Acylated iridoid glycosides with hyaluronidase inhibitory activity from the rhizomes of Picrorhiza kurroa Royle ex Benth

Article abstract of DOI:10.1016/j.phytochem.2019.112185

Seven new acylated iridoid glycosides, picrorhizaosides A–G (1–7), were isolated from the methanol extract of the rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae), in addition to six known iridoid glycosides (8–13). The structures of these new iridoids, including their stereochemistry, were determined based on chemical and physicochemical evidence derived from NMR and MS analysis. Of the isolates, picrorhizaosides D (4, IC₅₀ = 43.4 μM) and E (5, 35.8 μM); picrosides I (8, 60.7 μM), II (9, 22.3 μM), and IV (11, 59.2 μM); and minecoside (13, 57.2 μM), exhibited a similar or stronger hyaluronidase inhibitory activity than those of the antiallergic medicines disodium cromoglycate (64.8 μM), ketotifen fumarate (76.5 μM), and tranilast (227 μM).

Full text of DOI:10.1016/j.phytochem.2019.112185

Phytochemistry169(2020)112185
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Acylated iridoid glycosides with hyaluronidase inhibitory activity from the
rhizomes of Picrorhiza kurroa Royle ex Benth
Toshio Morikawaa,b,∗, Yusuke Nakanishia, Naoki Inouea, Yoshiaki Mansea, Hideyuki Matsuuraa,
Shinya Hamasakia, Masayuki Yoshikawaa, Osamu Muraokaa,b, Kiyofumi Ninomiyaa,b
a Pharmaceutical Research and Technology Institute, 3-4-1 Kowakae, Higashi-osaka, Osaka, 577-8502, Japan
b Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, 577-8502, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Picrorhiza kurroa Royle ex Benth.
Plantaginaceae
Iridoids
Picrorhizaosides
Hyaluronidase inhibitors
Seven new acylated iridoid glycosides, picrorhizaosides A–G (1–7), were isolated from the methanol extract of
the rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae), in addition to six known iridoid glycosides
(8–13). The structures of these new iridoids, including their stereochemistry, were determined based on che-
mical and physicochemical evidence derived from NMR and MS analysis. Of the isolates, picrorhizaosides D (4,
IC50 = 43.4 μM) and E (5, 35.8 μM); picrosides I (8, 60.7 μM), II (9, 22.3 μM), and IV (11, 59.2 μM); and
minecoside (13, 57.2 μM), exhibited a similar or stronger hyaluronidase inhibitory activity than those of the
antiallergic medicines disodium cromoglycate (64.8 μM), ketotifen fumarate (76.5 μM), and tranilast (227 μM).
1. Introduction
1993), and phenolic compounds (Kumar and Sharma, 2014; Stuppner
and Wagner, 1989a). In addition, biological effects of the extract and/
Picrorhiza kurroa Royle ex Benth., formerly of the Scrophulariaceae
family, but now classified as a member of the Plantaginaceae family, is
a small perennial herb found mainly in the northwest Himalayan re-
gion, from Kashmir to Kumaun, and Garhwal regions of India, and
Nepal growing at an elevation of 3000–5000 m a.s.l. (Kant et al., 2013;
Zhang et al., 2004). It is an unresolved name on www.theplantlist.org,
and is currently classified in the Plantaginaceae family (The Plant List,
http://www.theplantlist.org/tpl1.1/record/kew-2549267, accessed on
August 16, 2019) (Win et al., 2019). The leaves of this plant are flat,
oval and sharply serrated (Kant et al., 2013) and the morphological
characteristics of the species are published on the internet (Plants of the
World online, http://powo.science.kew.org/taxon/808408-1, accessed
on August 16, 2019). It is an important plant, commonly known as
“kutki”, in traditional medicinal systems, such as the Ayurvedic and
Unani systems, that has been used for the treatment of liver and lung
diseases, fever, skin lesions, worm infections, rheumatic diseases, ur-
inary disorders, heart failure, and snake and scorpion bites (Mehra
et al., 2017; Rathee et al., 2016; Sultan et al., 2017; Zahiruddin et al.,
2017). Previous studies on the chemical constituents of the rhizomes of
this plant have led to the isolation and characterization of a number of
compounds, such as iridoids (Jia et al., 1999; Laurie et al., 1985; Singh
and Rastogi, 1972), triterpenes (Laurie et al., 1985; Stuppner and
Wangner, 1989b; Stuppner et al., 1990, 1991; Stuppner and Müller,
or constituents have been reported, such as hepatoprotective (Han
et al., 2018; Jia et al., 2015; Rastogi et al., 2000; Visen et al., 1993),
anti-ischemic (Seth et al., 2000; Singh et al., 2000), anti-inflammatory
(Kumar et al., 2016; Marin et al., 2013; Wang et al., 2008; Zhang et al.,
2005), antitumor (Rathee et al., 2013), and antioxidative (Chander
et al., 1992; Krupashree et al., 2014) effects. During our characteriza-
tion of the bioactive constituents of plants in Tibet and Xinjiang, both
autonomous regions in China, a methanol extract of the rhizomes of P.
kurroa was found to inhibit hyaluronidase activity. In order to in-
vestigate new hyaluronidase inhibitors, we conducted a search for the
bioactive constituents from the rhizomes of P. kurroa. Here, we have
discussed the isolation, structural elucidation, and identification of the
constituents from the extract, and assessed their hyaluronidase in-
hibitory activity.
2. Results and discussion
2.1. Isolation
Dried rhizomes of P. kurroa were extracted with methanol under
reflux to yield the methanol extract. The methanol extract was parti-
tioned into ethyl acetate (EtOAc)-H2O yielding an EtOAc-soluble frac-
tion and an aqueous phase. The latter was subjected to Diaion HP-20
∗ Corresponding author. Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka, 577-8502, Japan.
E-mail address: morikawa@kindai.ac.jp (T. Morikawa).
https://doi.org/10.1016/j.phytochem.2019.112185
Received 23 June 2019; Received in revised form 15 October 2019; Accepted 22 October 2019
0031-9422/©2019ElsevierLtd.Allrightsreserved.

T. Morikawa, et al.
Phytochemistry169(2020)112185
Fig. 1. Structures of picrorhizaosides A–G (1–7).
column chromatography (H2O → MeOH), which yielded H2O- and
MeOH-eluted fractions. The EtOAc-soluble and MeOH-eluted fractions
were subjected to normal-phase silica gel and reversed-phase ODS
column chromatographic purification steps, and finally HPLC, which
yielded picrorhizaosides A–G (1–7), as shown in Fig. 1. In addition, six
known iridoid glycosides, picrosides I (8) (Kitagawa et al., 1969, 1971),
II (9) (Weinges et al., 1972), III (10) (Weinges and Künstler, 1977), and
IV (11) (Li et al., 1998); 6-feruloylcatalpol (12) (Stuppner and Wagner,
1989a); and minecoside (13) (Sticher and Afifi-Yazar, 1979), were
isolated, as shown in Fig. 2.
