, CH3 1.34, s
179.9, C
17.0, CH3 1.19, s
19.4, CH3 1.07, s
23.8, CH3 1.43, s
29.1, CH3 0.77, s
29.0, CH3 1.37, s
179.5, C
15.9, CH3 0.79, s
17.2, CH3 0.92, s
26.5, CH3 1.30, s
28.9, CH3 0.89, s
29.5, CH3 1.39, s
180.0, C
65.7, CH2 3.80, d (10.8) 67.1, CH2 4.29, m
3.72, d (10.8)
4.11, d (11.0)
31
1′
2′
162.1, CH 8.39s
105.5, CH 5.08, d (7.6)
84.8, CH 4.32, m
105.5, CH 5.08, d (7.8)
84.9, CH 4.33, m
104.7, CH 4.85, d (8.0)
82.1, CH 4.15, m
105.5, CH 5.11, d (7.8)
79.7, CH 4.55, dd (9.0,
7.8)
105.5, CH 5.11, d (7.7)
79.7, CH 4.55, dd (9.2,
7.6)
3′
4′
78.1, CH 4.43, t (8.8)
73.4, CH 4.60, m
78.1, CH 4.43, pt (8.7)
73.4, CH 4.60, m
78.8, CH 4.31, m
73.6, CH 4.31, m
79.1, CH 4.67, m
73.8, CH 4.47, m
79.1, CH 4.68, t (9.0)
73.9, CH 4.50, dd (9.8,
9.0)
5′
6′
1″
2″
77.8, CH 4.63, m
172.9, C
107.3, CH 5.47, d (7.8)
77.2, CH 4.28, m
77.8, CH 4.61, m
172.9, C
107.3, CH 5.47, d (7.6)
77.2, CH 4.29, m
76.5, CH 4.28, m
175.5, C
105.2, CH 5.52, d (7.9)
76.1, CH 4.09, dd (8.8,
7.8)
77.9, CH 4.62, m
172.9, C
103.1, CH 5.97, d (7.9)
78.5, CH 4.48, m
77.8, CH 4.68, d (9.6)
173.0, C
103.1, CH 5.97, d (7.8)
78.5, CH 4.48, dd (9.1,
7.7)
3″
4″
78.0, CH 4.35, m
73.7, CH 4.68, m
78.0, CH 4.36, m
73.7, CH 4.65, m
78.0, CH 4.20, t (9.1)
70.3, CH 4.38, t (9.1)
79.3, CH 4.35, m
74.0, CH 4.62, m
79.3, CH 4.35, pt (9.1)
74.0, CH 4.62, dd (9.8,
8.9)
5″
78.8, CH 4.65, m
172.5, C
78.8, CH 4.68, m
172.6, C
78.6, CH 3.71, dt (9.6,
3.8)
61.9, CH2 4.34, 4.38, m
77.8, CH 4.48, m
77.9, CH 4.48, d (9.7)
6″
172.6, C
172.5, C
1‴
2‴
102.6, CH 6.47, d (2.3)
72.8, CH 4.8, dd (3.1,
2.1)
102.5, CH 6.46, d (1.9)
72.8, CH 4.80, dd (3.2,
1.6)
D
DOI: 10.1021/acs.jnatprod.8b00022
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Table 1. continued
1
2
3
4
5
position δC, type
3‴
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
73.1, CH 4.73, dd (9.0,
3.3)
73.1, CH 4.74, dd (9.2,
3.7)
4‴
5‴
74.7, CH 4.38, t (9.0)
70.0, CH 5.08, dd (8.8,
6.3)
74.7, CH 4.37, pt (9.2)
70.0, CH 5.08, dd (9.3,
6.2)
6‴
19.1, CH3 1.84, d (6.2)
19.4, CH3 1.84, d (6.0)
glucopyranosyl-β-D-glucopyranosiduronic acid (24-hydroxyglu-
coglycyrrhizin).
HRESIMS analysis of compound 4 afforded a molecular
formula of C48H72O20, based on a deprotonated molecular ion
peak [M − H]− at m/z 967.4570, calcd for C48H71O20 at m/z
967.4539. In comparison to glycyrrhizin (C42H62O16, 11)
compound 4 showed a molecular formula that was increased
by C6H10O4, indicating a further methylpentose moiety, which
could be substantiated by ESIMS/MS of this compound. The
protonated molecule of saponin 4 (m/z 969, [M + H]+)
exhibited a fragmentation pattern with m/z 823, 647, and 453,
thus indicating a methyl-pentose and two uronic acid moieties.
Besides the typical 1H and 13C NMR shifts for the
glycyrrhetinic acid moiety, all HSQC, HMBC, and ROE
correlations corresponded to the glycyrrhizin (11) aglycone
(Table 1 and Table S1, Supporting Information). Further, by
means of NMR spectroscopy as well as by determination of the
absolute configuration of the monosaccharide moieties after
acidic hydrolysis, the presence was demonstrated of two 1″ →
2′ linked β-D-glucuronic acids attached to an additional 1‴ →
2″ glycosylated α-L-rhamnose unit. This was substantiated by
means of the observed COSY and HMBC correlations: H-1′
(δH 5.11)/C-3 (δC 89.8), H-1′ (δH 5.11)/H-2′ (δH 4.55), H-2′
(δH 4.55)/C-1″ (δC 103.1), H-1″ (δH 5.97)/H-2″ (δH 4.48),
H-2″ (δH 4.48)/C-1‴ (δH 6.47). α-L-Rhamnose could be
identified as the terminal sugar moiety as a result of the COSY
correlations at H-1‴ (δH 6.47)/H-2‴ (δH 4.80), H-2‴ (δH
4.80)/H-3‴ (δH 4.73), H-3‴ (δH 4.73)/H-4‴ (δH 4.38), H-4‴
(δH 4.38)/H-5‴ (δH 5.08), and H-5‴ (δH 5.08)/H-6‴ (δH
1.84) and the presence of a methyl group resonating at δH 1.84
(3H, d, J = 6.2 Hz, H-6‴) and by comparison of the coupling
Figure 3. Key HMBC and COSY (left) as well as ROE (right)
correlations of selected compounds.
