Palhinosides A-H: Flavone Glucosidic Truxinate Esters with Neuroprotective Activities from Palhinhaea cernua

Neuroprotective Activities from Palhinhaea cernua

Article

Palhinosides A−H: Flavone Glucosidic Truxinate Esters with

Jing Li, Lei-Hong Tan, Hui Zou, Zhen-Xing Zou, Hong-Ping Long, Wen-Xuan Wang, Ping-Sheng Xu,

Li-Fei Liu, Kang-Ping Xu,* and Gui-Shan Tan*

Cite This: https://dx.doi.org/10.1021/acs.jnatprod.9b00470

Read Online

ACCESS

Metrics & More

Article Recommendations

sı Supporting Information

ABSTRACT: Palhinosides A−H (1−8), new ﬂavone glucosidic

truxinate esters, including β-truxinate and μ-truxinate forms, were

isolated from Palhinhaea cernua. Their structures were elucidated by

extensive spectroscopic methods and chemical analyses. The ﬂavone

glucoside cyclodimers possess a unique cyclobutane ring in their

carbon scaﬀolds. Compounds 2−7 represent three pairs of stereo-

isomers (2/3, 4/5, 6/7). The protective eﬀects of 1−8 against the

damage of HT-22 cells induced by L-glutamate were evaluated, and

compounds 4 and 5 showed better neuroprotective eﬀects than the

positive control, Trolox.

lavone glucosidic truxinate esters are cyclodimeric

structure elucidation, and neuroprotective eﬀects of the new

derivatives containing an unusual cyclobutane ring in

compounds are described.

their carbon scaﬀolds. Flavonoid glucoside cyclodimers in

truxillate or truxinate forms are unusual in plants, and to date

only 13 derivatives have been reported from Monochaetum

RESULTS AND DISCUSSION

■

Compound 1 was obtained as a yellowish, amorphous powder

with a molecular formula of C H O according to the

multiflorum, Stachys aegyptiaca, Itoa orientalis, Paepalanthus

geniculatus, Ginkgo biloba, Chamaerhodos altaica, and

Potentilla anserine.

important pharmacological activities, including antioxidant,

antineuroinﬂammatory, and neuroprotective activities.

Palhinhaea cernua (family Lycopodiaceae) is widely

distributed in southern and eastern China. The whole plants

of P. cernua, called “Pu-Di-Wu-Gong” in Chinese, are

traditionally used as an herbal medicine for relaxing tendons,

healing contusions, and treating rheumatism.

phytochemical investigations of P. cernua indicated the

presence of ﬂavones, neolignans, lycopodium alkaloids,

and serratene-type triterpenoids.

structurally unique and biologically interesting natural products

from plants of the family Lycopodiaceae,

52 24

positive ion at m/z 1157.2926 [M + H] (calcd for 1157.2921)

in the HRESIMS data, indicating 35 indices of hydrogen

deﬁciency. The UV absorption maxima at 271 and 324 nm

Some of these derivatives possess

suggested a ﬂavone skeleton. In the H NMR spectrum, signals

at δ 7.94/7.73 (each 2H, d, J = 8.5 Hz, H-2′/6′), 6.93/6.84

(

each 2H, d, J = 8.5 Hz, H-3′/5′), 6.37/6.16 (each 1H, d, J =

.0 Hz, H-6), 6.74/6.42 (each 1H, d, J = 2.0 Hz, H-8), 6.66/

.64 (each 1H, s, H-3), and 12.80 (2H, br s) were assigned to

,10

two sets of apigenin moieties. Proton signals at δ 6.32/6.24

(

each 2H, d, J = 8.5 Hz, H-2a/6a/3a/5a) and 6.34 (4H, d, J =

.5 Hz, H-2b/6b/3b/5b) indicated the presence of two p-

substituted aromatic moieties. Additionally, two anomeric

13−20

As part of a search for

proton signals at δ 5.34/5.00 (each 1H, d, J = 7.5 Hz, H-1″)

were observed, suggesting the presence of two β-glucopyr-

12,21,22

the chemical

anosyl groups. The C NMR data of 1 revealed 60 resonances.

components in the n-BuOH portion of the extract of P. cernua

were investigated. As a result, eight new truxinate derivatives,

palhinosides A−H (1−8), together with two known

compounds, apigenin-7-O-(6″-O-p-coumaroyl)-β-D-glucopyra-

noside (9) and methyl 4-hydroxycinnamate (10), have been

isolated. Their structures were identiﬁed by analysis of 1D/2D

NMR and MS data, as well as chemical analyses. These ﬂavone

glucoside cyclodimers were isolated from the family

Lycopodiaceae for the ﬁrst time. Herein, the isolation,

In addition to the carbon signals of two apigenin 7-O-β-

glucoside moieties and two 4-hydroxyphenyl groups, the

remaining six signals were attributed to four methine carbons

Received: May 17, 2019

XXXX American Chemical Society and

American Society of Pharmacognosy

Journal of Natural Products

J. Nat. Prod. XXXX, XXX, XXX−XXX

arrangement of the four methine proton signals (Figure 3 ).

Article

Figure 1. Structures of compounds 1−10 identiﬁed from P. cernua.