4), 6.38 (1H, dd, J = 6.0, 1.7 Hz, H-3)]; α- and β-glucopyranosyl moi-
eties [δ 4.79 (1H, d, J = 8.1 Hz, Glc-H-1), 4.88 (1H, d, J = 3.4 Hz, Glc-
H-1′)]; and a vanillyl group [δ 3.90 (3H, s, –OCH3), 6.85 (1H, d,
J = 8.3 Hz, Van-H-5), 7.56 (1H, d, J = 2.0 Hz, Van-H-2), 7.60 (1H, dd,
J = 8.3, 2.0 Hz, Van-H-6)]. In the heteronuclear multiple-bond corre-
lation (HMBC) spectrum of 1, long-range correlation was observed
between the H-6 and the vanillyl ester carbonyl carbon (δC 168.0), as
shown in Fig. 3. Enzymatic deacylation of 1 with tannase yielded de-
sacyl scalbidoside (1a) (Çalis et al., 1993) as shown in Fig. 3. Treatment
of 1 with 5% potassium hydroxide (KOH), vanillic acid was identified
by HPLC analysis (Pan et al., 2010) and then the residue was treated
with 1.0 M hydrochloric acid (HCl), which led to release of D-glucose,
which was identified by HPLC analysis using an optical rotation de-
tector (Morikawa et al., 2018b). The proton and carbon signals in the
1H and 13C NMR spectra of 1 were superimposable on those of the
principal iridoid constituent, picroside II (9 = 6-O-vanillylcatalpol),
except for the signals due to an additional α-D-glucopyranosyl moiety.
This evidence allowed us to confirm the structure of 1.
2.2. Structures of picrorhizaosides A–G (1–7)
Picrorhizaoside A (1) was obtained as a white powder with negative
29
optical rotation ([α]D –65.0 in MeOH). The IR spectrum contained
absorption bands at 1720, 1599, and 1516 cm−1 that were attributable
to ester carbonyl and aromatic functions, and broad bands at 3486 and
1104 cm−1 that were suggestive of a glycoside moiety. The molecular
formula of 1 was assigned as C29H38O18 on the basis of the quasimo-
lecular ion peak as a sodium adduct at m/z 697.1963 [M + Na]+ (calcd
for C29H38O18Na+, 679.1956) from positive-ion high-resolution FABMS
(HRFABMS) measurement. The 1H (Table S1) and 13C NMR (Table S2)
spectra (CD3OD) of 1, which were assigned with the aid of DEPT, 1H–1H
COSY, HMQC, and HMBC experiments, showed signals assignable to: a
hydroxymethyl group [δ 4.02 and 4.06 (1H each, both d, J = 13.1 Hz,
H2-10)]; five methines, including an acetal group [δ 2.63 (1H, dd,
J = 9.7, 7.7 Hz, H-9), 2.69 (1H, m, H-5), 3.78 (1H, br s, H-7), 5.03 (1H,
dd, J = 8.0, 1.2 Hz, H-6), 5.08 (1H, d, J = 9.7 Hz, H-1)]; a pair of di-
substituted olefin functional group [δ 5.01 (1H, dd, J = 6.0, 4.6 Hz, H-
Picrorhizaoside B (2) was obtained as a white powder with a ne-
29
gative optical rotation ([α]D –12.3 in MeOH). The IR spectrum
showed absorption bands at 1715 and 1636 cm−1 that were attribu-
table to ester carbonyl and olefin functional groups, and broad bands at
3398 and 1073 cm−1 that were suggestive of a glycoside structure. In
the HRFABMS analysis of 2 indicated that the molecular formula was
C
30H38O16. As shown in Fig. 3, the enzymatic hydrolysis of 2 with
tannase liberated its desacyl derivative, rehmannioside B (2a) (Oshio
and Inouye, 1981). Through a similar procedure as described above
(vide ante), the alkaline and acid hydrolyses of 2, trans-cinnamic acid, D-
glucose, and D-galactose were identified by HPLC analyses. The 1H
2

T. Morikawa, et al.
Phytochemistry169(2020)112185
Fig. 2. Known iridoids (8–13) isolated from the rhizomes of P. kurroa.
Fig. 3. Enzymatic deacylation of 1 and 2.
(Table S1) and 13C NMR (Table S2) spectra (pyridine-d5) showed signals
assignable to the rehmannioside B part: a hydroxyl methyl {δ [4.34
(1H, d, J = 13.1 Hz) and 4.58 (1H, m), H2-10]}; four methines [δ 2.80
(1H, dd, J = 9.5, 8.0 Hz, H-9), 2.86 (1H, m, H-5), 4.08 (1H, m, H-6),
4.22 (1H, m, H-7)]; an acetal [δ 5.43 (1H, d, J = 9.5 Hz, H-1)]; a pair of
di-substituted olefins [δ 5.24 (1H, dd, J = 6.0, 4.3 Hz, H-4), 6.38 (1H,
dd, J = 6.0, 1.8 Hz, H-3)]; α-D-galactopyranosyl [δ 5.39 (1H, d,
J = 3.9 Hz, H-Gal-1)] and β-D-glucopyranosyl moieties [δ 5.47 (1H, d,
J = 7.7 Hz, H-Glc-1)] together with a trans-cinnamoyl group {δ [7.30
(1H, m), 7.34 (2H, m), 7.55 (2H, br d, J = ca. 9 Hz), arom.-H], 6.88 and
7.89 (1H each, both d, J = 16.0 Hz, Cin-H-8 and 7)}. The position of the
acyl group in 2 was determined on the basis of the HMBC experiment,
which showed a long-range correlation between the 6-position of the β-
D-glucopyranosyl moiety [δ 4.37–4.40 (2H, m)] and the trans-cinna-
moyl ester carbonyl carbon (δC 166.9). Thus, the connected position
was clarified unambiguously, and the structure of 2 was elucidated.