3
3
3
constants J1‴,2‴ = 2.3 Hz, J2‴,3‴ = 3.2 Hz, J3‴,4‴ = 9.0 Hz,
3.39 (1H, d, J = 12.3 Hz), δH 4.33 (1H, m) for δC 63.6 and δH
4.34 (1H, m), δH 4.38 (1H, m) for δC 61.9 in the HSQC
spectrum, respectively, indicating the occurrence of two
CH2OH groups. HMBC correlations of H-3 (δH 3.54) with
δC 63.6 and H-5 (δH 0.87) with δC 63.6 indicated that one
CH2OH group is located at C-24. The ROE correlation of H-
25 (δH 1.06)/H-24 (δH 3.39) substantiated this observation
(Figure 3). The additional hydroxy group at the triterpenoid A
ring resulted in a downfield shift of H-3 at δH 3.54 (1H, dd, J =
12.3, 5.4 Hz) in contrast to glycyrrhizin (11) at δH 3.37 (1H,
dd, J = 12.0, 4.5 Hz) (Figure 3). Further, specification of the
second CH2OH group could be carried out by the COSY
correlation of H-5″ (δH 3.71)/δH 4.34 and H-5″ (δH 3.71)/δH
4.38. These signals correlated with δC 61.9 (CH2) in the
HSQC spectrum, revealing the second CH2OH group to occur
3
3J4‴,5‴ = 9.0 Hz, and J5‴,6‴ = 6.2 Hz with those reported
earlier.48 Consequently, compound 4 was assigned as
(3β,20β)-20-carboxy-11-oxo-30-norolean-12-en-3-yl 2-O-α-L-
rhamnopyranosyl-2-O-β-D-glucopyranuronosyl-β-D-glucopyra-
nosiduronic acid (rhaoglycyrrhizin). For the first time, the
structure of this compound proposed previously by mass
spectrometry was confirmed unequivocally.49
Compound 5 demonstrated a molecular formula of
C48H74O19 based on the deprotonated molecular ion peak
[M − H]− at m/z 953.4777, calcd for C48H73O19 at m/z
953.4746. In contrast to saponin 4 (C48H72O20), compound 5
exhibited a molecular formula diminished by one oxygen and
increased by two hydrogen atoms, indicating the substitution
of a carbonyl group by a methylene group. Furthermore, the
ESIMS/MS data of 5 showed fragments at m/z 655 and 439,
representing cleavages of 322 Da (uronic acid + methyl-
pentose) and 216 Da (uronic acid). Compared to glycyrrhizin
(11) and rhaoglycyrrhizin (4), the aglycone fragment of 5 at
m/z 439 Da indicated a mass difference of 14 Da,
demonstrating the substitution of a carbonyl group by a
3
3
at C-6″. The observed coupling constants J2″,3″, J3″,4″, and
3J4″,5″ with 8.8 to 9.6 Hz as well as acidic hydrolysis leading to
the determination of the absolute configuration of the
monosaccharides revealed the terminal sugar moiety to be β-
D-glucose. Thus, compound 3 was identified as (3β,20β)-20-
carboxy-24-hydroxy-11-oxo-30-norolean-12-en-3-yl 2-O-β-D-
E
DOI: 10.1021/acs.jnatprod.8b00022
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 2. NMR Spectroscopic Data for Compounds 6−10 in Pyridine-d5 (500 MHz for H and 125 MHz for 13C, δ Values in
ppm, J Values in Hz)
6
7
8
9
10
δH (J in Hz)
position δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
1
2
3
39.8, CH2 3.06, dd (9.5,
3.7)
1.11, m
26.9, CH2 2.25, m
39.9, CH2 3.07, dd (9.5, 39.1, CH2 0.82, m
3.7)
39.9, CH2 3.09, dd (9.3,
3.7)
1.09, m
27.1, CH2 2.34, dd (13.5,
4.8)
2.13, m
89.1, CH 3.39, dd (12,
4.9)
39.8, CH2 3.05, dd (3.7,
3.4)
1.11, m
26.9, CH2 2.26, m
1.12, m
1.40, m
26.9, CH2 2.27, m
26.8, CH2 1.88, m
2.05, m
2.08, m
2.24, m
2.04, m
90.2, CH 3.37, dd
(11.8, 4.5)
90.1, CH 3.35, dd (12, 4.6) 90.1, CH 3.37, dd (12, 89.7, CH 3.30, dd
4.6) (12.0, 5.0)
4
5
40.4, C
55.8, CH 0.82, m
40.4, C
55.9, CH 0.81, m
39.9, C
56.1, CH 0.70, m
40.2, C
55.7, CH 0.73, dd (12,
2.3)
40.4, C
55.8, CH 0.80, d (11.7)
6
7
8
18.0, CH2 1.58, 1.38, m
33.2, CH2 1.65, 1.30, m
45.9, C
18.0, CH2 1.57, 1.38, m 18.8, CH2 1.43, 1.26, m 18.0, CH2 1.52, 1.33, m
33.3, CH2 1.61, 1.30, m 33.1, CH2 1.40, 1.22, m 33.3, CH2 1.54, 1.23, m
18.0, CH2 1.56, 1.37, m
33.2, CH2 1.61, 1.25, m
45.9, C
45.9, C
40.4, C
45.9, C
9
10
11
62.5, CH 2.51, s
37.6, C
199.9, C
62.5, CH 2.50, s
37.6, C
199.9, C
48.1, CH 1.56, s
37.2, C
24.0, CH2 1.83 dd (8.9, 199.8, C
3.4)
62.4, CH 2.42, s
37.6, C
62.4, CH 2.43, s
37.6, C
199.8, C
12
13
14
15
16
17
18
129.0, CH 5.97, s
170.0, C
43.8, C
27.2, CH2 1.72, 1.11, m
27.0, CH2 2.09, 0.97, m
32.5, C
49.1, CH 2.54, dd (13.3,
3.5)
129.0, CH 5.98, s
169.9, C
43.8, C
123.1, CH 5.27, t (3.7)
145.0, C
42.2, C
129.2, CH 5.81, s
169.5, C
43.9, C
129.1, CH 5.79, s
169.4, C
43.9, C
27.0, CH2 1.69, 1.06, m
26.9, CH2 2.05, 0.90, m
32.9, C
27.2, CH2 1.72, 1.10, m 27.0, CH2 1.74, 0.92, m 27.0, CH2 1.68, 1.04, m
27.0, CH2 2.06, 0.95, m 27.6, CH2 2.14, 0.88, m 26.9, CH2 2.12, 0.89, m
32.5, C
28.7, C
32.9, C
–
49.1, CH 2.54, dd
46.9, CH 2.16, dd
47.0, CH 2.25, dd (14,
3.8)
47.0, CH 2.23, m
(13.3, 3.5)
(13.0, 3.5)
19
42.0, CH2 2.13, 1.75, m
42.0, CH2 2.14, 1.74, m 41.8, CH2 2.56, 1.70, m 40.2, CH2 2.49, t (13.8),
1.62, m
40.1, CH2 2.47, 1.61, m
20
21
22
23
24
25
26
27
28
29
30
1′
44.5, C
44.5, C
43.2, C
42.9, C
42.9, C
31.9, CH2 2.27, 1.48, m
38.8, CH2 1.73, 1.45, m
28.7, CH3 1.40, s