[

δ 43.6 (7b), 43.9 (7a), 42.7 (8b), and 43.4 (8a)] and two

8a,8b-cis-conﬁgurations, which establishment a β-truxinate

ester carbonyls [δ 172.4 (×2)]. In the C NMR spectra, the

signals appeared in pairs and were similar to those of the

known apigenin coumaroyl glucoside 9, except for the

absence of the characteristic trans-oleﬁnic proton signals (δ_H

.64 and 6.36, J = 16.0 Hz) in 9. Instead, four upﬁeld methine

proton signals at δ 4.02 (1H, dd, J = 10.0, 5.0 Hz, H-7b), 3.86

(

1H, dd, J = 10.0, 5.0 Hz, H-7a), 3.67 (1H, t, J = 9.5 Hz, H-

b), and 3.39 (1H, t, J = 9.5 Hz, H-8a) were observed in 1.

Analysis of the HSQC and H− H COSY data revealed that

the four methine groups (C-7a, C-8a, C-7b, and C-8b) were

connected to form a cyclobutane ring (Figure 2 ) for the

Figure 3. Selected NOESY correlations of compounds 1−8.

Additionally, the D-conﬁguration of the glucosyl moieties were

conﬁrmed via hydrolysis, derivation, and HPLC analysis.

Therefore, the structure of 1 was deduced to be a β-truxinic-

type dimeric ﬂavone glucosidic truxinate ester (Figure 1).

Palhinoside B (2) was puriﬁed as a yellowish amorphous

powder, with a molecular formula of C H O via the

62 29

HRESIMS (m/z 1319.3396 [M + H] , calcd for 1319.3450)

data, suggesting that it has one more glucosyl moiety

(

C H O ) than 1. The NMR data of 2 exhibited spectroscopic

6 10 5

features similar to 1 (Tables 1 and 3), except for the signals of

a β-glucopyranosyl group connected at C-4′, which was

deduced by the HMBC correlation of H-1‴ [δ 5.00 (1H, d,

.5)] to C-4′ (δ 161.0). The H NMR spectrum showed the

cyclobutyl proton signals at δ 4.00 (1H, dd, J = 10.0, 5.0 Hz,

H-7b), 3.86 (1H, dd, J = 10.0, 5.0 Hz, H-7a), 3.65 (1H, t, J =

9.5 Hz, H-8b), and 3.36 (1H, t, J = 9.0 Hz, H-8a). Based on

the splitting patterns and coupling constants the relative

conﬁguration of the cyclobutane ring in 2 was also determined

as the β-truxinate arrangement. Additionally, the glucopyr-

anosyl moieties were deduced to be D-conﬁgured via the same

method as for 1. The structure of 2 was supported by the 2D

NMR data as shown in Figure 1.

Figure 2. Key COSY and HMBC correlations of compounds 1 and 4.

remaining index of hydrogen deﬁciency. Thus, compound 1

was considered to be a dimer of 9, formed by a [2+2]

cycloaddition reaction between two α,β-unsaturated carbonyl

moieties. The key HMBC correlations (Figure 2) from H-2a/

H-6a to C-7a and from H-2b/H-6b to C-7b, together with the

COSY data, conﬁrmed that the two p-substituted aromatic

moieties were vicinal and established a head-to-head cyclo-

Palhinoside C (3) was obtained as a yellowish amorphous

powder, whose molecular formula was determined to be

4,25

butane derivative.

C H O by the HRESIMS (m/z 1319.3458 [M + H] , calcd

62 29

The key NOE cross-peaks between H-8a and H-2a(6a)/H-

b(6b) and between H-8b and H-2a(6a)/H-2b(6b) suggested

the 7a,8a- and 7b,8b-trans-conﬁgurations and the 7a,7b- and

for 1319.3450) data. Compound 3 had similar HRESIMS and

NMR data to 2, indicating that 3 was an isomer of 2. Analysis

of the NMR data of these two compounds showed only slight

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. H NMR Data for Compounds 1, 2, 4, and 6

Table 2. H NMR Data for Compounds 3, 5, 7, and 8

(

DMSO-d₆)

(DMSO-d₆)

carbon

no.

carbon

no.

Apigenin Moiety

6.66/6.64, s

6.37/6.16, d

6.75/6.59, s

6.36/6.15, d (2.0) 6.46, br s

6.89, s

6.97, s

6.48, d (2.0)

6.76/6.60, s

6.18/6.40, d

(1.5)

6.73/6.43, d

(1.5)

7.98/7.73, d

(8.5)

6.89, s

7.00, s

6.76, s

6.49, d (2.0) 6.49, d (2.0) 6.17/6.43, d

(2.0)

6.84, d (2.0) 6.84, d (2.0) 6.76/6.40, d

(2.0)

8.00, d (8.5) 8.11, d (9.0) 8.00/7.83, d

(8.5)

6.95, d (8.5) 7.21, d (9.0) 7.19/7.10, d

(8.5)

(

2.0)

6.74/6.42, d

2.0)

7.94/7.73, d

8.5)

6.93/6.84, d

8.5)

6.81/6.36, d (2.0) 6.81, br s

6.85, d (2.0)

(

′/6′

′/5′

7.80/7.93, d (8.5) 8.00, d (9.0) 8.09, d (9.0)

7.06/6.94, d (8.5) 6.94, d (9.0) 7.21, d (9.0)

2′/6′

3′/5′

(

7.17/6.85, d

(8.5)

(

Glucose Moiety

1″

5.34/5.00, d

7.5)

5.40/5.00, d (7.5) 5.12, d (7.5) 5.16, d (7.5)

1″

5.30/4.97, d

(7.5)

5.16, d (7.5) 5.16, d (7.2) 5.34/4.97, d

(7.0)