Picrorhizaoside C (3) was obtained as a white powder with negative
vanillyl group [δ 3.90 (3H, s, –OCH3), 7.22 (1H, d, J = 8.4 Hz, Van-H-
5), 7.60 (1H, d, J = 2.0 Hz, Van-H-2), 7.63 (1H, dd, J = 8.4, 2.0 Hz,
Van-H-6)]. As shown in Fig. S1, the 1H–1H COSY analysis of 3 indicated
the presence of the partial structures presented with bold lines. In the
HMBC analysis of 3, long-range correlations were observed between the
following protons and carbons: H-1 and C-3 (δC 141.7); H-3 and C-1 (δC
95.4); H-7 and C-8 (δC 66.0); H-9 and C-8; H2-10 and C-8; Glc-H-1 and
C-1; Glc-H2-6 and Van-C-7 (δC 167.4); and Glc-H-1′ and Van-C-4 (δC
152.4). Next, the stereostructure of 3 was characterized by phase-sen-
sitive nuclear Overhauser enhancement spectroscopy (NOESY) experi-
ment, which showed NOE correlations between the following proton
pairs: H-1 and H-6, H2-10; H-5 and H-9; H-6 and H-7; and H-7 and H2-
10, as shown in Fig. S1. Thus, the stereostructure of 3 was elucidated as
shown.
Picrorhizaoside D (4) was obtained as a white powder with negative
29
optical rotation ([α]D –35.0 in MeOH). The molecular formula of 4
26
was determined as C37H46O22 by HRFABMS analysis. The acid and al-
kaline hydrolysis of 4, which was conducted using the same procedure
as for 3, led to the identification of D-glucose and vanillic acid, re-
spectively. The 1H and 13C NMR spectra of 4 (CD3OD, Tables S1 and S2)
showed signals assignable to: a hydroxymethyl [δ 3.88 and 3.99 (1H
each, both d, J = 11.9 Hz, H2-10)]; five methines, including an acetal
group [δ 2.69 (1H, dd, J = 9.5, 5.3 Hz, H-9), 3.07 (1H, m, H-5), 5.11
(1H, dd, J = 4.6, 4.3 Hz, H-6), 5.55 (1H, d, J = 4.3 Hz, H-7), 5.62 (1H,
d, J = 5.3 Hz, H-1)]; a pair of di-substituted olefins [δ 5.23 (1H, dd,
J = 6.2, 3.6 Hz, H-4), 6.36 (1H, dd, J = 6.2, 2.0 Hz, H-3)]; two β-glu-
copyranosyl moieties [δ 4.74 (1H, d, J = 8.1 Hz, Glc-H-1), 5.01 (1H, d,
J = 7.6 Hz, Glc-H-1′)]; and two vanillyl groups [δ 3.89 and 3.90 (3H
each, both s, –OCH3), 6.84 (1H, d, J = 8.2 Hz, Van-H-5), 7.25 (1H, d,
optical rotation ([α]D –45.6 in MeOH). The molecular formula of 3
was determined as C29H38O18 by HRFABMS analysis. The acid hydro-
lysis of 3 with 1.0 M HCl liberated D-glucose and then the residue was
treated with 5.0% KOH to obtain vanillic acid; both were identified by
HPLC analysis. The 1H and 13C NMR spectra of 3 (CD3OD, Tables S1 and
S2) showed signals assignable to the catalpol moiety: a hydroxyl methyl
[δ 3.50 and 4.08 (1H each, both d, J = 13.1 Hz, H2-10)]; four methines
[δ 2.18 (1H, m, H-5), 2.48 (1H, dd, J = 9.8, 7.6 Hz, H-9), 3.21 (1H, br s,
H-7), 3.52 (1H, m, H-6)]; an acetal [δ 4.75 (1H, d, J = 9.8 Hz, H-1)]; a
pair of di-substituted olefins [δ 5.01 (1H, dd, J = 6.0, 4.6 Hz, H-4), 6.28
(1H, dd, J = 6.0, 1.9 Hz, H-3)]; and a β-D-glucopyranosyl moiety [δ
4.77 (1H, d, J = 7.9 Hz, Glc-H-1)] together with an additional β-D-
glucopyranosyl moiety [δ 5.04 (1H, d, J = 7.7 Hz, Glc-H-1′)] and a
3

T. Morikawa, et al.
Phytochemistry169(2020)112185
J = 8.6 Hz, Van-H-5′), 7.58 (1H, d, J = 2.0 Hz, Van-H-2), 7.59 (1H, dd,
J = 8.2, 2.0 Hz, Van-H-6), 7.62 (1H, d, J = 2.0 Hz, Van-H-2′), 7.66 (1H,
dd, J = 8.6, 2.0 Hz, Van-H-6′)]. As shown in Fig. S1, the 1H–1H COSY
analysis of 4 indicated the presence of the partial structures shown in
bold lines, and in the HMBC experiment long-range correlations were
observed between the following proton and carbon pairs: H-1 and C-3
(δC 141.4); H-3 and C-1 (δC 93.3); H-6 and Van-C-7 (δC 167.5); H-7 and
C-8 (δC 82.3), Van-C-7' (δC 166.7); H-9 and C-8, H2-10 and C-8; H-Glc-1
and C-1; and H-Glc-1′ and Van-C-4' (δC 152.4). The stereostructure of 4
was characterized by phase-sensitive NOESY experiment, which re-
vealed NOE correlations between the following proton pairs of H-1 and
H-6, H2-10; H-5 and H-9; and H-7 and H2-10, as shown in Fig. S1. Thus,
the stereostructure of 4 was elucidated as shown.
in D-glucose. The 1H (Table S3) and 13C NMR (Table S4) spectroscopic
properties (in CD3OD) were quite similar to those of 6 except for the
signals due to the trans-feruloyl moiety [δ 3.84 (3H, s, –OCH3), 6.38 and
7.63 (1H each, both d, J = 16.0 Hz, Fer-H-8′ and Fer-H-7′), 6.75 (1H, d,
J = 8.0 Hz, Fer-H-5′), 7.03 (1H, dd, J = 8.0, 1.6 Hz, Fer-H-6′), 7.15
(1H, d, J = 1.6 Hz, Fer-H-2′)] instead of the trans-cinnamoyl group
connected at the 6′-position of the β-D-glucopyranosyl part in 6. The
connectivity of the trans-feruloyl part was characterized on the basis of
the HMBC spectrum, in which long-range correlations were observed
between the following proton and carbon pairs: H-1 [δ 5.08 (d,
J = 9.6 Hz)] and C-3 (δC 142.5); H-3 [δ 4.99 (dd, J = 5.6, 3.2 Hz)] and
C-1 (δC 93.3); H-6 [δ 5.09 (dd, J = 8.0, 0.8 Hz)] and Van-C-7 (δC
167.8); H-Glc-1 [δ 4.82 (d, J = 8.0 Hz)] and C-1 (δC 95.6); H2-Glc-6 [δ
3.95 (m) and 4.03 (dd, J = 11.2, 4.8 Hz)] and C-3' (δC 98.8); H-1' [δ
5.23 (d, J = 8.0 Hz)] and C-3'; H-3' [δ 4.93 (dd, J = 8.8, 2.4 Hz)] and C-
Glc-6 (δC 68.7); H-6' [δ 5.30 (dd, J = 8.0, 0.8 Hz)] and Fer-C-9' (δC
169.2); H-Glc-1' [δ 4.79 (d, J = 8.0)] and C-1' (δC 95.4), as shown in
Fig. S1. Thus, the connected position was clarified unambiguously, and
the stereostructure of 7 was elucidated. Previously, similar dimeric ir-
idoid glycosides were isolated from several plant resources such as
Eriophyton wallichii Benth. (Fan et al., 2011), Rehmannia glutinosa Li-
bosch (Liu et al., 2012), and Rehmannia chingii H. L. Li (Liu et al., 2016).