17.1, CH3 1.16, s
17.0, CH3 1.22, s
19.1, CH3 1.08, s
23.8, CH3 1.44, s
29.0, CH3 0.79, s
29.1, CH3 1.37, s
179.5, C
105.7, CH 5.05, d (7.5)
78.8, CH 4.57, m
79.2, CH 4.65, m
73.9, CH 4.47, pt (9.3)
77.8, CH 4.64, m
172.9, C
32.0, CH2 2.28, 1.47, m 30.3, CH2 2.25, 1.71, m 30.1, CH2 2.20, 1.73, m
38.8, CH2 1.73, 1.46, m 36.9, CH2 1.57, 1.38, m 36.2, CH2 1.53, 1.36, m
30.0, CH2 2.21, 1.72, m
36.2, CH2 1.52, 1.39, m
28.8, CH3 1.46, s
17.3, CH3 1.27, s
17.0, CH3 1.20, s
19.2, CH3 1.07, s
23.8, CH3 1.37, s
28.9, CH3 0.86, s
20.1, CH3 1.42, s
181.1, C
105.4, CH 5.1, d (7.3)
79.7, CH 4.55, t (8.3)
79.1, CH 4.67, m
73.8, CH 4.47, t (9.0)
77.7, CH 4.63, m
173.0, C
28.8, CH3 1.45, s
17.2, CH3 1.22, s
17.1, CH3 1.24, s
19.2, CH3 1.08, s
23.9, CH3 1.43, s
29.1, CH3 0.80, s
29.0, CH3 1.38, s
179.5, C
105.6, CH 5.08, d (7.6)
79.6, CH 4.54, m
79.2, CH 4.66, m
73.9, CH 4.52, m
77.8, CH 4.64, m
173.1, C
28.4, CH3 1.39, s
17.2, CH3 1.24, s
16.0, CH3 0.83, s
17.3, CH3 0.92, s
26.5, CH3 1.25, s
28.7, CH3 0.94, s
20.3, CH3 1.49, s
181.6, C
105.5, CH 5.07, d (8.0)
84.9, CH 4.29, m
78.2, CH 4.44, t (8.9)
73.4, CH 4.58, m
77.8, CH 4.62, m
172.5, C
28.5, CH3 1.45, s
17.1, CH2 1.23, s
17.2, CH3 1.26, s
19.2, CH3 1.08, s
23.8, CH3 1.38, s
28.9, CH3 0.87, s
20.1, CH3 1.43, s
181.1, C
105.6, CH 5.08, d (7.7)
84.3, CH 4.40, m
77.7, CH 4.42, m
73.6, CH 4.60, m
77.6, CH 4.65, m
172.8, C
2′
3′
4′
5′
6′
1″
2″
102.4, CH 5.89, d (7.6)
79.0, CH 4.34, m
103.1, CH 5.76, d (7.6)
77.1, CH 4.68, dd (7.5, 77.2, CH 4.27, m
9.5)
107.3, CH 5.47, d (7.9)
107.0, CH 5.31, d (7.8)
74.8, CH 4.68, dd (9.8,
7.8)
103.1, CH 5.97, d (7.6)
78.4, CH 4.47, pt (9.0)
3″
4″
5″
6″
79.8, CH 4.27, t (9.0)
73.1, CH 4.1, t (9.2)
76.6, CH 4.21, dd (9.5, 78.0, CH 4.32, m
3.5)
74.8, CH 4.35, dd (9.8,
3.3)
72.0, CH 5.05, dd (3.3,
1.9)
79.3, CH 4.35, m
74.0, CH 4.62, m
77.8, CH 4.47, m
172.7, C
70.9, CH 4.50, m
73.7, CH 4.64, m
78.2, CH 3.89, ddd (9.2,
5.9, 3.0)
63.7, CH2 4.54, m
4.35, m
76.7, CH 3.93, dd (1.6, 78.9, CH 4.65, m
5.4)
77.1, CH 4.81, d (1.9)
62.3, CH2 4.49, m
4.42, dd
172.8, C
171.8, C
(11.0, 5.4)
1‴
2‴
102.5, CH 6.43, d (1.5)
72.8, CH 4.79, dd (3.4,
1.6)
102.5, CH 6.35, d (2.3)
72.8, CH 4.8, dd (3.2,
2.2)
102.5, CH 6.48, d (1.5)
72.7, CH 4.80, d (2.0)
3‴
73.1, CH 4.73, dd (9.2,
3.4)
73.1, CH 4.75, dd (9.1,
3.2)
73.1, CH 4.73, dd (9.2,
3.5)
F
DOI: 10.1021/acs.jnatprod.8b00022
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. continued
6
7
8
9
10
δH (J in Hz)
position δC, type
δH (J in Hz)
74.7, CH 4.37, m
69.8, CH 5.09, m
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
4‴
5‴
74.8, CH 4.34, pt (9.5)
69.8, CH 5.07, m
74.7, CH 4.37, m
70.0, CH 5.08, dd (9.1,
6.3)
6‴
19.4, CH3 1.84, d (6.2)
19.4, CH3 1.80, d (6.0)
1
19.4, CH3 1.84, d (6.2)
methylene group in the aglycone moiety. The H and 13C
NMR signal assignment, as well as analysis of the
monosaccharide units after acidic hydrolysis, showed for
compound 5 the same sugar pattern as for 4, with two 1″ →
2′ linked β-D-glucuronic acids attached to an additional 1‴ →
2″ glycosylated α-L-rhamnose unit. The 13C NMR spectrum of
compound 5 showed in contrast to rhaoglycyrrhizin (4) no
signal at δC 199.9 (C-11) (Table 1). Although 5 had no
carbonyl group signal at C-11, it exhibited an additional
6 was assigned as (3β,20β)-20-carboxy-11-oxo-30-norolean-
12-en-3-yl 2-O-α-L-rhamnopyranosyl-2-O-β-D-glucopyranosyl-
β-D-glucopyranosiduronic acid (rhaoglucoglycyrrhizin).
Compound 7 afforded a molecular formula of C48H74O19
based on the [M − H]− peak at m/z 953.4781 in the
HRESIMS, calcd for C48H73O19 (m/z 953.4746), revealing the
same empirical formula as 6. Moreover, the ESIMS/MS of 7
showed fragments at m/z 647 and m/z 453 with similar neutral
mass losses of 308 Da (146 + 162 Da) and 194 Da to saponin
methylene group with H NMR chemical shifts of δH 1.81
6. The H and 13C NMR resonances for the aglycone, uronic
1
1
(2H, m, H-11) and showed COSY and HMBC correlations
between H-11 (δH 1.81)/H-9 (δH 1.64), H-11 (δH 1.81)/H-12
(δH 5.46), H-11 (δH 1.81)/C-9 (δC 48.2), H-11 (δH 1.81)/C-
12 (δC 123.2), and H-11 (δH 1.81)/C-13 (δC 145.5).
Moreover, upfield shifts for H-9 from δH 2.48 (1H, s, 4) to
δH 1.64 (1H, t, J = 8.9 Hz, 5), for H-12 from δH 5.94 (1H, s, 4)
to δH 5.46 (1H, t, J = 4.0 Hz, 5), and for H-18 from δH 2.53
(1H, dd, J = 16.9, 3.3 Hz, 4) to δH 2.41 (1H, dt, J = 13.8, 4.3
Hz, 5) (Figure 2) occurred. Thus, compound 5 was assigned as
(3β,20β)-20-carboxy-30-norolean-12-en-3-yl 2-O-α-L-rhamno-
pyranosyl-2-O-β-D-glucopyranuronosyl-β-D-glucopyranosidur-
onic acid (11-deoxo-rhaoglycyrrhizin).
acid, and methyl pentose units could be assigned as almost
identical, establishing glycyrrhetinic acid, glucuronic acid,
rhamnose, and an unknown sugar as partial structures.