(

2″

3″

4″

5″

6″

3.35, m

3.30, m

3.35, m

3.19, m

3.73, m

4.39, d

(10.7)

3.29, m

3.33, m

3.20, m

3.75, m

2″

3″

4″

5″

6″

3.40, m

3.31, m

3.34, m

3.20, m

3.76, m

4.48, d

(10.0)

3.30, m

3.33, m

3.20, m

3.75, m

4.38, d (8.8) 4.27/4.71, d

(11.0)

3.40, m

3.37/3.43, m

3.19, m

3.43/3.45, m

3.16/3.28, m

3.76/4.01, m

4.31/4.67, d

3.35/3.45, m

3.17/3.27, m

4.06/3.76, m

4.28/4.67, d

(11.0)

3.41/3.39, m

3.18/3.20, m

3.70/3.97, m

4.27/4.70, d

(10.0)

4.00/3.70, m

4.47, d (8.8)

(

10.7)

.03/3.29, d

10.7)

4.02/3.90, d

(11.0)

4.10, br d

(10.7)

4.02, d (8.8)

4.06/3.87, d

(10.0)

4.00, d

(10.0)

4.15, d (8.8) 4.08/3.88, d

(11.0)

(

‴

5.00, d (7.5)

3.33, m

3.43, m

3.22, m

3.35, m

5.04, d (7.2)

3.29, m

3.40, m

3.20, m

3.33, m

1‴

5.00, d (7.5)

5.02, d (7.2) 5.04/5.01, d

(7.0)

2‴

3‴

4‴

5‴

3.34, m

3.42, m

3.21, m

3.34, m

3.77, m

3.53, m

3.30, m

3.40, m

3.20, m

3.33, m

3.72, m

3.49, m

3.33, m

3.45, m

3.23, m

3.39, m

3.76, d (11.0)

3.53, d (11.0)

3.74, d (11.0)

3.72, m;

3.49, m

.54, d (11.0)

6‴

Truxinyl Moiety

a/6a 6.32, d (8.5)

a/5a 6.24, d (8.5)

6.31, d (8.5)

6.28, d (8.5)

6.60, d (8.5) 6.42, d (8.5)

6.41, d (8.5) 6.68, d (8.5)

Truxinyl Moiety

2a/6a

3a/5a

6.43, d (8.5)

6.28, d (8.5)

6.65, d (8.5) 6.36, d (8.5) 6.43, d (8.0)

6.41, d (8.5) 6.65, d (8.5) 6.29, d (8.0)

3.86, dd (10.0, 3.86, dd (10.0,

3.96, m

4.00,

overlapped

.0)

5.0)

3.36, t (9.5)

3.95,

overlapped

4.00,

overlapped

4.00,

overlapped

3.95,

overlapped

3.39, t (9.5)

3.67,

overlapped

3.75,

overlapped

3.54,

overlapped

3.78,

overlapped

3.78,

overlapped

3.52,

overlapped

b/6b 6.34, br s

b/5b 6.34, br s

6.33, d (8.5)

6.35, d (8.5)

6.56, d (8.5) 6.43, d (8.5)

6.31, d (8.5) 6.68, d (8.5)

2b/6b 6.40, br s

3b/5b 6.40, br s

6.63, d (8.5) 6.46, d (8.5) 6.40, br s

6.39, d (8.5) 6.65, d (8.5) 6.40, br s

4.02, dd (10.0, 4.00, dd (10.0,

3.98, m

4.00,

overlapped

.0)

5.0)

3.65, t (9.5)

3.97,

overlapped

3.72,

overlapped

4.00,

overlapped

4.00,

overlapped

3.98,

overlapped

3.67, t (9.5)

3.71,

overlapped

3.75,

overlapped

3.70,

overlapped

3.70,

overlapped

3.71,

overlapped

3.60,

overlapped

3.52, s

3.50, s

Recorded at 500 MHz. Recorded at 400 MHz.

Recorded at 500 MHz. Recorded at 400 MHz

diﬀerences in the methine signals of the cyclobutane ring,

which also suggested that the 2D structure of 3 was the same

as 2, whereas the conﬁgurations of the cyclobutane ring were

(1H, m, H-7b), 3.96 (1H, m, H-7a), 3.71 (1H, overlapped, H-

8b), and 3.67 (1H, overlapped, H-8a). Protons resonating at

diﬀerent. The NOE correlations of H-7a (δ 3.95)/H-8a (δ_H

δ 8.00 (2H, d, J = 9.0 Hz, H-2′/6′), 6.94 (2H, d, J = 9.0 Hz,

.54), H-7b (δ 3.97)/H-8b (δ 3.72), H-8a/H-2b, H-8a/H-

b, H-8b/H-2a, and H-8b/H-86a indicated that H-7a/7b and

H-3′/5′), 6.46 (1H, br s, H-6), 6.81 (1H, br s, H-8), and 6.89

(1H, s, H-3) indicated the presence of an apigenin moiety.

H-8a/8b were trans-related, while H-7a/8a and H-7b/8b were

cis-oriented. Therefore, the substituents on the cyclobutane

ring had a μ-truxinate arrangement. The conﬁgurations of

the glucosyl moieties in 3 were the same as in 2, deduced by

hydrolysis and HPLC analysis.

