New hexalactone derivatives and a pair of new oxaspiro-carbon epimeric glycosides from the fruits of Illicium lanceolatum

Article abstract of DOI:10.1016/j.bioorg.2019.103113

Five new compounds (1–5), including three hexalactone derivatives (1–3) and a pair of new oxaspiro-carbon epimeric glycosides (4 and 5), and six known compounds (6–11) were obtained from the fruits of Illicium lanceolatum. The structures of the new compounds were elucidated using extensive spectroscopic data. The absolute configurations of compounds 1–3 were determined by an analysis of their CD spectra. It was determined that compounds 4 and 5, which are epimeric at C-5, possess the same 1-oxaspiro[4,5]decane-7α,8α,9β-triol moiety. Plausible biogenetic pathways for 4 and 5 derived from the key precursor shikimic acid were proposed. Compounds 1–11 were all assayed on monosodium glutamate-induced human neuroblastoma SH-SY5Y cell damage. The results demonstrated that compounds 4, 5, and 8–10 possess potential neuroprotective effects. The anti-inflammatory, antiviral, and cytotoxic activities of 1–11 were also evaluated.

Full text of DOI:10.1016/j.bioorg.2019.103113

Accepted Manuscript
New hexalactone derivatives and a pair of new oxaspiro-carbon epimeric gly-
cosides from the fruits of Illicium lanceolatum
Yang-Lan Liu, Xiao-Jing Wang, Ru-Bing Wang, Mi Li, Wen-Rui Li, Jian-Pei
Zhang, Xiu-Qi Bao, Dan Zhang, Shuang-Gang Ma
PII:
S0045-2068(19)30725-4
https://doi.org/10.1016/j.bioorg.2019.103113
103113
DOI:
Article Number:
Reference:
YBIOO 103113
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 May 2019
24 June 2019
8 July 2019
Please cite this article as: Y-L. Liu, X-J. Wang, R-B. Wang, M. Li, W-R. Li, J-P. Zhang, X-Q. Bao, D. Zhang, S-
G. Ma, New hexalactone derivatives and a pair of new oxaspiro-carbon epimeric glycosides from the fruits of
Illicium lanceolatum, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103113
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New hexalactone derivatives and a pair of new oxaspiro-carbon epimeric glycosides from the fruits
of Illicium lanceolatum
Yang-Lan Liu†, Xiao-Jing Wang†, Ru-Bing Wang, Mi Li, Wen-Rui Li, Jian-Pei Zhang, Xiu-Qi Bao, Dan Zhang, Shuang-Gang
Ma
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of MateriaMedica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Corresponding author. Tel.: +86-10-63165324; fax: +86-10-63017757; e-mail: mashuanggang@imm.ac.cn
†These authors contributed equally to this work.
1

New hexalactone derivatives and a pair of new oxaspiro-carbon epimeric glycosides from the fruits
of Illicium lanceolatum
Yang-Lan Liu†, Xiao-Jing Wang†, Ru-Bing Wang, Mi Li, Wen-Rui Li, Jian-Pei Zhang, Xiu-Qi Bao, Dan Zhang, Shuang-Gang
Ma
ABSTRACT
Five new compounds (1–5), including three hexalactone derivatives (1–3) and a pair of new oxaspiro-carbon epimeric
glycosides (4 and 5), and six known compounds (6–11) were obtained from the fruits of Illicium lanceolatum. The structures of
the new compounds were elucidated using extensive spectroscopic data. The absolute configurations of compounds 1-3 were
determined by an analysis of their CD spectra. It was determined that compounds 4 and 5, which are epimeric at C-5, possess
the same 1-oxaspiro[4.5]decane-7α,8α,9β-triol moiety. Plausible biogenetic pathways for 4 and 5 derived from the key
precursor shikimic acid were proposed. Compounds 1–11 were all assayed on monosodium glutamate-induced human
neuroblastoma SH-SY5Y cell damage. The results demonstrated that compounds 4, 5, and 8-10 possess potential
neuroprotective effects. The anti-inflammatory, antiviral, and cytotoxic activities of 1–11 were also evaluated.