Although saponins 6 and 7 showed similar NMR shifts and
fragment patterns by ESIMS/MS analysis, the 1H and J-
resolved NMR spectra suggested somewhat different sugar
1
patterns. The H NMR resonances and coupling constants of
the second carbohydrate moiety of compound 7 showed
resonances and coupling constants of δH 5.76 (1H, d, J = 7.6
Hz, H-1″), δH 4.21 (1H, dd, J = 9.5, 3.5 Hz, H-3″), and δH
3.93 (1H, dd, J = 5.4, 1.6 Hz, H-5″) (Table 2). After the
determination of monosaccharides after acidic hydrolysis and
their observed NMR data, a β-D-galactose moiety was
identified as a second carbohydrate moiety.48 Thus, compound
7 was identified as (3β,20β)-20-carboxy-11-oxo-30-norolean-
12-en-3-yl 2-O-α-L-rhamnopyranosyl-2-O-β-D-galactopyrano-
syl-β-D-glucopyranosiduronic acid (rhaogalactoglycyrrhizin).
HRESIMS analysis of compound 8 revealed a molecular
formula of C42H64O15, based on the deprotonated molecular
ion peak [M − H]− at m/z 807.4183, calcd for C42H63O15
(m/z 807.4167), having the same empirical formula as
compounds 1, 12, and 15. ESIMS/MS revealed a fragment
at m/z 439 with a neutral mass loss of 370 Da due to a possible
elimination of two uronic acid substituents. The same fragment
at m/z 439 was detected for compounds 1, 5, and 12 bearing a
CH2OH group at C-30 or a methylene group at C-11.
HRESIMS analysis of 6 revealed a molecular formula of
C48H74O19, based on the deprotonated molecule [M − H]−
peak at m/z 953.4775, calcd for C48H73O19 (m/z 953.4805),
indicating the same molecular formula as saponin 5 or a
molecular formula reduced by one oxygen and increased by
two hydrogen atoms when compared to 4 (C48H72O20).
Compound 6 showed ESIMS/MS fragments at m/z 809, 647,
and 453, representing neutral mass losses of 146, 162, and 194
Da, respectively, which were assigned to sugar fragmentations
comprising a methyl-pentose, a hexose, and a uronic acid
residue. Similar aglycone fragments of m/z 453 for 6 and
glycyrrhizin (11) as well as similar 1H and 13C NMR
resonances, e.g., for the junction positions at δH 3.35 (1H,
dd, J = 12.0, 4.6 Hz, H-3), 2.51 (1H, s, H-9), and 2.54 (1H, dd,
J = 13.3, 3.5 Hz, H-18), were measured, as occurred in the
glycyrrhizin aglycone (11) [δH 3.37 (1H, dd, J = 12.0, 4.5 Hz,
H-3), δH 2.46 (1H, s, H-9), and δH 2.54 (1H, dd, J = 13.1, 3.7
Hz, H-18)] (Table 2, Figure 2). Thus, the aglycone of 6 was
identified as glycyrrhetinic acid. The determination of the
carbohydrate moieties as a 1′ → 3 linked β-D-glucuronic acid, a
1″ → 2′ linked hexose, and a 1‴ → 2″ glycosylated α-L-
rhamnose unit was carried out by the HMBC and COSY
correlations of H-1′ (δH 5.05)/C-3 (δC 90.1), H-1′ (δH 5.05)/
H-2′ (δH 4.57), H-2′ (δH 4.57)/C-1″ (δC 102.4), H-1″ (δH
5.89)/H-2″ (δH 4.34), and H-2″ (δH 4.34)/C-1‴ (δC 102.5).
Further, the 1H NMR resonances and coupling constants of δH
5.89 (1H, d, J = 7.6 Hz, H-1″), 4.27 (1H, t, J = 9.0 Hz, H-2″),
4.10 (1H, t, J = 9.2 Hz, H-3″), 3.89 (1H, ddd, J = 9.2, 5.9, 3.0
Hz, H-5″), 4.35 (1H, m, H-6″), and 4.54 (1H, m, H-6″), as
well as determination of the monosaccharide moieties after
acidic hydrolysis, substantiated the presence of a 1″ → 2′
linked β-D-glucose moiety (Figure 2).48 Consequently, saponin
1
Compound 8 showed similar H NMR resonances at δH 1.83
(2H, dd, J = 8.9, 3.4 Hz, H-11) and δH 5.27 (1H, t, J = 3.7 Hz,
H-12) to saponins 5 and 12 with δH 1.79−1.85 (2H, H-11)
and δH 5.46 (1H, H-12) and a methylene group at C-11
(Tables 1, 2, and S2, Supporting Information, Figure 2). With
COSY and HMBC correlations of H-11 (δH 1.83)/H-9 (δH
1.56), H-11 (δH 1.83)/H-12 (δH 5.27), H-11 (δH 1.83)/C-9
(δC 48.1), H-11 (δH 1.83)/C-12 (δC 123.1), and H-11 (δH
1.83)/C-13 (δC 145.0), this inference could be substantiated.
1
Further structure elucidation exhibited for H-18 a H NMR
resonance at δH 2.16 (1H, dd, J = 13.0, 3.5 Hz), an upfield
shift, in contrast to δH 2.41 (1H, dd, J = 13.7, 3.3, 5) and 2.41
(1H, dd, J = 13.5, 4.5 Hz, 12), respectively (Figure 2).
Although similar COSY and HMBC correlations of compound
8 and glycyrrhizin (11) for the triterpenoid E-ring could be
observed, ROE correlations for the proton pairs H-28 (δH
0.94)/H-18 (δH 2.16) and H-18 (δH 2.16)/H-29 (δH 1.49)
were different. While compounds 1−7, 11−18, and 21
G
DOI: 10.1021/acs.jnatprod.8b00022
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Journal of Natural Products
Article
exhibited a β-orientation of the carboxylic group C-30,
compound 8 shared like saponins 19 and 20 an α-orientation
(Figure 3). Since two 1″ → 2′ linked β-D-glucuronic acids were
shown to be present, saponin 8 was elucidated as (3β,20α)-20-
carboxy-30-norolean-12-en-3-yl 2-O-β-D-glucopyranuronosyl-
β-D-glucopyranosiduronic acid (11-deoxo-20α-glycyrrhizin).