This article is the first report on the isolation and structural determi-
nation of dimeric iridoid glycosides (5–7) from the genus Picrorhiza. As
for the produce of the artifacts from natural occurring products, addi-
tion of the alcoholic fragment during the extraction procedure to the
double bond in the 3,4-position of the iridane skeleton has been re-
ported recently (Venditti, 2018). However, the isolation studies of the
dimeric iridoid glycosides of E. wallichii have been performed on the
95% aqueous ethanol extract under the non-heating extraction condi-
tions (extracted at room temperature) (Fan et al., 2011) and from the
non-alcoholic hot water extract from R. chingii (Liu et al., 2016). The
details of the dimerization process of the iridoid glycosides require
further study.
Picrorhizaosides E (5) and F (6) were also obtained as white pow-
24
24
ders with negative optical rotations (5: [α]D –89.0; 6: [α]D –94.0
both in MeOH). The HRFABMS analysis of 5 and 6 showed that the
common molecular formula was C47H56O24. The alkaline hydrolysis of
5 and 6 liberated two organic acids, vanillic acid and trans-cinnamic
acid, and the residues that were then treated with 1.0 M HCl and re-
sulted in D-glucose. The 1H (Table S3) and 13C NMR (Table S4) spec-
troscopic properties (CD3OD) of 5 showed signals assignable to two
iridoid moieties: {two hydroxymethyl groups [δ 3.62 and 4.04 (1H
each, both d, J = 13.2 Hz, H2-10′), 3.84 and 4.60 (1H each, both d,
J = 12.9 Hz, H2-10)]; a methylene [δ 1.66 (1H, ddd, J = 13.7, 8.6,
7.2 Hz), 1.87 (1H, ddd, J = 13.7, 2.3, 2.3 Hz), H2-4']; 10 methines, in-
cluding two acetal groups [δ 2.04 (1H, dddd, J = 8.0, 7.2, 7.2, 2.3 Hz,
H-5′), 2.39 (1H, dd, J = 8.0, 8.0 Hz, H-9′), 2.67 (1H, m, H-5), 2.68 (1H,
m, H-9), 3.38 (1H, d, J = 3.2 Hz, H-7′), 3.73 (1H, d, J = 0.8 Hz, H-7),
3.93 (1H, m, H-6′), 4.85 (1H, dd, J = 8.6, 2.3 Hz, H-3′), 4.90 (1H, d,
J = 8.0 Hz, H-1′), 5.11 (1H, dd, J = 8.0, 0.8 Hz, H-6), 5.21 (1H, d,
J = 8.9 Hz, H-1)]; and a pair of di-substituted olefins [δ 4.98 (1H, dd,
J = 6.0, 4.0 Hz, H-4), 6.38 (1H, dd, J = 6.0, 1.2 Hz, H-3)]} together
with two β-glucopyranosyl moieties [δ 4.79 (1H, d, J = 7.5 Hz, Glc-H-
1), 4.77 (1H, d, J = 8.0 Hz, Glc-H-1′)] and vanillyl [δ 3.86 (3H, s,
–OCH3), 6.83 (1H, d, J = 8.3 Hz, Van-H-5), 7.53 (1H, d, J = 2.0 Hz,
Van-H-2), 7.56 (1H, dd, J = 8.3, 2.0 Hz, Van-H-6)] and trans-cinnamoyl
moieties {δ [7.38 (3H, m), 7.61 (2H, m), arom.-H], 6.56 and 7.69 (1H
each, both d, J = 16.0 Hz, Cin-H-8 and 7)}. The connectivity of the acyl
groups and glucopyranosyl linkages was characterized in HMBC ex-
periment, which showed long-range correlations between the following
proton and carbon pairs: H-1 and C-3 (δC 142.6); H-3 and C-1 (δC 95.2);
H-6 and Van-C-7 (δC 167.9); H-7 and C-8 (δC 65.6); H-9 and C-8; H2-10
and C-8, C-3' (δC 98.9); H-1′ and C-3'; H-3′ and C-10 (δC 67.4), C-1' (δC
95.8); H-7′ and C-8' (δC 66.0); H-9′ and C-8'; H2-10′ and C-8'; Glc-H-1
and C-1; Glc-H-1′ and C-1'; Glc-H2-6′ and Cin-C-9' (δC 168.4). The ste-
reostructure of 5 was characterized by the phase-sensitive NOESY
analysis, which showed the NOE correlations between the following
proton pairs: H-1 and H-6, H2-10; H-5 and H-9; H-6 and H-7; H-7 and
H2-10; H-1′ and H-3′, H-4′α (δ 1.66); H-3′ and H-4′α; H-4′β (δ 1.87) and
H-5'; H-5′ and H-9'; H-6′ and H-7'; and H-7′ and H2-10′, as shown in Fig.
S1. This evidence allowed us to confirm the stereostructure of 5. The
proton and carbon signals in the 1H (Table S3) and 13C NMR (Table S4,
in CD3OD) of 6 were quite similar to those of 5, except for the signals
arising from the 10- and Glc-6-positions. Thus, the signals due to the 10-
position in 6 [δ 3.88 and 4.06 (1H each, both d, J = 12.8 Hz); δC 61.36]
were shifted higher than those of 5, whereas the signals due to the Glc-
6-position in 6 [δ 3.90 (1H, m) and 4.05 (1H, dd, J = 11.2, 4.0 Hz); δC
68.6] were observed at a lower shift as compared with those of 5 [δ
3.67 (1H, dd, J = 11.8, 6.0 Hz) and 3.95 (1H, m); δC 64.4]. The con-
nectivity of the acyl groups and glucopyranosyl linkages was char-
acterized on the basis of the HMBC spectrum, as shown in Fig. S1.
Consequently, the structure of 6 was determined.