Key words: Illicium lanceolatum; hexalactone derivatives; oxaspiro carbon epimers; glycosides; neuroprotective activity
Corresponding author. Tel.: +86-10-63165324; fax: +86-10-63017757; e-mail: mashuanggang@imm.ac.cn
†These authors contributed equally to this work.
2

1. Introduction
The genus Illicium, the sole genus currently classified in the family Illiciaceae, are widely distributed in southeastern Asia
and America with more than 40 species.[1] In the phytochemical studies of those species, the unique seco-prezizaane
sesquiterpenes and prenylated C6-C3 compounds were the focus.[2, 3] Notably, the fruits of the plants of the genus Illicium are
a rich source of (-)-shikimic acid possessing useful inherent carbon chirality and considered an excellent chiral precursor for
biosynthesis and chemosynthesis (e.g., in Tamiflu).[4-6]
Illicium lanceolatum A. C. Smith is a folk medicinal plant with the Chinese name ‘Mangcao’.[7] Its roots and root bark have
historically been used as folk medicine to treat bruises, internal injuries, and rheumatoid osteoarthritis.[8] Previous
investigations of the plant resulted in the isolation of menthane monoterpenes, germacrane sesquiterpenes, santalane
sesquiterpenoids and tetranor sesquiterpenoids.[9-11] In our research on the bioactive compounds from the fruits of
I.lanceolatum, five new compounds, including three hexalactone derivatives, (4S,6R)-4-hydroxy-4,6-dimethyltetrahydro-2H-
pyran-2-one (1, lanceolactone A), (4S,6R)-4-O-β-D-glucopyranosyl-4,6-dimethyltetrahydro-2H-pyran-2-one (2,
lanceolactonoside A), and methyl 2-((2S,4R)-4-hydroxy-4-methyl-6-oxotetrahydro-2H-pyran-2-yl)acetate (3, lanceolactone B),
and a pair of structurally unique epimeric1-oxaspiro[4,5]decane glycosides, 5S*-(7α,8α,9β)-8-O-β-D-glucopyranosyl-7,9-
dimethoxy-1-oxaspiro[4,5]dencane (4, spirolancinoside A) and 5R*-(7α,8α,9β)-8-O-β-D-glucopyranosyl-7,9-dimethoxy-1-
oxaspiro[4,5]decane (5, spirolancinoside B), together with six known compounds (6-11), have been isolated from the H2O-
soluble fraction of the 95% ethanol extract of the fruits of I. lanceolatum. Notably, compounds 4 and 5 were the first examples
of 1-oxaspiro[4,5]decane glycosides and possess the same 1-oxaspiro[4,5]decane-7α,8α,9β-triol moiety, and they could be
biosynthesized from the key precursor (-)-shikimic acid, which has been obtained on a kilogram scale from the H2O-soluble
fraction. Additionally, the neuroprotective, anti-inflammatory, antiviral, and cytotoxic activities of 1–11 were evaluated.
2. Materials and Methods
2.1. General experimental procedures
The UV data were measured on a JASCO V-650 spectrophotometer, and the CD spectra were obtained on a JASCO J-815
spectrophotometer. Optical rotations were recorded on a Rudolph Autopol V automatic polarimeter at 20℃. IR spectra were
recorded on a Thermo Nicolet 5700 spectrometer through FT-IR Microscope Transmission. NMR spectra were obtained on a
VNS-600 spectrometer for 1D NOE and a BRUKER-AV-Ⅲ-500 spectrometer for 1H, 13C, 1H-1H COSY, HSQC, HMBC,
ROESY and TOCSY. Chemical shifts are given in δ (ppm) with solvent signals (CD3OD: δH 3.31/ δC 49.0) as references. LC-
MS and HRESIMS data were recorded on a Thermo Fisher Scientific Q-Exactive Focus Orbitrap LC-MS/MS spectrometer.