The molecular formula of compound 9 was analyzed by
HRESIMS for C42H62O16, based on the [M − H]− peak at m/z
821.3965, calcd for C42H61O16 (m/z 821.3960), identical to
the molecular formula of glycyrrhizin (11). Moreover, neutral
mass losses of 176 and 194 Da were observed in the ESIMS/
MS of 9, based on fragments of m/z 647 and 453, thus
indicating the presence of two uronic acid moieties. Structure
elucidation by 1D and 2D NMR experiments of 9 revealed
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-2000 polarimeter at 25 °C using a 600 μL
1
cell with 1 dm path length. The H, 13C, COSY, HSQC, HMBC, J-
RESOLVED, ROE-, and selective TOCSY-NMR spectra were
acquired on a Bruker 500 MHz Avance III spectrometer (Bruker,
Rheinstetten, Germany) using pyridine-d5 as solvent. Chemical shifts
are reported in parts per million, relative to the solvent signals. Liquid
chromatography/mass spectrometry (ESIMS/MS) for MS/MS
experiments was carried out by acquisition of mass and product ion
spectra on an API 4000 QTrap mass spectrometer (Sciex, Darmstadt,
Germany). Fragmentation of the molecular ions [M + H]+ into
specific product ions was induced by collision with nitrogen (4.5 ×
10−5 Torr). For analyzing the monosaccharides after hydrolysis of the
saponins a Nexera X2 UHPLC system (Shimadzu Europe GmbH,
Duisburg, Germany) connected to a QTRAP 5500 mass spectrometer
(Sciex) was used. UPLC/time-of-flight mass spectrometry (HRE-
SIMS) was achieved by an Acquity UPLC core system (Waters UK
Ltd., Manchester, UK) connected to a SYNAPT G2 HDMS
spectrometer (Waters) operating in the negative electrospray
(ESI−) mode. Medium-pressure liquid chromatography of the
MeOH−H2O extract prepared from licorice roots was performed
1
similar H NMR resonances for the triterpenoid E-ring at δH
2.25 (1H, dd, J = 14.0, 3.8 Hz, H-18), 2.49 (1H, t, J = 13.8 Hz,
H-19), and 1.62 (1H, m, H-19) compared to saponins 8, 19,
and 20 (Figure 2). Moreover, the ROE correlations of H-28
(δH 0.87)/H-18 (δH 2.24) and H-18 (δH 2.24)/H-29 (δH 1.43)
in a similar manner to saponins 8, 19, and 20 supported an α-
orientation of the carboxylic acid group at C-30. Although
compound 9 showed in the ESIMS/MS a comparable
fragmentation pattern and had the same COSY and HMBC
correlations as found for glycyrrhizin (11), the proton
resonances δH 4.68 (1H, dd, J = 9.8, 7.8 Hz, H-2″), 5.05
(1H, dd, J = 3.3, 1.9 Hz, H-4″), and 4.81 (1H, d, J = 1.9 Hz, H-
5″) were more downfield shifted compared to 11, at δH 4.28
(1H, dd, J = 9.5, 7.6 Hz, H-2″), 4.64 (1H, m, H-4″), and 4.65
(1H, m, H-5″) (Table 2). In particular, the coupling constants
on a Sepacore chromatography system (Buchi, Flawil, Switzerland).
̈
All preparative and semipreparative separations of fractions II/III and
IV and all subsequent subfractions were conducted on an LC-
2000Plus system (JASCO, Groß-Umstadt, Germany).
The following compounds were purchased from the sources given
in parentheses: glycyrrhizin ammonical (Extrasynthese, Genay,
́
France) and formic acid (Merck, Darmstadt, Germany). Deuterated
solvents for NMR spectroscopy were obtained from Euriso-Top (Gif-
Sur-Yvette, France), and solvents for HPLC separation (CH3CN and
MeOH) were of HPLC grade (Mallinckrodt Baker, Griesheim,
Germany), with the solvents used for HRESIMS and ESIMS/MS
analysis being of LC-MS grade (Honeywell, Seelze, Germany).
Ultrapure water for HPLC separation and mass spectrometry was
purified by means of a Milli-Q Water Advantage A 10 water system
(Millipore, Molsheim, France).
Plant Material. Dried and peeled G. glabra roots (Radix Liquiritae
mundat conc EU. AB. 7.3) were obtained from Heinrich Klenk
GmbH, Schwebheim, Germany. A sample (6381-A-140109) was
deposited at the authors’ laboratory at the Technical University
Munich, Freising, Germany.
Extraction and Isolation. Peeled G. glabra roots (1.5 kg) were
ground in a laboratory blender (Grindomix GM300, Retsch, Haan,
Germany) at 4000 U/min for 120 s. Extraction of the ground roots
was achieved six times with 15 L of MeOH and eight times with 15 L
of MeOH−H2O (7:3 v/v) with ultrasonification, followed by filtration
(Schleicher and Schuell filter, 24 cm). The combined filtrates were
collected, freed from solvent in a vacuum at 38 °C, and freeze-dried
(yield: 470 g, 31%). For MPLC experiments, the MeOH−H2O
extract was dissolved in H2O (336 g/840 mL). An aliquot (20 mL) of
this solution was injected onto a self-packed MPLC polypropylene
3
3
3J2″,3″ = 9.8 Hz, J3″,4″ = 3.3 Hz, and J4″,5″ = 1.9 Hz and the
determination of monosaccharides after acidic hydrolysis led to
identification of β-D-galacturonic acid as the terminal
carbohydrate moiety of saponin 9. Thus, saponin 9 was
elucidated as (3β,20α)-20-carboxy-11-oxo-30-norolean-12-en-
3-yl 2-O-β-D-galactopyranuronosyl-β-D-glucopyranosiduronic
acid (20α-galacturonoylglycyrrhizin).
HRESIMS analysis of compound 10 showed a deprotonated
molecular ion peak [M − H]− at m/z 967.4586 with a
molecular formula of C48H72O20 (calcd for m/z 967.4539,
C48H71O20). The same fragmentation pattern in the ESIMS/
MS with m/z 823, 647, and 453 indicated in a similar manner
to rhaoglycyrrhizin (4) a methylpentose and two uronic acid
1
moieties as the saccharide units. The H NMR resonances of
10 at δH 2.23 (1H, m, H-18), 2.47 (1H, m, H-19), and 1.61
(1H, m, H-19) were supporting the configuration of the
triterpenoid E-ring similar to saponins 8, 19, and 20 (Table 2,
Figure 3). ROE cross signals of H-28 (δH 0.86)/H-18 (δH
2.23) and H-18 (δH 2.23)/H-29 (δH 1.42) revealed an α-
orientation of the C-30 carboxylic acid group. COSY and
HMBC NMR experiments demonstrated the following
correlations: H-1′ (δH 5.10)/C-3 (δC 90.2), H-1′ (δH 5.10)/
H-2′ (δH 4.55), H-2′ (δH 4.55)/C-1″ (δC 103.1), H-1″ (δH
5.97)/H-2″ (δH 4.47), and H-2″ (δH 4.47)/C-1‴ (δH 6.48).