2.3. Inhibitory effects on hyaluronidase
Hyaluronidases are enzymes that break down hyaluronic acid, a
mucopolysaccharide related to inflammation through the release of
histamine from mast cells. Hyaluronidase inhibitors appear to be ef-
fective therapies for the suppression of allergies and inflammation
(Girish and Kemparaju, 2007; Kakegawa et al., 1992; Murata et al.,
2010). It is known that the antiallergic medicine disodium cromogly-
cate (DSCG) exhibits a strong inhibitory effect against hyaluronidase
(Kakegawa et al., 1992). Therefore, a close relationship has been
identified between allergic reactions and hyaluronidase inhibitory ac-
tivity (Kakegawa et al., 1992; Murata et al., 2010). We have reported a
phenylethanoid glycoside isolated from the flowers of Mimusops elengi
L., an accepted name on www.theplantlist.org, which is currently
classified as a member of the Sapotaceae family (The Plant List, http://
www.theplantlist.org/tpl1.1/record/kew-128461, accessed on August
16, 2019) (Morikawa et al., 2018c), and aporphine- and benzylisoqui-
noline-type alkaloids from the flowers of Nelumbo nucifera Gaertn.,
which is the accepted name on www.theplantlist.org, which is currently
classified as a member of the Nelumbonaceae family (The Plant List,
http://www.theplantlist.org/tpl1.1/record/kew-2384945, accessed on
August 16, 2019) (Morikawa et al., 2019), with hyaluronidase in-
hibitory activity. Through our continued studies on antiallergic prin-
ciples with hyaluronidase inhibitory activity, the methanol extract of
the rhizomes of P. kurroa was found to show the inhibitory activity
[inhibition (%): 55.1
1.7 at 100 μg/mL; IC50 = 47.7 μg/mL] as pre-
sented in Table 1. To identify the active constituents of this plant ma-
terial, the inhibitory effects of the principal iridoids (1–13) against
hyaluronidase were examined. As shown in Table 1, picrorhizaosides D
(4, IC50 = 43.4 μM) and E (5, 35.8 μM), picrosides I (8, 60.7 μM), II (9,
22.3 μM), and IV (11, 59.2 μM), and minecoside (13, 57.2 μM),
The molecular formula of picrorhizaoside G (7) was determined to
be C48H58O26 by HRFABMS measurement. Treatment of 7 with 5%
KOH led to the release of vanillic acid and trans-ferulic acid together
with the residue; the residue was then treated with 1.0 M HCl, resulting
4

T. Morikawa, et al.
Phytochemistry169(2020)112185
Table 1
Effects of methanol extract, its fractions, and iridoid constituents from the rhizomes of P. kurroa on hyaluronidase activity.
Inhibition (%)
IC50
0 μg/mL
3 μg/mL
10 μg/mL
30 μg/mL
100 μg/mL
(μg/mL)
47.7
MeOH extract
0.0
0.0
0.0
0.0
7.6
15.3
10.3
14.9
8.1
2.9
4.8a
37.8
14.6
29.1
−3.6
6.3b
5.4
2.3b
8.4
47.0
6.3b
55.1
34.6
60.1
7.9
1.7b
4.1b
3.6b
EtOAc-soluble fraction
MeOH-eluted fraction
H2O-eluted fraction
3.1
2.3
3.1
17.3
46.6
6.4
2.0b
44.1
1.4
0.8
−10.0
8.4
4.7
Inhibition (%)
IC50
0 μM
3 μM
14.3
5.8
10 μM
20.5
13.9
14.2
33.5
37.3
28.3
45.6
27.8
39.1
29.9
40.8
30 μM
21.1
23.1
16.4
43.9
48.2
33.9
59.2
36.9
41.0
38.4
46.4
100 μM
41.0
40.5
32.2
60.7
61.8
61.1
63.1
52.3
54.6
53.7
52.8
(μM)
Picrorhizaoside A (1)
Picrorhizaoside B (2)
Picrorhizaoside C (3)
Picrorhizaoside D (4)
Picrorhizaoside E (5)
Picroside I (8)
Picroside II (9)
Picroside III (10)
Picroside IV (11)
6-Feruloylcatalpol (12)
Minecoside (13)
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
3.3
1.1
2.4
4.5
7.6
7.2
1.9
3.6
5.7
6.3
4.6
3.0b
5.5
3.6
1.1
2.2b
4.7
4.8
2.1b
3.5b
6.3
2.1b
2.9b
6.7b
5.0b
6.1b
7.5b
4.2b
5.4b
7.6b
2.8b
0.8b
7.1
17.3
14.6
17.2
19.4
17.6
11.8
23.2
32.0
7.7b
5.0b
3.1b
4.5b
4.4b
1.7b
2.9b
4.1b
5.3b
6.9b
2.6b
5.5b
2.8b
3.4b
2.3b
3.8b
43.4
35.8
60.7
22.3
92.7
59.2
85.0
57.2
6.5
4.2
6.0a
3.8b
3.1
3.9b
2.7b
Disodium cromoglycate
Ketotifen fumarate
0.0
0.0
2.0
6.1
4.0
11.9
2.4
1.9
14.4
26.7
0.4a
4.9
39.0
36.4
4.9b
2.9b
69.1
54.6
2.2b
2.5b
64.8
76.5
Inhibition (%)
0 μM
30 μM
100 μM
300 μM
1000 μM
Tranilast
0.0
4.5
17.7
5.5
32.0
5.0b
53.7
7.0b
77.8
3.8b
227
Each value represents the mean
S.E.M. (N = 4).
Significantly different from the control, ap < 0.05, bp < 0.01.
exhibited similar or stronger hyaluronidase inhibitory activity than that
of the antiallergic medicine disodium cromoglycate (DSCG, 64.8 μM),
as well as those of ketotifen fumarate (76.5 μM) and tranilast (227 μM),
which are recognized as effective antiallergic agents owing to their
inhibitory activity on degranulation (Morikawa et al., 2018a).
NMR spectra, JNM-ECA800 (800 MHz), JNM-ECA600 (600 MHz), JNM-
EAC500 (500 MHz), and JNM-ECS400 (400 MHz) spectrometers (JEOL
Ltd., Tokyo, Japan); 13C NMR spectra, JNM-ECA800 (200 MHz), JNM-
ECA600 (150 MHz), JNM-EAC500 (125 MHz), and JNM-ECS400
(100 MHz) spectrometers (JEOL Ltd.) in CD3OD at room temperature or
in pyridine-d5 at 40 °C with tetramethylsilane as an internal standard;
FABMS and HRFABMS, JMS-SX102A mass spectrometer (JEOL Ltd.);
HPLC detector, SPD-10Avp UV–Vis detector (Shimadzu Co.); HPLC
column, Cosmosil 5C18-MS-II and Cosmosil πNAP (Nacalai Tesque, Inc.,
Kyoto, Japan) 4.6 mm i.d. × 250 mm and 20 mm i.d. × 250 mm for
analytical and preparative purposes, respectively.