Preparative HPLC was performed on a Shimadzu LC-6AD instrument with a dual detector of Shimadzu SPD-20A and
Shimadzu RID-20A, using a CAPCELL PAK C-18 column (10mm × 250mm, 5 μm). ODS (50 μm, YMC.GEL ODS-A-HG,
Japan), Sephadex LH-20 (GE Healthcare, Sweden), polyamide (30 – 60 mesh, Sinopharm Chemical Reagent Co., China),
macroporous resin (D101, Zhengzhou Qinshi Technology Co., China) and silica gel (160 – 200, 200 – 300 mesh, Qingdao
Marine Chemical Factory, China) were used for column chromatography. HSGF254 (Yantai Institute of Chemical Industry) was
used for TLC. Snailase (Yuanye Biotechnology Co., Ltd., Shanghai, China) was used for hydrolysis.
3

2.2. Plant material
Fruits of Illicium lanceolatum A. C. Smith were collected from Guilin of Guangxi Province, China, in October 2012 and
identified by Prof. Lin Ma (Institute of Materia Medica, Chinese Academy of Medical Sciences). A voucher specimen (ID-S-
2832) was deposited in the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy
of Medical Sciences.
2.3. Extraction and isolation
The air-dried, pulverized fruits of I. lanceolatum (100 kg) were extracted three times with 95% EtOH, and the solvent was
evaporated in vacuo to obtain 19.8 kg of residue. The residue was suspended in H2O and extracted with EtOAc to yield an
EtOAc-soluble part (3.2 kg) and a H2O-soluble part (2.2 kg). The H2O-soluble part was chromatographed on a D101
macroporous resin column, eluting with gradient EtOH-H2O (0%, 30%, 60% and 95% EtOH). The 30% EtOH fraction (625 g)
was subjected to a column of polyamide (6 L) with gradient elution using H2O, 30% EtOH, 60% EtOH and 95% EtOH. Then,
the H2O fraction (400 g) was chromatographed on the D101 macroporous resin column (5 L) again, using the gradient EtOH-
H2O as a mobile phase. This 30% EtOH fraction (220 g) was separated on silica gel (3.5 kg) column chromatography using
CH2Cl2-MeOH (30:1 to 1:1, with 5‰ HCOOH) to yield twenty-three fractions (G1-G23). Fraction G4 (1.31 g) was separated
by column chromatography (CC) on silica (30 g) with CH2Cl2/MeOH (30:1) to afford seven subfractions, of which subfraction
5 (462 mg) was purified by HPLC using 15% CH3CN-H2O to yield 1 (50 mg, tR = 13 min) and 3 (59 mg, tR = 19 min). Fraction
G12 (5 g) was separated by column chromatography on Sephadex LH-20 with MeOH and gave seven subfractions.
Subfraction 5 (1.3 g) was purified by HPLC to yield 7 (7 mg, tR = 26 min) with 17% CH3CN-H2O and 6 (662 mg, tR = 15 min)
with 13% CH3CN-H2O. Fraction G16 (21 g) was separated on a column of Sephadex LH-20 and eluted with CH3OH to
provide seven fractions. Fraction 3 (12.4 g) afforded five subfractions upon being passed through another Sephadex LH-20
column with H2O as the mobile phase. Subfraction 1 (700 mg) was further purified by HPLC to yield 4 (21 mg, tR = 35 min)
and 5 (57 mg, tR = 51 min) with 10% CH3CN-H2O (with 5‰ TFA) and 2 (31 mg, tR = 21 min) with 5% CH3CN-H2O (with 5‰
TFA). Subfraction 2 (7.6 g) was subjected to an ODS column with a gradient of MeOH-H2O (with 5‰ TFA) and then purified
by HPLC to give 10 (34 mg, tR = 46 min) and 11 (3 mg, tR = 51 min) with 25% MeOH-H2O (with 0.1% TFA), 9 (10 mg, tR = 70
min) with 20% MeOH-H2O (with 0.1% TFA) and 8 (20 mg, tR = 41 min) with 30% MeOH-H2O (with 0.1% TFA).