Altogether, after the determination of the absolute config-
uration of the monosaccharides after acidic hydrolysis, the
presence of two 1″ → 2′ linked β-D-glucuronic acids attached
to an additional 1‴ → 2″ glycosylated α-L-rhamnose unit could
be established. Consequently, the structure of compound 10
was determined as (3β,20α)-20-carboxy-11-oxo-30-norolean-
12-en-3-yl 2-O-α-L-rhamnopyranosyl-β-D-glucopyranuronosyl-
β-D-glucopyranosiduronic acid (20α-rhaoglycyrrhizin).
cartridge (150 × 40 mm i.d., Buchi) filled with LiChroprep RP-18
̈
25−40 μm bulk material (Merck, Darmstadt, Germany). Chromatog-
raphy was performed using H2O as solvent A and MeOH as solvent B
as well as the following gradient at a flow rate of 40 mL/min: 0 min,
0% B; 13.5 min, 0% B; 43.5 min, 50% B; 58.5 min, 50% B; 58.6 min,
60% B; 73.5 min, 60% B; 73.6 min, 70% B; 88.5 min, 70% B; 88.6
min, 80% B; 103.5 min, 80% B; 103.6 min, 100% B; 114 min, 100% B.
Before each separation run, a conditioning step was conducted with 0
min, 100% B; 5 min, 100% B; 10 min, 0% B; 20 min, 0% B. Then, four
fractions, I (42.5−58.5 min), II (58.5−73.5 min), III (73.5−88.5
min), and IV (88.5−103.5 min), were collected by monitoring the
effluent using UV detection at 250 nm. Each fraction was freed from
solvent under a vacuum and lyophilized. Combined fractions II and
III were dissolved in CH3CN−H2O (3:7, 5.2 g/45 mL). HPLC
separations were performed using a Nucleodur C18 Pyramid column
(250 × 21 mm i.d., 5 μm, Macherey-Nagel, Duren, Germany) and
̈
0.1% formic acid in H2O (A v/v) and CH3CN (B) as solvents.
H
DOI: 10.1021/acs.jnatprod.8b00022
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Separation was performed at a flow rate of 24 mL/min, starting with 0
min, 30% B, followed by the following steps: 3 min, 30% B; 5 min,
35% B; 45 min, 35% B; 50 min, 100% B; 55 min, 100% B; 58 min,
30% B; 65 min, 30% B. By monitoring the effluent at 250 nm, the
extract was separated into fractions II/III-1 to II/III-24 to yield 11
(1.88 g, tR 27.3 min, II/III-19) and 19 (178 mg, tR 35.5 min, II/III-
22).
min to yield fractions II/III-21-3-1 to II/III-21-3-3, from which
compound 10 (II/III-21-3-3, 3.8 mg, tR 16.1 min) was obtained.
Fractionation of II/III-21-4 was conducted with a gradient using 0
min, 30% B; 3 min, 30% B; 5 min, 35% B; 40 min, 38% B followed by
a conditioning step of 30% B for 5 min to obtain fractions II/III-21-4-
1 and II/III-21-4-2, from which saponin 1 (II/III-21-4-2, 10.1 mg, tR
28.4 min) occurred.
Subfractionation of fractions II/III-15 (57 mg), II/III-16 (29 mg),
II/III-17 (95 mg), II/III-18 (50 mg), II/III-20 (36 mg), II/III-21 (56
mg), and II/III-23 (64 mg) was carried out using semipreparative
HPLC analysis with 0.1% formic acid (v/v) as solvent A and CH3CN
as solvent B. Fractions II/III-20, II/III-21, and II/III-23 were
dissolved in CH3CN−H2O (3:7, 10 mL) individually and analyzed
by means of a Synergi Hydro-RP 80A column (250 × 10 mm i.d., 4
μm, Phenomenex, Aschaffenburg, Germany). For HPLC analysis of
fractions II/III-15, II/III-16, II/III-17, and II/III-18 a Nucleodur C18
Pyramid column (250 × 10 mm i.d., 5 μm, Macherey Nagel) was
used. For chromatography of fractions II/III-15, II/III-16, and II/III-
17 the following gradient was used: 0 min, 30% B; 15 min, 30% B; 40
min, 35% B; 45 min, 100% B. Semipreparative analysis of fraction II/
III-18 was effected by the gradient 0 min, 30% B; 6 min, 30% B; 40
min, 38% B; 45 min, 100% B. In turn, fractionation of II/III-20 was
conducted with the gradient 0 min, 30% B; 3 min, 30% B; 5 min, 35%
B; 50 min, 50% B; 52 min, 100% B; 57 min, 100% B; 60 min, 30% B;
65 min, 30% B. For further purification of fractions II/III-21 and II/
III-23 the following gradient was used: 0 min, 30% B; 3 min, 30% B; 5
min, 35%B; 50 min, 75% B; 52 min, 100% B; 57 min, 100% B; 60
min, 30% B; 65 min, 30% B. The effluent of each fractionation was
monitored by UV detection at a wavelength of 250 nm. The following
subfractions were collected: II/III-15-1 to II/III-15-10; II/III-16-1 to
II/III-16-9; II/III-17-1 to II/III-17-8; II/III-18-1 to II/III-18-5; II/
III-20-1 to II/III-20-8; II/III-21-1 to II/III-21-9; II/III-23-1 and II/
III-23-2. From fractions II/III-15-6, II/III-21-9, and II/III-23-2
compounds 13 (36 mg, tR = 27.1 min), 2 (2.2 mg, tR = 25.7 min),
and 9 (26 mg, tR = 19.1 min) were obtained.
Further purification of fractions II/III-15-7 (9.2 mg), II/III-16-8
(23 mg), II/III-17-4 (53 mg), II/III-17-6 (22 mg), II/III-18-2 (22
mg), II/III-20-4 (28 mg), II/III-21-3 (7.2 mg), and II/III-21-4 (16.6
mg) was carried out by additional semipreparative HPLC steps. After
dissolving the fractions in CH3CN−H2O (3:7 v/v) (2−6 mL),
separately, fractionation was carried out using a Nucleodur C18
Pyramid column (250 × 10 mm i.d., 5 μm, Macherey Nagel), with
a flow rate of 4.8 mL/min and solvents consisting of 0.1% formic acid
in H2O (v/v) as solvent A and CH3CN as solvent B. All preparative
HPLC analysis was carried out by UV detection at a wavelength of
250 nm. Separation of II/III-15-7 was conducted with the following
solvent gradient: 0 min, 30% B; 3 min, 30% B; 5 min, 32% B; 40 min,
32% B. Accordingly, two subfractions, II/III-15-7-1 and II/III-15-7-2,
were collected, in which 3 (4.3 mg, tR 30.3 min) was afforded in II/
III-15-7-1. Further separation of II/III-16-8 using the solvent gradient
0 min, 30% B; 3 min, 30% B; 5 min, 35% B; 50 min, 65% B; 52 min,
100% B; 57 min, 100% B; 60 min, 30% B; and 65 min, 30% B was
carried out, yielding in subfractions II/III-16-8-1 and II/III-16-8-2
saponin 7 (3.7 mg, tR 15.2 min) in fraction II/III-16-8-1.