Proton signals at δ 6.60 (2H, d, J = 8.5 Hz, H-2a/6a), 6.41

(2H, d, J = 8.5 Hz, H-3a/5a), 6.56 (2H, d, J = 8.5 Hz, H-2b/

6b), and 6.31 (2H, d, J = 8.5 Hz, H-3b/5b) suggested the

presence of two 4-hydroxyphenyl groups. The signal for a

methoxy group was observed at δ 3.60 (3H, overlapped, H-

Palhinoside D (4) was obtained as a yellowish amorphous

powder. The molecular formula, C H O , was determined

10b). Additionally, an anomeric proton signal at δ 5.12 (1H,

d, J = 7.5 Hz, H-1″) indicated the presence of a β-

glucopyranosyl group. The acid hydrolysis of 4 aﬀorded D-

glucose, as was veriﬁed by HPLC analysis. The NMR data

revealed that 4 possessed two 4-hydroxyphenyl groups, an

apigenin 7-O-β-glucoside moiety, four methine groups, and a

36 15

by the HRESIMS (m/z 757.2125 [M + H] , calcd for

57.2127) data. A detailed comparison of the NMR

spectroscopic data of 4 and 1 disclosed that they are analogues.

The H NMR data showed four methine signals at δ 3.98

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 3. ¹³C NMR Data for Compounds 1−8 (DMSO-d₆)

carbon no.

164.6

103.4/103.3

182.1

163.7/164.6

104.2/103.4

182.1/182.0

161.2/161.6

99.9/99.8

163.2/162.5

94.4/94.3

157.1/156.9

105.6/105.5

124.3/121.5

128.5/129.1

116.8/116.6

161.0/161.7

128.9

128.8

115.0

155.7

43.8

43.5

172.3

128.9

128.8

163.8/164.5

104.3/103.3

182.2/182.0

161.7/161.4

99.7/99.5

163.1/162.7

94.7/94.8

157.1/156.8

105.6/105.5

124.3/121.5

128.7/128.9

117.1/116.4

161.0/161.7

129.3/129.1

128.9

115.0

155.7

43.8

43.3

172.3

129.1

128.8

115.0

164.8

103.5

182.4

161.2

100.0

163.1

95.1

157.4

105.8

121.3

129.1

116.6

161.6

129.3

129.1

115.0

155.7

43.3

164.8

103.5

182.5

162.0

100.0

163.2

95.2

157.4

105.9

121.4

129.1

116.5

161.6

129.4

129.2

115.0

155.8

44.4

164.0

104.6

182.7

161.8

100.0

163.4

95.2

157.4

106.0

124.3

128.7

117.1

160.9

129.5

129.2

115.1

155.9

44.4

164.0

104.6

182.5

161.8

100.0

163.3

95.2

157.4

106.0

124.3

128.7

117.0

160.9

129.4

129.2

115.0

155.8

44.4

163.8

104.3/104.2

182.2/182.0

160.7/161.0

99.5/99.7

163.2/162.6

94.7

157.1/156.8

105.5

124.3

128.5/128.7

117.1/116.9

161.3/161.7

129.3

128.9

115.1

155.7

43.8

43.4

172.3

129.1

128.8

115.0

155.6

161.3/161.1

99.7/99.9

163.1/162.6

94.6/94.5

157.2/157.0

105.6/105.5

121.5

128.9/128.8

116.6/116.3

162.1/161.8

129.2

129.0

115.0

155.7

43.9

43.4

172.4

129.1

129.0

′

′/6′

′/5′

′

a/6a

a/5a

b/6b

b/5b

42.9

43.0

43.2

43.0

172.8

129.3

129.1

114.9

155.7

43.3

42.9

173.2

52.2

99.9

73.3

76.6

70.5

172.7

129.5

129.2

115.0

155.8

44.4

42.8

173.3

52.1

99.9

73.5

76.6

70.4

172.7

129.5

129.2

115.0

155.9

44.3

42.9

173.3

52.2

99.9

73.4

76.7

70.3

74.2

64.4

100.4

73.7

77.7

70.2

77.0

61.2

172.8

129.5

129.1

115.0

155.8

44.3

43.0

173.3

52.2

99.9

73.5

76.6

70.4

74.3

64.5

100.5

73.6

77.7

70.3

77.0

61.1

115.0

155.7

43.6

42.7

115.0

155.7

43.5

42.7

155.7

43.7

43.0

173.1

43.6

42.9

173.2

172.9

173.0

″

‴

99.5/99.3

73.3

77.1/76.4

70.9

74.3/74.2

65.0/65.6

99.4/99.2

73.3

77.1/76.4

70.9

99.5/99.4

73.3/73.2

77.1/76.4

71.1/70.7

74.3/74.2

64.7/65.6

100.8

73.7

77.6

70.0

77.0

99.3

73.2

77.1/76.4

70.8/71.1

74.2/74.3

64.8/65.8

100.3/100.7

73.7

77.5/77.6

70.1

76.9/77.0

61.2

74.2

64.4

74.3

64.5

65.0/65.7

100.6

73.7

77.7

70.2

77.0

61.2

Recorded at 125 MHz. Recorded at 100 MHz.

methoxy group. Compared with 1, one of the two apigenin 7-

O-β-glucoside moieties in 1 was replaced by a methoxy group.

This conclusion was supported by the HMBC correlations

the stereocenters of the cyclobutane ring. The relative

conﬁguration was deﬁned as a μ-truxinate arrangement,

which was conﬁrmed by the NOESY cross-peaks between H-

7a/H-8a, H-7b/H-8b, and H-2a(6a)/7b(8b), suggesting the

(^Fⁱ^g^u^r^e²⁾^o^f^H^-¹⁰^b⁽^δH ³^.⁶⁰⁾^wⁱ^t^h^C^-⁹^b⁽^δC 173.2).