2.4. Lanceolactone A (1)
20
A colorless oil, [α]D − 41.2 (c 0.94, MeOH); IR: vmax 3422, 2979, 2935, 1725, 1458, 1384, 1256, 1084, 1024, 984 cm-1; CD
(MeOH) max (Δε) 213 (− 1.54) nm; 1H NMR (500 MHz, Methanol-d4) and 13C NMR (125 MHz, Methanol-d4) data, see Table
1; ( + )-HRESIMS m/z 145.08586 [M + H]+ (calcd for C7H13O3, 145.08592).
2.5.Lanceolactonoside A (2)
20
A colorless oil, [α]D − 15.2 (c 0.77, MeOH); IR: vmax 3383, 2978, 2922, 1727, 1450, 1387, 1264, 1077, 1041, 865, 813 cm-
1; CD (MeOH) max (Δε) 212 (− 1.03) nm;1H NMR (600 MHz, Methanol-d4) and 13C NMR (150 MHz, Methanol-d4) data, see
Table 1; ( + )-HRESIMS m/z 329.12054 [M + Na]+ (calcd for C13H22O8Na, 329.12069).
Compound 2a: Compound 2 (4mg) was stirred in H2O (500 μl) with snailase (20 mg) at 37 °C for 3 d, and then the
hydrolysate was extracted with EtOAc to afford 2a as a colorless oil; 1H NMR (Methanol-d4, 500 MHz) δH 4.47 (1H, m, H-6),
4

2.69 (1H, d, J = 16.0 Hz, H-3a), 2.47 (1H, d, J = 16.0 Hz, H-3b), 2.07 (1H, dd, J = 14.0, 3.5 Hz, H-5α), 1.71 (1H, dd, J = 14.0,
11.5 Hz, H-5β), 1.37 (3H, d, J = 6.0 Hz, H-7), 1.35 (3H, s, H-8); ( + )-HRESIMS m/z 145.08585 [M + H]+ (calcd for C7H13O3,
145.08592).
2.6.Lanceolactone B (3)
20
A colorless oil, [α]D + 10.0 (c 1.27, MeOH); IR: vmax 3436, 2962.1, 1737, 1440, 1384, 1259, 1025 cm-1; CD (MeOH) max
(Δε) 221 (− 0.86) nm; 1H NMR (500 MHz, Methanol-d4) and 13C NMR (125 MHz, Methanol-d4) data, see Table 1; ( + )-
HRESIMS m/z 203.09138 [M + H]+ (calcd for C9H15O5, 203.09140).
2.7. Spirolancinoside A (4)
20
A colorless oil; [α]D − 10.0 (c 4.01, MeOH); IR: vmax 3385, 2930, 2885, 1679, 1452, 1365, 1085, 827 cm-1; 1H NMR (600
MHz, Methanol-d4) and 13C NMR (150 MHz, Methanol-d4) data, see Table 2; ( + )-HRESIMS m/z 401.17743 [M + Na]+ (calcd
for C17H30O9Na, 401.17820).
2.8. Spirolancinoside B (5)
20
A colorless oil; [α]D − 22.0 (c 0.81, MeOH); IR: vmax 3393, 2925, 2878, 1679, 1450, 1365, 1076, 801 cm-1; 1H NMR (600
MHz, Methanol-d4) and 13C NMR (150 MHz, Methanol-d4) data, see Table 2; ( + )-HRESIMS m/z 401.17780 [M + Na]+ (calcd
for C17H30O9Na, 401.17820).
2.9. Hydrolysis and determination of the absolute configuration of the sugar moieties of compounds 2, 4, and 5[12]
Compound 2 (4 mg) was stirred in H2O (500 μL) with snailase (20 mg, Yuanye Biotechnology Co., Ltd., Shanghai, China)
at 37 °C for 3 d. Then, the solution was extracted with EtOAc five times, and MeOH was added into the aqueous layer for the
precipitation of the snailase. After evaporation under reduced pressure and drying in vacuo, the aqueous layer yielded a
monosaccharide residue.