Chromatography of fraction II/III-17-4 and II/III-17-6 was achieved
by applying the following gradient: 0 min, 30% B; 3 min, 30% B; 5
min, 35% B; 50 min, 52%, followed by a conditioning step with 30% B
for 5 min, for II/III-17-4 and 0 min, 30% B; 3 min, 30% B; 23 min,
75% B, followed by a conditioning step with 30% B for 5 min for II/
III-17-6, obtaining fractions II/III-17-4-1, II/III-17-4-2, II/III-17-6-1,
and II/III-17-6-2 with 4 (32 mg, tR 18.0 min) in II/III-17-4-2 and 6
(10.6 mg, tR = 14.2 min) in II/III-17-6-2. Purification of fractions II/
III-18-2 (22 mg) and II/III-20-4 (28 mg) was conducted by means of
the gradient 0 min, 30% B; 3 min, 30% B; 5 min, 35% B; 35 min, 75%
B followed by a conditioning step of 30% B for 5 min. Separation of
fractions II/III-18-2-1, II/III-18-2-2, II/III-20-4-1, and II/III-20-4-2
led to compounds 20 (10.6 mg, tR 15.8 min, II/III-18-2-2) and 15 (13
mg, tR 15.5 min, II/III-20-4-1). For separation of fraction II/III-21-3
the following gradient was used: 0 min, 30% B; 3 min, 30% B; 5 min,
35% B; 35 min, 75% B, followed by a conditioning step of 30% B for 5
A solution of fraction IV in CH3CN−H2O (3:7 v/v, 1.4 g/18 mL)
was separated by means of preparative HPLC using a Nucleodur C18
Pyramid column (250 × 21 mm i.d., 5 μm, Macherey Nagel) and the
following gradient: 0 min, 30% B; 30 min, 70% B; 33 min, 100% B; 38
min, 100% B, 40 min, 30% B; 45 min, 30% B. For this, solvent A was
0.1% formic acid in H2O (v/v) and solvent B CH3CN (flow rate: 21
mL/min). The effluent was detected using a DAD detector at 200/
250 nm and separated into 18 subfractions (IV-1 to IV-18). After
evaporation of the solvent under a vacuum and freeze-drying,
compound 12 (78 mg, tR 17.7 min) was obtained from fraction IV-11.
For further purification of compounds 14, 16, 17, 5, 18, 8, and 21
fractions IV-6 (23 mg), IV-7 (24 mg), IV-9 (79 mg), IV-14 (41 mg),
IV-15 (25 mg), and IV-16 (21 mg) were separated by means of
semipreparative HPLC. For the chromatography of fractions IV-6, IV-
7, IV-9, IV-11, and IV-14 a Nucleodur C18 Pyramid column (250 × 21
mm i.d., 5 μm, Macherey Nagel) was used. Fractions IV-15 and IV-16
were fractionated using a Nucleodur C18 Pyramid column (250 × 10
mm i.d., 5 μm, Macherey Nagel). Both columns were run using 0.1%
formic acid in H2O (v/v) as solvent A and CH3CN as solvent B.
Chromatography of fractions IV-6, IV-7, and IV-9 was performed
using a DAD detector at 200 and 250 nm and the following gradient
operated at 21 mL/min: 0 min, 35% B; 3 min, 35% B; 5 min, 40% B;
30 min, 55% B; 33 min, 100% B; 38 min, 100% B; 40 min, 35% B; 45
min, 35% B, yielding subfractions IV-6-1, IV-6-2, IV-7-1, IV-7-2, IV-9-
1, IV-9-2, and IV-9-3 with saponins 14 (8.6 mg, tR 28.5 min) in
fraction IV-6-2, 16 (7.2 mg, tR 33.9 min) in IV-7-2, 17 (5.4 mg, tR
39.4 min) in IV-7-3, and 21 (3.6 mg, tR 35.3 min) in IV-9-1.
Purification of fraction IV-14 was carried out by means of preparative
HPLC (21 mL/min) using the following conditions: 0 min, 30% B; 3
min, 30% B; 18 min, 100% B; 22 min, 100% B; 25 min, 30% B; 27
min, 30% B. The effluent was monitored using a UV detector (200
nm), and subfractions IV-14-1 and IV-14-2 with compound 5 (6.1
mg, tR 15.8 min) in fraction IV-14-1 were obtained. Separation of
fractions IV-15 and IV-16 was accomplished by semipreparative
HPLC at a flow rate of 4.8 mL/min and applying the gradient 0 min,
30% B; 3 min, 30% B; 22 min, 100% B; 25 min, 100% B; 28 min; 30%
B; 30 min, 30% B. Collection of subfractions IV-15 and IV-16 was
performed by monitoring the effluent using a UV detector (250 nm)
and resulted in the following subfractions: IV-15-1, IV-15-2, and IV-
16-1 to IV-16-3, leading to saponins 18 (9.7 mg, tR 15.4 min) in
fraction IV-15-1 and 8 (12 mg, tR 16.8 min) in fraction IV-16-3.
30-Hydroxyglycyrrhizin (1): white powder; [α]25 +17 (c 0.05,
D
MeOH); 1H and 13C NMR data, see Table 1; ESIMS m/z 809 [M +
H]+; ESIMS/MS (DP 20 V; CE 8 V) m/z 809 (8), 647 (3), 633 (6),
457 (4), 453 (2), 439 (100); HRESIMS m/z 807.4166 [M − H]−
(calcd for C42H63O15, 807.4167.
Glycyrrhizin-20-methanoate (2): white powder; [α]25 +22 (c
D
1
0.08, MeOH); H and 13C NMR data, see Table 1; ESIMS m/z 837
[M + H]+; ESIMS/MS (DP 20 V; CE 11 V) m/z 837 (67), 809 (30),
661 (35), 457 (100), 442 (100); HRESIMS m/z 807.4195 [M −
COHH]− (calcd for C42H63O15, 807.4167).
24-Hydroxy-glucoglycyrrhizin (3): white powder; [α]25 +21 (c
D
0.05, MeOH); H and 13C NMR data, see Table 1; ESIMS m/z 825
1
[M + H]+; ESIMS/MS (DP 40 V; CE 20 V) m/z 825 (14), 664 (4),
663 (5), 649 (3), 645 (15), 627 (52), 487 (62), 469 (100), 451 (20);
HRESIMS m/z 823.4122 [M − H]− (calcd for C42H63O16, 823.4116).
Rhaoglycyrrhizin (4): white powder; [α]25 −9 (c 0.18, MeOH);
D
1H and 13C NMR data, see Table 1; ESIMS m/z 969 [M + H]+;
ESIMS/MS (DP 40 V; CE 16 V) m/z 969 (5), 823 (6), 647 (35),
671 (25), 455 (55), 453 (100), 319 (14), 275 (10), 242 (9), 141
(18), 130 (15); HRESIMS m/z 967.4570 [M − H]− (calcd for
C48H71O20, 967.4539).