According to the molecular formula, compound 4 was thus

deduced to be a cyclobutane-type dimer formed by [2+2]-

cycloaddition of 9 and 10 at the coumaroyl double bonds. The

β-truxinate arrangement of the cyclobutane ring in compound

a,8a- and 7b,8b-cis-conﬁgurations and the 7a,7b- and 8a,8b-

trans-conﬁgurations.

The molecular formula of palhinoside F (6) was determined

as C H O on the basis of the HRESIMS (m/z 919.2661 [M

glucosyl unit (C H O ) than 4. Comparison of the NMR data

of 4 and 6 suggested that they are similar compounds. The

additional β-glucopyranosyl moiety proved to be connected to

46 20

was deduced by the NOESY data. The key NOESY

H] , calcd for 919.2655) data, which suggested one more

correlations between H-8a/8b and H-2a(6a)/2b(6b) con-

ﬁrmed the 7a,8a-trans-conﬁguration and the 7a,7b- and 8a,8b-

cis-conﬁgurations. From these data, the structure of palhinoside

D (4) was fully identiﬁed (Figure 1).

C-4′ by the HMBC correlation of H-1‴ (δ 5.04)/C-4′ (δ

Palhinoside E (5) was obtained as a yellowish amorphous

160.9). The relative conﬁguration of the core structure was

powder with the molecular formula C H O deduced from

36 15

its HRESIMS data. The 2D structure was the same as that of 4

according to its NMR data (Tables 2 and 3). The only

diﬀerence between 5 and 4 was the relative conﬁguration of

deﬁned as a β-truxinic-type by the NOESY experiment. The

structure of palhinoside F (6) was thus elucidated as shown in

Figure 1.

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Figure 4 , the protective eﬀects of compounds 4 and 5 were

Article

The molecular formula of palhinoside G (7) was determined

to be the same as 6 by the HRESIMS data. Interpretation of

the 1D and 2D NMR data showed that 7 and 6 possess the

same 2D structure. The conﬁguration of the cyclobutane ring

in 7 was determined to be the same as 5 by analysis of the

NOESY data (Figure 3).

According to the HRESIMS data (m/z 1479.3833 [M −

−

H] , calcd for 1479.3832), the molecular formula of 8 was

determined to be C H O , suggesting two more glucosyl

72 34

moieties (C H O ) than 1. The NMR data of 8 showed

20 10

similar signals to 1, indicating that they have similar structures.

In the H NMR spectra, four anomeric protons were observed

at δ 5.34/4.97 (each 1H, d, J = 7.0 Hz, H-1″) and 5.04/5.01

(

each 1H, d, J = 7.0 Hz, H-1‴), which indicated the presence

of four β-glucopyranosyl groups. The two additional β-

Figure 5. Inhibitory eﬀect of compound 4 on ROS generation in HT-

2 cells.

glucopyranosyl groups were connected to C-4′ (δ 161.3/

61.7) of the constituent apigenin units, as evidenced by the

HMBC correlation of H-1‴/C-4′. Moreover, the cyclobutane

moiety was established as a μ-truxinate arrangement by the key

NOESY correlations of H-8a/2b(6b), H-7a/7b, H-8a/8b, and

H-8b/2a(6a). Thus, the structure of palhinoside H (8) was

determined as shown in Figure 1.

using a Q-TOF mass spectrometer (Agilent 6540, CA, USA) and a

Thermo Scientiﬁc Q-Exactive Orbitrap mass spectrometer (Thermo

Fisher, USA). NMR spectra were recorded on a Bruker AV-400 MHz

spectrometer and a Bruker AV-500 MHz spectrometer (Bruker

Karlsruhe, Germany). Toyopearl HW-40c (TOYOPEARL TOSOH,

Tokyo, Japan) and Macroporous resin HP-20 (CangZhou Bon

Adsorber Technology Co. Ltd., Hebei, China) were used for open

column chromatography (CC). Semipreparative HPLC experiments

were carried out using an Agilent 1200 liquid chromatograph; the

columns used were preparative YMC Pack ODS-A columns (10 μm,

Flavonol diglucosidic truxinate esters (biginkgoside F) from

Ginkgo biloba leaves were reported to have a neuroprotective

eﬀect against Aβ₂₅₋₃₅-induced decreased cell viability of SH-

SY5Y neuroblastoma cells. Therefore, the new cyclodimeric

derivatives were tested for their neuroprotective activity against

L-glutamate-induced neurotoxicity in HT-22 cells. As shown in

250 × 10 mm, YMC Co. Ltd., Kyoto, Japan). All solvents were

analytical grade.

Plant Material. Whole herbs of P. cernua were collected in the

town of Wutong, Lingui district, Guangxi Province, People’s Republic

of China, in August 2013. A voucher specimen (No. 20130805) has

been deposited in the Xiangya School of Pharmaceutical Sciences,

Central South University, and was authenticated by Prof. Jin-Ping Li

(

Central South University, Changsha, China).

Extraction and Isolation. The air-dried whole herbs of P. cernua

15.0 kg) were extracted with 75% aqueous EtOH (×3) under reﬂux

(

to give a concentrated residue (0.8 kg). The residue was suspended in

H O and successively partitioned with petroleum ether, EtOAc, and

n-BuOH. The n-BuOH layer (130.0 g) was chromatographed over

macroporous resin (HP-20) with a gradient elution of H O and 10,

0, 50, 75, and 95% EtOH−H O to yield six fractions. The 50%

EtOH portion (21.0 g) was subjected to Toyopearl HW-40c CC

Figure 4. Eﬀect of compounds 1−8 against HT-22 cell damage

induced by L-glutamate.