The residue was dissolved in pyridine (2 mL), and L-cysteine methyl ester hydrochloride was added. The mixture was
maintained at 60 °C for 2 h and evaporated and dried in vacuo. Then, the residue was reacted with N-trimethylsilylimidazole
(for gas chromatography, 500 μL) at 60 °C for 2 h, and the solution was transferred into H2O (2 mL) and extracted with n-
hexane (2 mL) five times to give the trimethylsilylated derivative of the monosaccharide. A standard D-glucose derivative was
prepared in a similar way. Finally, the derivative of the monosaccharide was analyzed by GC-MS and compared with the
standard D-glucose derivative under the following conditions. Capillary column: SE-54 (30 m × 0.25 mm, i.d.); detection: FID;
initial temperature: 200°C; final temperature: 280 °C (10 °C/min) for 35 min; carrier: N2 gas. The retention times of the
monosaccharide derivative and standard D-glucose derivative were the same, 29.4 min. The monosaccharide absolute
configurations of compounds 4 and 5 were determined in the same way.
2.10. Bioactivity assay
Neuroprotective Assay. Human neuroblastoma SH-SY5Y cells were cultured in 96-well plates at a concentration of 8×10-3
for 24 h. Then, mixtures of monosodium glutamate without and with the test compounds were added to the culture medium to
give final concentrations of 120 mM and 10 μM, respectively. All the experiments were carried out in triplicate and incubated
for 24 h. Then, 100 μL MTT (0.5 mg/mL) was added to each well, and the plates were incubated for 4 h at 37 °C. Finally, the
absorbances at 570 nm were measured after adding 150 μL DMSO.
5

Protection rate % = (OD test group – OD model group) ∕ (OD control group – OD model group) × 100%
According to previously published methods, the cytotoxic,[13] anti-inflammatory,[14] and antiviral activities[15] of all
compounds were assayed.
3. Results and discussion
Compound 1 was obtained as a colorless oil. Its molecular formula, C7H12O3, was assigned based on the HRESIMS and
NMR data. Its IR spectrum showed typical absorption bands for OH (3422 cm−1) and carbonyl (1725 cm−1) groups. The 13C
(Table 1) and HSQC NMR data displayed seven carbons, including a carbonyl (δC 174.6), a tertiary oxygenated carbon (δC
69.4), an oxygenated methine (δC 75.1), two methylenes (δC 45.9 and 45.2), and two methyls (δC 29.4 and 21.4). These 13C
NMR data were similar to those of 3,6-dimethyl-3-hydroxy-tetrahydro-2H-pyran-2-one.[16] A careful comparison of their
NMR data revealed that a hydroxyl and a singlet methyl (δH 1.35) were both located at the C-4 of the hexalactone ring instead
of C-3 similar to in the latter, which is supported by the HMBC correlations from H-8 to C-3, C-4, and C-5 in the HMBC
spectrum. The planar structure of 1, as shown in Fig. 1, was further confirmed by the key HMBC correlations from H-6 to C-2
and C-7 and from CH3-7 to C-5 and C-6 (Fig. 2).
The relative configuration of 1 was determined by the NOE experiment, and the absolute configuration was elucidated by
the circular dichroism (CD). The NOE correlation from H-6 to H-8 indicated that H-6 and CH3-8 were on the same face of the
six-membered lactone ring (Fig. 3). The CD spectrum showed a negative Cotton effect at 213 nm for the n→π* transition of
the lactone group, indicating that the C-β of the lactone carbonyl was below the C-CO-O-C lactone plane (Fig. 4) based on
Beecham’s rules[17, 18]. Based on the above mentioned relative configuration and the α-oriented C-β of the lactone ring, the
absolute configuration of 1 was established as 4S,6R. Thus, the structure of compound 1was assigned as (4S,6R)-4-hydroxy-
4,6-dimethyltetrahydro-2H-pyran-2-one, and it was named lanceolactone A.