The following experimental conditions were used for column chro-
matography (CC): normal-phase silica gel CC, silica gel 60N (Kanto
Chemical Co., Ltd., Tokyo, Japan; 63–210 mesh, spherical, neutral);
reversed-phase ODS CC, Chromatorex ODS DM1020T (Fuji Silysia
Chemical, Ltd., Aichi, Japan; 100–200 mesh); TLC, pre-coated TLC
plates with silica gel 60F254 (Merck, Darmstadt, Germany, 0.25 mm,
normal-phase) and silica gel RP-18 WF254S (Merck, Darmstadt,
Germany, 0.25 mm, reversed-phase); reversed-phase HPTLC, pre-
coated TLC plates with silica gel RP-18 WF254S (Merck, 0.25 mm); de-
tection was performed by spraying 1% Ce(SO4)2–10% aqueous H2SO4,
followed by heating.
2.4. Concluding remarks
In conclusion, seven new acylated iridoid glycosides, picrorhizao-
sides A–G (1–7), having hyaluronidase inhibitory activity were isolated
from the methanol extract of the rhizomes of P. kurroa. Of the isolated
iridoids, picrorhizaosides D (4) and E (5), picrosides I (8), II (9), and IV
(11),
and
minecoside
(13)
were
active
inhibitors
(IC50 = 22.3–60.7 μM). These hyaluronidase inhibitory activities were
similar to or stronger than those of the antiallergic medicines DSCG
(64.8 μM), ketotifen fumarate (76.5 μM), and tranilast (227 μM), but
weaker than that of the alkaloids isolated from N. nucifera, such as
asimilobine (11.7 μM) and coclaurine (11.4 μM) (Morikawa et al.,
2019). In addition, inhibitory activity against degranulation of several
iridoids, such as aucubin (Jeong et al., 2002) and oleuropein (Persia
et al., 2014) has been reported. These data demonstrated that the me-
thanol extract and several iridoid constituents from the rhizomes of P.
kurroa may be new candidates for the development of antiallergic
medicines. The detailed mechanism of action, including the de-
granulation inhibitory activity and the structural requirements, should
be studied further.
3.2. Plant material
The rhizomes of P. kurroa, which were cultivated in the Tibet
Autonomous Region, China, were obtained in November 2006 from
China Meheco Corporation, Beijing, China. Identification of the plant
material was confirmed by the Garden of Medicinal Plants, Kindai
University and by one of the authors (M.Y.). A voucher specimen (lot.
no. 1230HX) of this plant material is on file in our laboratory.
3. Experimental
3.1. General experimental procedures
The following instruments were used to obtain physical data: spe-
cific rotations, SEPA-300 digital polarimeter (Horiba Ltd., Kyoto,
Japan, l = 5 cm); UV spectra, UV-1600 spectrometer (Shimadzu Co.,
Kyoto, Japan); IR spectra, FTIR-8100 spectrometer (Shimadzu Co.); 1H
3.3. Extraction and isolation
Dried rhizomes of P. kurroa (2800 g) were extracted three times
5

T. Morikawa, et al.
Phytochemistry169(2020)112185
with MeOH (10 L) under reflux for 3 h. Evaporation of the combined
extracts under reduced pressure yielded the MeOH extract (1318 g,
47.07%). An aliquot (1150 g) of the MeOH extract was partitioned in an
EtOAc-H2O (1:1, v/v) mixture, which yielded an EtOAc-soluble fraction
(203.6 g, 8.33%) and an aqueous phase. The aqueous phase was sub-
jected to Diaion HP-20 CC (3.0 kg, H2O → MeOH, applied in three di-
visions), which yielded H2O-eluted (519.1 g, 21.25%) and MeOH-eluted
(427.3 g, 17.49%) fractions.
3.3.1. Picrorhizaoside A (1)
A white powder; [α]D29 −65.0 (c 0.66, MeOH); UV (MeOH) λmax nm
(log ε): 220 (4.28), 266 (4.08), 295 (3.95); IR (KBr) νmax cm−1: 3486,
1720, 1599, 1516, 1223, 1104; 1H and 13C NMR spectroscopic data, see
Tables S1 and S2; Positive-ion FABMS m/z 697 [M + Na]+; Positive-
ion HRFABMS m/z 697.1963 [M + Na]+ (calcd for C29H38O18Na+
697.1956).
,
An aliquot (117.0 g) of the EtOAc-soluble fraction was subjected to
normal-phase silica gel CC [3.0 kg, CHCl3–MeOH–H2O (20:3:1 →
10:3:1 → 7:3:1, v/v/v, lower layer) → MeOH], which yielded 10 frac-
tions [Fr. 1 (18.70 g), Fr. 2 (10.44 g), Fr. 3 (4.39 g), Fr. 4 (9.15 g), Fr. 5
(19.97 g), Fr. 6 (4.62 g), Fr. 7 (14.37 g), Fr. 8 (15.55 g), Fr. 9 (6.82 g),
and Fr. 10 (6.77 g)]. Fraction 5 (19.97 g) was subjected to reversed-
phase ODS CC [600 g, MeOH–H2O (20:80 → 40:60 → 60:40 → 70:30, v/
v) → MeOH], which yielded six fractions {Fr. 5-1 (1337.2 mg), Fr. 5-2
(618.0 mg), Fr. 5-3 [ = picroside I (8), 13.07 g, 0.931%], Fr. 5-4
(862.5 mg), Fr. 5-5 (514.4 mg), and Fr. 5–6 (2726.4 mg}}. Fraction 8
(15.55 g) was subjected to reversed-phase ODS CC [700 g, MeOH–H2O
(20:80 → 40:60 → 60:40, v/v) → MeOH], which yielded five fractions
[Fr. 8-1 (811.8 mg), Fr. 8-2 (2269.4 mg), Fr. 8-3 (= 9, 5739.5 mg,
0.409%), Fr. 8-4 (2655.7 mg), and Fr. 8-5 (3392.1 mg)]. Fraction 8-2
(572.4 mg) was purified by HPLC [Cosmosil πNAP, UV (230 nm),
CH3CN-1% aqueous AcOH (15:85, v/v)] to give picrosides II (9,
60.6 mg, 0.0171%), III (10, 63.8 mg, 0.0180%), and IV (11, 133.8 mg,
0.0378%). Fraction 9 (6.82 g) was subjected to reversed-phase ODS CC
[220 g, MeOH–H2O (40:60 → 60:40 → 80:20, v/v) → MeOH] to give
four fractions [Fr. 9-1 (1798.4 mg), Fr. 9-2 (1315.9 mg), Fr. 9-3
(771.7 mg) and Fr. 9-4 (2288.3 mg)]. Fraction 9-3 (515.4 mg) was
purified by HPLC [Cosmosil 5C18-MS-II, RI, MeOH-1% aqueous AcOH
(50:50, v/v)] to give picrorhizaosides E (5, 28.3 mg, 0.0030%), F (6,
27.7 mg, 0.0030%), and G (7, 15.5 mg, 0.0017%).