[H O−MeOH (10:3 → 0:1), v/v] to yield 12 fractions (Fr. A−L).

Fraction E (0.2 g) was puriﬁed by semipreparative HPLC (MeCN−

H O, 25:75, v/v) to yield 1 (8.0 mg, t = 18.5 min) and 9 (20.0 mg, t

15.8 min). Fraction F (0.25 g) was further puriﬁed by Toyopearl

better than that of Trolox at concentrations ranging from 1.3

to 15 μM, while compounds 6 and 7 showed eﬀects similar to

Trolox. The rest of the compounds exerted weak protective

eﬀects. High concentration glutamate can induce overgenera-

tion of reactive oxygen species (ROS), resulting in the death of

HW-40c and preparative HPLC (MeCN−H O, 20:80, v/v) to yield

compounds 2 (7.5 mg, t = 35.2 min) and 3 (11 mg, t = 35.8 min).

Fraction G (2.1 g) was further separated by Toyopearl HW-40c CC

with 60% MeOH−H O isocratic elution to aﬀord 10 fractions (G1−

G10). Compounds 4 (3.2 mg, t = 23.3 min) and 5 (3.0 mg, t = 23.7

min) were obtained from fraction G4 (20 mg) by semipreparative

HPLC (MeCN−H O, 21:79, v/v). Compounds 6 (6.0 mg, t = 19.5

min) and 7 (5.5 mg, t = 20.0 min) were obtained from fraction G5

neuronal cell. It is generally known that ﬂavones blocked the

intracellular ROS accumulation by its antioxidant properties.

Therefore, we examined the eﬀects of compound 4 against the

glutamate-induced accumulation of intracellular ROS in HT-

(

25 mg) by semipreparative HPLC (MeCN−H O, 22:78, v/v).

Fraction H (1.3 g) was puriﬁed by a Toyopearl HW-40c column and

2 cells. Compound 4 eﬀectively decreased ROS generation in

semipreparative HPLC (MeCN−H O, 28:72, v/v) to aﬀord 8 (6.5

mg, t = 17.4 min) and 10 (8.0 mg, t = 20.8 min).

a dose-dependent manner (Figure 5), indicating that it could

be the mode of action of its neuroprotective eﬀect.

Palhinoside A (1): yellowish, amorphous powder; [α]

−16 (c

0.1, MeOH); UV (MeOH) λ (log ε) 271 (3.61), 324 (3.70) nm;

max

EXPERIMENTAL SECTION

■

for H and C NMR (DMSO-d

(+)-HRESIMS m/z 1157.2926 [M + H] (calcd for C H O ,

1157.2921).

) data, see Tables 1 and 3;

General Experimental Procedures. UV spectra were collected

on a Shimadzu UV-2450 spectrometer (Kyoto, Japan). Optical

rotations were recorded on a Jasco model 1020 polarimeter (Horiba,

Tokyo, Japan). ECD spectra were measured on a Chirascan

instrument (New Haven, CT, USA). HRESIMS data were obtained

53 24

Palhinoside B (2): yellowish, amorphous powder; [α]²⁵−27 (c

0.1, MeOH); UV (MeOH) λ (log ε) 271 (3.62), 319 (3.64) nm;

for H and C NMR (DMSO-d ) data, see Tables 1 and 3;

max

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Republic of China; orcid.org/0000-0001-8041-6185 ;

Article

(

+)-HRESIMS m/z 1319.3396 [M + H]⁺(calcd for C H O ,

1D ( H, ¹³C, and DEPT135 NMR) and 2D NMR

(

66 63 29

319.3450).

H− H COSY, HSQC, HMBC, and NOESY), ECD,

Palhinoside C (3): yellowish, amorphous powder; [α]²⁵_D−27 (c

and HRESIMS spectra of compounds 1−8 (PDF)

.1, MeOH); UV (MeOH) λ (log ε) 273 (3.68), 319 (3.59) nm;

max

for H and C NMR (DMSO-d ) data, see Tables 2 and 3;

(

+)-HRESIMS m/z 1319.3458 [M + H] (calcd for C H O ,

AUTHOR INFORMATION

Corresponding Authors

63 29

■

319.3450).

Palhinoside D (4): yellowish, amorphous powder; [α]²⁵_D−54 (c

Kang-Ping Xu − Xiangya School of Pharmaceutical Sciences,

Central South University, Changsha 410008, People’s Republic

of China; Email: xukp395@csu.edu.cn

.1, MeOH); UV (MeOH) λ (log ε) 268 (3.27), 328 (3.27) nm;

max

for H and C NMR (DMSO-d ) data, see Tables 1 and 3;

(

+)-HRESIMS m/z 757.2125 [M + H] (calcd for C H O ,

40 37 15

Gui-Shan Tan − National Institution of Drug Clinical Trial,

National Clinical Research Center for Geriatric Disorders,

Xiangya Hospital, and Xiangya School of Pharmaceutical

Sciences, Central South University, Changsha 410008, People’s

Email: tgs395@csu.edu.cn

57.2127).