I
DOI: 10.1021/acs.jnatprod.8b00022
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
11-Deoxo-rhaoglycyrrhizin (5): white powder; [α]25D −23 (c 0.02,
MeOH); 1H and 13C NMR data, see Table 1; ESIMS m/z 977 [M +
Na]+; ESIMS/MS (DP 20 V, CE 50 V) m/z 977 (100), 655 (33), 521
(47), 439 (10), 345 (12), 261 (5), 191 (6); HRESIMS m/z 953.4777
[M − H]− (calcd for C48H73O19, 953.4746).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00022.
■
S
Rhaoglucoglycyrrhizin (6): white powder; [α]25 +35 (c 0.16,
D
MeOH); 1H and 13C NMR data, see Table 2; ESIMS m/z 955 [M +
H]+; ESIMS/MS (DP 20 V; CE 10 V) m/z 955 (7), 809 (5), 647
(41), 471 (27), 453 (100), 294 (4), 256 (6); HRESIMS m/z
953.4775 [M − H]− (calcd for C48H73O19, 953.4805).
MPLC, HPLC, LC-MS parameters; NMR, MS spectro-
scopic data for new and known compounds 1−21;
determination of absolute configurations of monosac-
charides after acidic hydrolysis (PDF)
Rhaogalactoglycyrrhizin (7): white powder; [α]25 −18 (c 0.2,
D
MeOH); 1H and 13C NMR data, see Table 2; ESIMS m/z 955 [M +
H]+; ESIMS/MS (DP 40 V; CE 21 V) m/z 955 (1), 647 (20), 629
(3), 471 (40), 453 (100); HRESIMS m/z 953.4781 [M − H]− (calcd
for C48H73O19, 953.4746).
AUTHOR INFORMATION
Corresponding Author
*Tel: +49-8161-712902. Fax: +49-8161-712949. E-mail:
thomas.hofmann@tum.de.
■
11-Deoxo-20α-glycyrrhizin (8): white powder; [α]25 −4 (c 0.14,
D
MeOH); 1H and 13C NMR data, see Table 2; ESIMS m/z 809 [M +
H]+; ESIMS/MS (DP 1 V, CE 110 V) m/z 809 (70), 439 (100), 205
(2), 203 (28), 191 (20), 189 (4), 149 (2) 147 (5), 141 (10), 95 (10),
93 (2); HRESIMS m/z 807.4183 [M − H]− (calcd for C42H63O15,
807.4167).
ORCID
Thomas Hofmann: 0000-0003-4057-7165
Notes
The authors declare no competing financial interest.
20α-Galacturonoylglycyrrhizin (9): white powder; [α]25 +24 (c
D
1
0.01, MeOH); H, and 13C NMR data, see Table 2; ESIMS m/z 823
[M + H]+ ESIMS/MS (DP 20 V; CE 7 V) m/z 823 (2), 805 (1), 647
(2), 471 (4), 453 (100); HRESIMS m/z 821.3965 [M − H]− (calcd
for C42H61O16, 821.3960).
ACKNOWLEDGMENTS
We thank A. Kirchhoff (Apotheke Stadtisches Klinikum
Munich) for her support in measuring the optical rotations.
■
̈
20α-Rhaoglycyrrhizin (10): white powder; [α]25 +41 (c 0.13,
D
MeOH); 1H and 13C NMR data, see Table 2; ESIMS m/z 969 [M +
H]+; ESIMS/MS (DP 20 V; CE 9 V) m/z 969 (1), 823 (3), 661 (2),
647 (11), 630 (1), 471 (11), 453 (100); HRESIMS m/z 967.4586 [M
− H]− (calcd for C48H71O20, 967.4539).
REFERENCES
■
(1) Attele, A. S.; Wu, J. A.; Yuan, C.-S. Biochem. Pharmacol. 1999,
58, 1685−1693.
(2) Fiore, C.; Eisenhut, M.; Ragazzi, E.; Zanchin, G.; Armanini, D. J.
Ethnopharmacol. 2005, 99, 317−324.
Acid Hydrolysis and Determination of the Absolute
Configuration of the Monosaccharides. According to a literature
protocol,50 the absolute configuration of the monosaccharides was
determined after acidic hydrolysis. A solution of each saponin (1−10,
50 μL of a 1 mM solution in CH3CN−H2O, 3:7) in HCl (2 M) was
heated for 1 h at 100 °C. Afterward, the mixture was evaporated to
dryness under reduced pressure. The residue was suspended in H2O
(750 μL) and extracted with EtOAc (2 × 750 μL). Next, the H2O
layer was evaporated to dryness to obtain the monosaccharide residue.
Each residue was dissolved in a solution of L-cysteine methyl ester
hydrochloride in anhydrous pyridine (2 mg/mL, Sigma-Aldrich,
Steinheim, Germany). The mixture was shaken for 1 h at 60 °C with
1400 rpm (Thermo-Shaker PHMT-PSC24N, Grant Bio, Cambridge,
UK). Then, to this solution was added phenylethyl isothiocyanate (5
μL, Sigma-Aldrich, Steinheim, Germany), and the resulting solution
was shaken for 1 h at 60 °C. The mixture was dried under a stream of
nitrogen and resolved in CH3CN−H2O (500 μL, 1/1 v/v), and each
solution was analyzed by means of UHPLC-MS/MS using a
Phenomenex Kinetex F5 column (100 × 2.1 mm i.d., 100 Å, 1.7
μm). Chromatography was performed with an injection volume of 0.5
μL and a flow rate of 0.4 mL/min. The mobile phase consisted of (A)
formic acid (1% in H2O) and (B) CH3CN (1% formic acid). The
following gradient was used: 0 min, 5% B; 3 min, 5% B; 5 min, 20% B;
25 min, 25% B; 27 min, 100% B; 30 min, 100% B; 31 min, 5% B; 35
min, 5% B. After optimizing instrument settings with authentic
samples (Sigma-Aldrich, Steinheim, Germany) treated the same way
as described above, the derivatized monosaccharides were analyzed
using the MRM transitions Q1/Q3 of m/z 461.0/298.1 (DP = 86 V,
CE = 17 V, CXP = 6 V) for D-glucose, Q1/Q3 of m/z 461.0/298.1
(DP = 71 V, CE = 17 V, CXP = 6 V) for L-glucose, Q1/Q3 of m/z
461.1/298.2 (DP = 71 V, CE = 17 V, CXP = 6 V) for D-galactose,
Q1/Q3 of m/z 475.0/312.1 (DP = 91 V, CE = 19 V, CXP = 6 V) for
D-galacturonic acid, Q1/Q3 of m/z 475.0/312.1 (DP = 61 V, CE = 19
V, CXP = 8 V) for D-glucuronic acid, and Q1/Q3 of m/z 445.0/282.1
(DP = 61 V, CE = 17 V, CXP = 6 V) for L-rhamnose. The following
retention times were observed for the corresponding derivatives: D-
glucose 13.28 min, L-glucose 13.04 min, D-galactose 12.38 min, D-
galacturonic acid 13.70 min, D-glucuronic acid 13.47 min, and L-
rhamnose 18.53 min.
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