An aliquot (133.8 g) of the MeOH-eluted fraction was subjected to
normal-phase silica gel CC [3.00 kg, CHCl3–MeOH–H2O (10:3:1 →
7:3:1, v/v/v, lower layer → 6:4:1, v/v/v) → MeOH] to yield nine
fractions [Fr. 1 (5.10 g), Fr. 2 (17.64 g), Fr. 3 (59.61 g), Fr. 4 (7.70 g),
Fr. 5 (5.45 g), Fr. 6 (15.03 g), Fr. 7 (5.00 g), Fr. 8 (2.49 g), and Fr. 9
(3.62 g)]. Fraction 2 (17.64 g) was subjected to reversed-phase silica gel
CC [510 g, MeOH–H2O (30:70 → 60:40, v/v) → MeOH] to yield five
fractions [Fr. 2-1 (2115.8 mg), Fr. 2-2 (= 8, 12.46 g, 1.629%), Fr. 2–3
(1014.2 mg), Fr. 2–4 (1693.7 mg), and Fr. 2–5 (221.1 mg)]. Fraction
2–3 (514.7 mg) was purified by HPLC [Cosmosil 5C18-MS-II, RI, MeOH-
1% aqueous AcOH (40:60, v/v)], which yielded 6-feruloylcatalpol (12,
109.0 mg, 0.0281%) and minecoside (13, 110.8 mg, 0.0285%). Fraction
5 (5.45 g) was subjected to reversed-phase ODS CC [180 g, MeOH–H2O
(5:95 → 20:80 → 30:70 → 40:60, v/v) → MeOH] to afford six fractions
[Fr. 5-1 (880.2 mg), Fr. 5-2 (1149.7 mg), Fr. 5-3 (896.0 mg), Fr. 5-4
(136.3 mg), Fr. 5-5 (1028.4 mg), and Fr. 5–6 (1242.2 mg)]. Fraction 5-3
(356.3 mg) was purified by HPLC [Cosmosil 5C18-MS-II, RI, MeOH–H2O
(40:60, v/v)] to give five fractions {Fr. 5-3-1 (20.0 mg), Fr. 5-3-2
[ = picrorhizaoside A (1, 59.8 mg, 0.0197%)], Fr. 5-3-3 (35.5 mg), Fr.
5-3-4 (31.9 mg), and Fr. 5-3-5 (193.4 mg)}. Fraction 5-3-4 (31.9 mg)
was further purified by HPLC [Cosmosil πNAP, RI, CH3CN–H2O (15:85,
v/v)] to give picrorhizaoside B (2, 6.4 mg, 0.0021%) and 9 (7.0 mg,
0.0023%). Fraction 6 (15.03 g) was subjected to reversed-phase ODS CC
[450 g, MeOH–H2O (20:80 → 30:70 → 50:50, v/v) → MeOH], which
yielded seven fractions [Fr. 6-1 (1254.3 mg), Fr. 6-2 (1555.1 mg), Fr. 6-
3 (5038.7 mg), Fr. 6-4 (2918.8 mg), Fr. 6-5 (1154.2 mg), Fr. 6-6
(2397.5 mg), and Fr. 6–7 (508.4 mg)]. Fraction 6-2 (517.2 mg) was
purified by HPLC [Cosmosil 5C18-MS-II, RI, MeOH-1% aqueous AcOH
(15:85, v/v)] to give picrorhizaoside C (3, 8.4 mg, 0.0033%). Fraction
6-5 (509.5 mg) was purified by HPLC [Cosmosil 5C18-MS-II, RI, MeOH-
1% aqueous AcOH (30:70, v/v)] to produce picrorhizaoside D (4,
8.7 mg, 0.0026%). The known compounds isolated were un-
ambiguously identified through the comparison of their physical and
spectroscopic data with those of reported values.
3.3.2. Picrorhizaoside B (2)
A white powder; [α]D29 −12.3 (c 0.35, MeOH); UV (MeOH) λmax nm
(log ε): 217 (3.90), 278 (4.03); IR (KBr) νmax cm−1: 3398, 1715, 1636,
1224, 1073; 1H and 13C NMR spectroscopic data, see Tables S1 and S2;
Positive-ion FABMS m/z 677 [M + Na]+; Positive-ion HRFABMS m/z
677.2064 [M + Na]+ (calcd for C30H38O16Na+, 677.2058).
3.3.3. Picrorhizaoside C (3)
A white powder; [α]D28 −45.6 (c 0.43, MeOH); UV (MeOH) λmax nm
(log ε): 216 (4.36), 257 (4.08), 291 (3.78); IR (KBr) νmax cm−1: 3415,
1719, 1649, 1605, 1509, 1275, 1075; 1H and 13C NMR spectroscopic
data, see Tables S1 and S2; Positive-ion FABMS m/z 697 [M + Na]+
;
Positive-ion HRFABMS m/z 697.1949 [M
+
Na]+ (calcd for
C29H38O18Na+, 697.1956).
3.3.4. Picrorhizaoside D (4)
A white powder; [α]D29 −35.0 (c 0.41, MeOH); UV (MeOH) λmax nm
(log ε): 218 (4.48), 261 (4.19), 293 (4.01); IR (KBr) νmax cm−1: 3433,
1717, 1655, 1638, 1509, 1279, 1076; 1H and 13C NMR spectroscopic
data, see Tables S1 and S2; Positive-ion FABMS m/z 865 [M + Na]+
;
Positive-ion HRFABMS m/z 865.2369 [M
+
Na]+ (calcd for
C
37H46O22Na+, 865.2378).
3.3.5. Picrorhizaoside E (5)
A white powder; [α]D24 −89.0 (c 0.67, MeOH); UV (MeOH) λmax nm
(log ε): 217 (3.55), 271 (3.44); IR (KBr) νmax cm−1: 3441, 1707, 1285,
1090, 1078; 1H and 13C NMR spectroscopic data, see Tables S3 and S4;
Positive-ion FABMS m/z 1027 [M + Na]+; Positive-ion HRFABMS m/z
1027.3055 [M + Na]+ (calcd for C47H56O24Na+, 1027.3059).