Palhinoside E (5): yellowish, amorphous powder; [α]²⁵_D−31 (c

.1, MeOH); UV (MeOH) λ (log ε) 268 (3.25), 331 (3.26) nm;

max

for H and C NMR (DMSO-d ) data, see Tables 2 and 3;

(

+)-HRESIMS m/z 757.2120 [M + H] (calcd for C H O ,

40 37 15

57.2127).

Palhinoside F (6): yellowish, amorphous powder; [α]²⁵_D−28 (c

.1, MeOH); HPLC-UV (MeCN−H O) λ 268, 320 nm; for H

max

Authors

and C NMR (DMSO-d ) data, see Tables 1 and 3; (+)-HRESIMS

m/z 919.2661 [M + H] (calcd for C H O , 919.2655).

Jing Li − National Institution of Drug Clinical Trial and

National Clinical Research Center for Geriatric Disorders,

Xiangya Hospital, Central South University, Changsha 410008,

People’s Republic of China

Lei-Hong Tan − Xiangya School of Pharmaceutical Sciences,

Central South University, Changsha 410008, People’s Republic

Hui Zou − Xiangya School of Pharmaceutical Sciences, Central

South University, Changsha 410008, People’s Republic of

China

Zhen-Xing Zou − Xiangya School of Pharmaceutical Sciences,

Central South University, Changsha 410008, People’s Republic

Hong-Ping Long − Xiangya School of Pharmaceutical Sciences,

Central South University, Changsha 410008, People’s Republic

Wen-Xuan Wang − Xiangya School of Pharmaceutical Sciences,

Central South University, Changsha 410008, People’s Republic

Complete contact information is available at:

Ping-Sheng Xu − National Institution of Drug Clinical Trial

and National Clinical Research Center for Geriatric Disorders,

Xiangya Hospital, Central South University, Changsha 410008,

People’s Republic of China

Li-Fei Liu − Xiangya School of Pharmaceutical Sciences, Central

South University, Changsha 410008, People’s Republic of

China

47 20

Palhinoside G (7): yellowish, amorphous powder; [α]

−61 (c

.1, MeOH); HPLC-UV (MeCN−H O) λ 268, 320 nm; for H

max

and C NMR (DMSO-d ) data, see Tables 2 and 3; (+)-HRESIMS

m/z 919.2657 [M + H] (calcd for C H O , 919.2655).

47 20

Palhinoside H (8): yellowish, amorphous powder; [α]

−26 (c

.2, MeOH); UV (MeOH) λ (log ε) 274 (3.48), 317 (3.47) nm;

max

for H and C NMR (DMSO-d ) data, see Tables 2 and 3;

−

(

−)-HRESIMS m/z 1479.3833 [M − H] (calcd for C H O ,

71 34

479.3832).

Sugar Identiﬁcation. The identiﬁcation of a glucosidic moiety

was performed via the methods reported by Badral et al. Each new

isolate (1.0 mg) was hydrolyzed in 1 mL of HCl solution (5%) for 2 h

at 60 °C; each residue was mixed with 5 mg of L-cysteine methyl ester

and 10 μL of o-tolyl isothiocyanate in 0.5 mL of a pyridine solution.

After reaction, the mixtures were analyzed by HPLC (Cosmosil 5C -

AR-II, 4.6 × 250 mm; MeCN−H O (1:3, v/v) containing 0.2%

triﬂuoroacetic acid, 1.0 mL/min; detection at 250 nm). The retention

times of the peaks of standard L-glucose and D-glucose are 15.2 and

6.5 min, respectively. The glucosidic moieties in 1−8 were

determined to be D-glucose by their corresponding retention times

(

16.5 min).

Neuroprotective Activity Assay. The highly diﬀerentiated HT-

2 cells (purchased from Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences) were inoculated into 96-well plates at

a density of 900 cells/well in medium containing 10% fetal bovine

serum (FBS) and 1% double antibiotics (penicillin, streptomycin),

then cultured in 5% CO with a 100% humidiﬁed atmosphere at 37

C for 24 h. The test compounds and the positive control (Trolox)

https://pubs.acs.org/10.1021/acs.jnatprod.9b00470

were prepared in serial dilutions from 0.6 to 15 μM in serum-free

medium. Afterward, each well was supplemented with various

concentrations of test compounds for 24 h before glutamate

treatment. Cell viability was detected by luminescent cell viability

assay (Promega, No. 0000126138) after the L-glutamate (50 mM)

treatment for 24 h.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

Reactive Oxygen Species Assay. For measurement of ROS,

■

HT-22 cells (1 × 10 cells/mL in 96-well plates) were treated with 50

This work was ﬁnancially supported by the National Natural

Sciences Foundation of China (No. 31700303), the Key

Projects of Science and Technology Plan of Changsha (No.

kq1801072), and the Science and Technology Innovation

Platform and Talent Special Project of Hunan Province (No.

mM glutamate for 8 h in the presence or absence of the test

compounds. The cells were incubated with 10 μM 2′,7′-dichloro-

ﬂuorescein diacetate for 30 min. The cells were washed with

phosphate-buﬀered saline, and their ﬂuorescence was recorded at a

wavelength of 525 nm with 490 nm excitation wavelength

017TP2013). The authors gratefully acknowledge the NMR

(

SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA).

measurements from the Modern Analysis and Testing Centre

of CSU.

ASSOCIATED CONTENT

■

sı Supporting Information

REFERENCES

■

327.

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00470.

(1) Isaza, J. H.; Ito, H.; Yoshida, T. Phytochemistry 2001, 58, 321−

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(

2) El-Ansari, M. A.; Nawwar, M. A.; Saleh, N. A. Phytochemistry

995, 40, 1543−1548.