8
7
7
(R)
9
HO
(S)
6
8
O
8
(R)
7
(S)
6
(R)
4
4
10
O
HO
4 (S)
5'
6
HO
O
O
HO
HO
1'
O
O
1
O
1
2
2
2
OH
3'
O
O
O
1
3
2
12
12
H3CO
H3CO
HO
HO
7
9
7
9
6
6
5
4
3
O1
O
O
2
5
2
O
O
HO
HO
HO
8
8
O
1
HO
3
4
10
10
OH
OH
H3CO
H3CO
11
11
4
5
O
HO
COOCH3
H3CO
O
OH
HO
HO
HO
O
O
HO
HO
O
O
HO
OH
O
HO
OCH3
OH
OH
8
7
6
O
O
OH
HO
O
HO
HO
OH
OH
O
OH
O
O
HO
HO
HO
OH
OH
O
O
O
OH
9
11
10
6

Fig. 1. Chemical structures of compounds 1–11.
HO
O
O
HO
O
HO
HO
HO
O
O
O
O
OH
O
O
O
3
2
1
HO
HO
O
O
O
O
O
HO
HO
O
O
HO
O
HO
OH
OH
O
O
4
5
1H-1H COSY and TOCSY
HMBC
Fig. 2. Key HMBC correlations of compounds 1–5 and 1H-1H COSY and TOCSY of compounds 4 and 5.
H
H
H
-
-
-
6
6
6
C
H3
-
8
C
H3
4
-
-
8
O
H
2
3
1
Fig. 3. Key NOE and ROESY correlations of compounds 1–3.
+
-
+
-
β
β
1
3
Fig. 4. The Beecham’s rules for lactones of compounds 1 and 3.
Compound 2 was a colorless oil with the molecular formula C13H22O8, as determined by the HRESIMS and NMR data. A
glucopyranosyl anomeric proton δH 4.46 (1H, d, J = 7.8 Hz, H-1') was observed in the 1H NMR spectrum (Table 1). The 13C
NMR spectrum showed 13 carbon signals, including six carbons (δC 98.5, 78.1, 77.8, 74.9, 72.0, 63.4) of a glucopyranosyl
moiety (Table 1). Aside from the hydrogen and carbon signals of the glucose unit in the 1H and 13C NMR, the remaining NMR
data of 2 were very similar to those of 1, suggesting that 2 might be a glycoside of 1. The aglycone (compound 2a) and
7

monosaccharide were obtained by snailase hydrolysis. The HRESIMS and 1H NMR data of 2a were the same as those of 1
(Supporting Information, Fig. S17, S18), and the monosaccharide was determined to be β-D-glucose by the coupling constant
(J = 7.8 Hz) of anomeric proton together with the GC analysis of its trimethylsilyl L-cysteine derivative.[12] Additionally, in
the HMBC spectrum, an HMBC correlation was observed from the glucose anomeric proton (δH 4.46) to the tertiary
oxygenated carbon (δC 76.7), indicating that the β-D-glucose moiety was located at the C-4 of the lactone ring (Fig. 2). The
absolute configuration (4S,6R) of 2 was determined by the NOE correlation from H-6 to H-8 and the negative Cotton effect at
212 nm in the CD spectrum, similar to in 1. On the basis of these data, compound 2 was determined as (4S,6R)-4-O-β-D-
glucopyranosyl-4,6-dimethyltetrahydro-2H-pyran-2-one and named lanceolactonoside A.
Table 1
1H and 13C NMR data in MeOH-d4 for 1–3.
1 a
2 b
δC
3 a
δC
position
δC
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
2
3
174.6
45.2
175.2
42.7
173.0
44.3
Hα 2.69 d (16.0)
Hα 2.82 d (15.6)
2.54 br s (2H)
Hβ 2.47 d (16.0)
Hβ 2.66 d (15.6)
4
5
69.4
45.9
76.7
45.2
68.3
41.1
Hα 2.07 dd (14.5, 4.0)
Hβ 1.71 dd (14.5, 12.0)
4.45 m
Hα 2.15 dd (14.4, 3.6)
Hβ 1.94 dd (14.4, 11.4)
4.50 m
Hα 1.94 dt (14.0, 3.0)
Hβ 1.77 dd (14.0, 12.0)
5.07 dtd (12.0, 6.5, 3.0)
2.72 d (6.5)
6
7
75.1
21.4
29.4
75.0
21.3
26.3
75.4
40.9
172.2
29.5
52.3
1.37 d (6.5)
1.38 d (6.6)
8
1.35 s
1.45 s
9
1.31 s
3.71 s
10
1'
2'
3'
4'
5'
6'
98.5
74.9
78.1
72.0
77.8
63.4
4.46 d (7.8)
3.12 dd (9.0, 7.8)
3.35 dd (9.0, 9.0)
3.18 dd (10.2, 9.0)
3.29 dd (7.2, 2.4)
3.83 dd (10.0, 2.4)
3.56 dd (10.0, 7.2)
a 1H NMR data were measured at 500MHz, 13C NMR data were measured at 125MHz.