3.3.6. Picrorhizaoside F (6)
A white powder; [α]D24 −94.0 (c 0.45, MeOH); UV (MeOH) λmax nm
(log ε): 218 (4.49), 270 (4.43); IR (KBr) νmax cm−1: 3426, 1709, 1287,
1110, 1074; 1H and 13C NMR spectroscopic data, see Tables S3 and S4;
Positive-ion FABMS m/z 1027 [M + Na]+; Positive-ion HRFABMS m/z
1027.3053 [M + Na]+ (calcd for C47H56O24Na+, 1027.3059).
3.3.7. Picrorhizaoside G (7)
A white powder; [α]D24 −69.7 (c 0.76, MeOH); UV (MeOH) λmax nm
(log ε): 218 (4.44), 266 (4.17), 295 (4.22); IR (KBr) νmax cm−1: 3422,
1701, 1287, 1101, 1074; 1H and 13C NMR spectroscopic data, see
Tables S3 and S4; Positive-ion FABMS m/z 1073 [M + Na]+; Positive-
ion HRFABMS m/z 1073.3119 [M + Na]+ (calcd for C48H58O26Na+
,
1073.3114).
3.4. Enzymatic deacylation of picrorhizaosides A (1) and B (2)
A solution of 1 (10.0 mg) in H2O (2.0 mL) was treated with tannase
(10.0 mg, from Aspergillus oryzae, Wako Pure Chemical Industry Ltd.,
Osaka, Japan) and the solution was stirred at 37 °C for 24 h. After EtOH
was added to the reaction mixture, the solvent was removed under
reduced pressure and the residue was purified by HPLC [Cosmosil 5C18
-
MS-II, RI, MeOH-1% aqueous AcOH (20:80, v/v)], which produced
desacylscalbidoside (1a, 6.0 mg, 78%). Through a similar procedure, a
solution of 2 (4.0 mg) in H2O (2.0 mL) was treated with tannase
(4.0 mg) and the solution was stirred at 37 °C for 24 h. The workup of
the reaction mixture described above yielded a residue that was sub-
sequently purified by HPLC [Cosmosil 5C18-MS-II, RI, MeOH-1%
6

T. Morikawa, et al.
Phytochemistry169(2020)112185
aqueous AcOH (20:80, v/v)] to produce rehmannioside B (2a, 2.0 mg,
62%).
Pure Chemical Industries Ltd., Osaka, Japan) acetate solution was
added to the reaction mixture and incubated at 37 °C for 20 min. The
optical density (O.D.) of the reaction mixture was measured by using a
microplate reader (SH-9000, CORONA) at a wavelength of 585 nm
(reference 670 nm). The final concentration of dimethyl sulfoxide
(DMSO) in the test solution was 1.0% and no influence of DMSO on the
inhibitory activity was detected. All experiments were performed in
quadruplicate and IC50 was determined graphically. Disodium cro-
moglycate (DSCG), ketotifen fumarate, and tranilast, clinically pre-
scribed antiallergic medicines, were used as the reference compounds.
Inhibition (%) = [(O.D. (C) – O.D. (N)) – (O.D. (T) – O.D. (B))]/
(O.D. (C) – O.D. (N)) × 100.
3.5. Alkaline and acid hydrolysis of picrorhizaosides A (1), B (2), and E–G
(5–7)
Solutions of 1, 2, and 5–7 (each 1.0 mg) in 5% aqueous potassium
hydroxide (KOH, 0.5 mL) were stirred at 40 °C for 1 h. Each mixture was
neutralized with Dowex HCR W2 (H+ form), and the resin was removed
by filtration. Removal of the solvent from the filtrate under reduced
pressure yielded the corresponding deacylated products, which were
subjected to HPLC analysis [column: Cosmosil πNAP, 250 × 4.6 mm
i.d.; mobile phase: CH3CN-1% aqueous AcOH (25:75, v/v); detection:
UV (254 nm); flow rate: 1.0 mL/min], which was identified as vanillic
acid (tR 6.5 min from 1 and 5–7), trans-cinnamic acid (tR 23.9 min from
2, 5, and 6), or trans-ferulic acid (tR 6.1 min from 7), respectively. Each
of the products was dissolved in 1.0 M HCl (1.0 mL) and heated at 80 °C
for 3 h. After cooling, the reaction mixture was neutralized with
Amberlite IRA-400 (OH− form), and then the resins were removed by
filtration. After removal of the solvent under reduced pressure, the re-
sidue was separated by Sep-Pak Plus Short C18 cartridge CC (Waters
Corporation, Milford, Massachusetts, USA, H2O → MeOH). The H2O-
eluted fraction was subjected to HPLC analysis under the following
conditions: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm
i.d. × 250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical
rotation [JASCO OR2090 Plus (JASCO Corporation, Tokyo, Japan);
mobile phase, CH3CN–H2O (80:20, v/v); flow rate 1.0 mL/min].
Identification of D-glucose (i) and D-galactose (ii) present in the H2O-
eluted fraction was conducted through a comparison of their retention
times and optical rotation with those of an authentic sample [i, tR
18.7 min (positive) from 1, 2, and 5–7] and [ii, tR 19.9 min (positive)
from 2].
Control (C): enzyme (+), test sample (−); Test (T): enzyme (+),
test sample (+); Blank (B): enzyme (−), test sample (+); Normal (N):
enzyme (−), test sample (−)
3.8. Statistics
The values were expressed as the mean
S.E.M. One-way analysis
of variance (ANOVA) followed by Dunnett's test was used for the sta-
tistical analysis. Probability (p) values less than 0.05 were considered
significant.
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgements
This work was supported in part by the MEXT-Supported Program
for the Strategic Research Foundation at Private Universities,
2014–2018, Japan (S1411037, T.M.), as well as the JSPS KAKENHI,
Japan [Grant Numbers 18K06726 (T.M.), 18K06739 (K.N.), and
16K08313 (O.M.)]. We would like to thank Editage (www.editage.com)
for English language editing.
3.6. Acid and alkaline hydrolysis of picrorhizaosides C (3) and D (4)
Solutions of 3 and 4 (both 1.0 mg) were dissolved in 1.0 M HCl
(1.0 mL) and heated at 80 °C for 3 h. After cooling, the reaction mixture
was neutralized with Amberlite IRA-400 (OH− form), and then the
resins were removed by filtration. After removal of the solvent under
reduced pressure, the residue was separated by Sep-Pak Plus Short C18
cartridge CC (H2O → MeOH). The HPLC analysis of the H2O-eluted
fraction led to the identification of the above as D-glucose. Both MeOH-
eluted fractions were dissolved in 5% KOH (0.5 mL) stirred at 40 °C for
1 h. The reaction mixtures were neutralized with Dowex HCR W2 (H+
form), and the resin was removed by filtration. The filtrates were
subjected to the HPLC analysis (vide supra) and identified as vanillic
acid.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.112185.
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