3) Chai, X. Y.; Song, Y. L.; Xu, Z. R.; Shi, H. M.; Bai, C. C.; Bi, D.;

Wen, J.; Li, F. F.; Tu, P. F. J. Nat. Prod. 2008, 71, 814−819.

4) Amaral, F. P.; Napolitano, A.; Masullo, M.; Santos, L. C.; Festa,

M.; Vilegas, W.; Pizza, C.; Piacente, S. J. Nat. Prod. 2012, 75, 547−

56.

5) Ma, G. L.; Xiong, J.; Yang, G. X.; Pan, L. L.; Hu, C. L.; Wang,

W.; Fan, H.; Zhao, Q. H.; Zhang, H. Y.; Hu, J. F. J. Nat. Prod. 2016,

9, 1354−1364.

6) Selenge, E.; Odontuya, G.; Murata, T.; Sasaki, K.; Kobayashi, K.;

Batkhuu, J.; Yoshizaki, F. J. Nat. Med. 2013, 67, 867−875.

7) Xu, J. F.; Zheng, X. P.; Liu, W. D.; Du, R. F.; Bi, L. F.; Zhang, P.

C. J. Asian Nat. Prod. Res. 2010, 12, 529−534.

8) Zhang, X. C.; Zhang, L. B. Flora of China; Science Press: Beijing,

004; Vol. 40, pp 69−72.

9) Xiao, P. G.Modern Chinese Material Medical; Chemistry and

Industry Press: Beijing, 2002; Vol. 3, pp 135−139.

10) Jiangsu College of New Medical. A Comprehensive Dictionary of

(

the Traditional Chinese Medicine; Shanghai Science and Technology

Press: Shanghai, 1996; pp 2394−2395.

(

11) Jiao, R. H.; Ge, H. M.; Shi, D. H.; Tan, R. X. J. Nat. Prod. 2006,

9, 1089−1091.

12) Li, J.; Xu, P. S.; Tan, L. H.; Zou, Z. X.; Wang, Y. K.; Long, H.

P.; Zhou, G.; Li, G.; Xu, K. P.; Tan, G. S. Fitoterapia 2017, 119, 45−

13) Dong, L. B.; Gao, X.; Liu, F.; He, J.; Wu, X. D.; Li, Y.; Zhao, Q.

S. Org. Lett. 2013, 15, 3570−3573.

14) Zhao, F. W.; Sun, Q. Y.; Yang, F. M.; Hu, G. W.; Luo, J. F.;

Tang, G. H.; Wang, Y. H.; Long, C. L. Org. Lett. 2010, 12, 3922−

925.

15) Tang, Y.; Xiong, J.; Zou, Y. K.; Zhang, H. Y.; Hu, J. F.

Phytochemistry 2016, 131, 130−139.

16) Zhang, D. B.; Chen, J. J.; Zhang, L.; Song, Q. Y.; Gao, K.

Phytochem. Lett. 2014, 10, 76−79.

17) Tang, Y.; Xiong, J.; Zou, Y. K.; Nay, B.; Wang, L. J.; Hu, J. F.

Chin. Chem. Lett. 2016, 27, 969−973.

18) Yan, J.; Sun, L.; Zhang, X.; Li, Z.; Zhou, L.; Qiu, M. H. Chem.

Pharm. Bull. 2009, 57, 1381−1384.

19) Yan, J.; Zhou, Z. Y.; Zhang, M.; Wang, J.; Dai, H. F.; Tan, J. W.

Planta Med. 2012, 78, 1387−1391.

20) Wei, J. J.; Wang, W. Q.; Song, W. B.; Xuan, L. J. Fitoterapia

018, 127, 151−158.

21) Weng, Y. M.; Yu, X.; Dong, Q. Z.; Li, F. S.; Cheng, F.; Zhang,

(

Y.; Yao, C. P.; Zou, Z. X.; Zhou, W. H.; Tan, G. S.; Xu, K. P.

Fitoterapia 2018, 128, 135−141.

(

22) Dong, Q. Z.; Zou, Z. X.; Jia, X. H.; Yu, X.; Li, J.; Zhou, W. H.;

Sun, H. H.; Wu, W.; Tan, G. S.; Xu, K. P. Bioorg. Chem. 2019, 87,

(

73−379.

23) Li, J.; Chen, Y. T. Acta Pharm. Sin. 2002, 37, 186−188.

24) Egerton, P. L.; Hyde, E. M.; Trigg, J.; Payne, A.; Beynon, P.;

Mijovic, M. V.; Reiser, A. J. Am. Chem. Soc. 1981, 103, 3859−3863.

25) Ben-Efraim, D. A.; Green, B. S. Tetrahedron 1974, 30, 2357−

364.

26) Badral, D.; Odonbayar, B.; Murata, T.; Munkhjargal, T.;

(

Tuvshintulga, B.; Igarashi, I.; Suganuma, K.; Inoue, N.; Brantner, A.

H.; Odontuya, G.; Sasaki, K.; Batkhuu, J. J. Nat. Prod. 2017, 80,

(

416−2423.

27) Tan, S.; Wood, M.; Maher, P. J. Neurochem. 1998, 71, 95−105.

28) Kling, B.; Bucherl, D.; Palatzky, P.; Matysik, F. M.; Decker, M.;

Wegener, J.; Heilmann, J. J. Nat. Prod. 2014, 77, 446−454.