b 1H NMR data were measured at 600MHz, 13C NMR data were measured at 150 MHz.
Compound 3 was isolated as a colorless oil. The molecular formula of 3 was determined as C9H14O5 from HRESIMS and
13C NMR data. A careful analysis of the 1H and 13C NMR data (Table 1) implied the similarity of the NMR data of 3 to those
of 1, and the differences between them were the disappearance of one doublet methyl signal (CH3-7) along with the presence
of a set of NMR signals of the 2-methoxy-2-oxoethyl group (CH3-O-CO-CH2) in 3 (Table 1). It was suggested that the 2-
methoxy-2-oxoethyl group was located at the C-6 of the lactone ring, which is supported by the key HMBC correlations from
H-7 to C-5, C-6, and C-8 and from CH3-10 (δH 3.71) to C-8.
8

Due to no NOE correlations between CH3-9 and H-6 or 2-methoxy-2-oxoethyl group were observed in the NOE difference
experiment (in MeOH-d4), a ROESY experiment on 3 was carried out in dry acetone-d6. In the ROESY spectrum, the observed
ROESY correlation from H-6 to 4-OH (Supporting Information, Fig. S26) revealed that 4-OH and H-6 were on the same side
of the lactone ring (Fig. 3). Additionally, the negative Cotton effect at 220 nm indicated that C-β was below the C-CO-O-C
lactone plane (Fig. 4). Therefore, the absolute configuration of 3 was established as 4R,6S. Thus, the structure of compound 3
was characterized as methyl 2-((2S,4R)-4-hydroxy-4-methyl-6-oxotetrahydro-2H-pyran-2-yl)acetate, and it was named
lanceolactone B.
Compound 4 was obtained as a colorless oil. Its molecular formula, C17H30O9 with 3 degrees of unsaturation, was
determined from 13C NMR data and the HRESIMS ion at m/z 401.17780 [M + Na]+. The 1H NMR spectrum of 4 indicated a β-
glucopyranosyl anomeric proton at δH 4.48 (1H, d, J = 7.8 Hz),The 13C NMR data (Table 2) showed 17 carbon signals,
including six carbons ascribed to an O-glucose unit that was confirmed by 1D TOCSY, HSQC, and HMBC data (Supporting
Information, Fig. 33). The monosaccharide, obtained by snailase hydrolysis, was determined to be β-D-glucose by GC analysis
in comparison with the trimethylsilyl L-cysteine derivative of standard glucose.[12]
Additionally, three oxymethine protons [δH 4.08 (dd, J = 4.2, 3.0 Hz, H-8), 3.84 (ddd, J = 4.2, 4.2, 4.2, H-9), 3.52 (ddd, J =
10.8, 4.2, 3.0, H-7)], one oxymethylene proton [δH 3.78 (t, J = 6.6 Hz, H-2)] and two oxymethyl protons [δH 3.39 (s, H-12),
3.36 (s, H-11)] were observed in the 1H NMR spectrum. In the high-field region (δH 2.00 – 1.75), eight overlapped proton
signals were unambiguously ascribed to four methylenes (δC 37.8, 37.7, 36.0, 26.8) by the aid of 1D TOCSY and HSQC data
(Table 1). Additionally, from a careful analysis of the 1H-1H COSY and 1D TOCSY spectra, two spin systems [CH2(2)-
CH2(3)-CH2(4) and CH2(6)-CH(7)-CH(8)-CH(9)-CH2(10)] were established (Fig. 2). In the HMBC spectrum, the correlations
from H-6/7/9 to the oxygenated quaternary carbon δC 83.6 (C-5) and from H-2/4 to C-5 suggested that 4 possesses a 1-
oxaspiro[4,5]decane skeleton (Fig. 2). These data suggest that 4 is a 1-oxaspiro[4,5]decane glycosidation derivative. The
HMBC correlations from H-8 to C-1′ and from H-1′ to C-8 indicate that the glucose moiety is located at C-8, and the
correlations from CH3-12 to C-7 and from CH3-11 to C-9 reveal that the two oxymethyls are located at C-7 and C-9,
respectively.
The relative configuration of hexatomic ring was determined based on the coupling constant of 3JH-H (Fig. 5). In the 1H NMR
spectrum, the large coupling constant between H-7 and H-6α (J = 10.8 Hz) and small coupling constant between H-7 and H-6β
(J = 4.2 Hz) suggest that H-7 is facing in the axial direction. An analysis of the coupling constants of H-9 (JH-9, H-10α = 4.2 Hz
and JH-9, H-10β = 4.2 Hz) indicates that H-9 is located at the equatorial bond, Similarly, H-8 was determined to be on the
equatorial bond by its two small coupling constants (JH-8, H-7 = 3.0 Hz, JH-8, H-9 = 4.2 Hz). These coupling constant data indicate
that the six-member carbon ring is in the pseudochair conformation. Furthermore, the relative configuration of the spiro carbon
(C-4) was determined by NOE experiments. The irradiation of H-7 enhanced H-4, indicating that C-4 and H-7 were on the
same side of the hexatomic ring, as shown in Fig. 5. Thus, the structure of compound 4 was determined to be 5S*-(7α,8α,9β)-8-
O-β-D-glucopyranosyl-7,9-dimethoxy-1-oxaspiro[4,5]decane, and it was named spirolancinoside A.
9

H
Ha
Hb
3
H
H
H
4
5
H
H
7
2
8
6
OCH3
10
H
1
GlcO
H3CO
O
H3CO
9
7
O
6
5
8
H
OCH3
H
H
9
H
10
H
H
H
GlcO
H
H
J
J
J
H-7, H-6 = 10.8 Hz JH-7, H-6 = 4.2 Hz
J
J
J
H-7, H-6 = 9.6 Hz JH-7, H-6 = 3.6 Hz
H-8, H-7 = 3.0 Hz H-8, H-9 = 5.4 Hz
H-8, H-7 = 3.0 Hz
J
J
H-8, H-9 = 4.2 Hz
J
H-9, H-10 =4.2 Hz
H-9, H-10 = 4.2 Hz
H-9, H-10 = 4.2 Hz JH-9, H-10 = 4.8 Hz
4
5
Fig.5. Key coupling constants of pseudochair conformation hexatomic ring and ROESY correlations of compounds 4 and 5.
Table 2 1H NMR (600 MHz) and 13C NMR (150 MHz) data in MeOH-d4 for 4 and 5.
4
5
position
δC
δH (J in Hz)
δC
δH (J in Hz)
2
3
4
67.2
26.8
37.7
3.78 t (6.6)
2.00 m b
68.1
26.1
39.5
3.82 t (7.2)
1.93 m a
Ha 2.00 mb
Hb 1.79 m a
1.80 m a
5
6
83.6
37.8
83.6
37.0
Hα 1.95 dd (12.0, 10.8) a
Hβ 1.75 m a
Hα 1.92 dd (13.2, 9.6) a
Hβ 1.74 dd (13.2, 3.6) a
3.76 mb
7
8
78.2
76.7
79.4
36.0
3.52 ddd (11.4, 4.2, 3.0)
4.08 dd (4.2, 3.0)
3.84 ddd (4.2, 4.2, 3.6)
Ha 1.88 dd (14.4, 3.6) a
Hb 1.75 m a
77.2
78.1
79.6
36.4
4.07 dd (5.4, 3.0)
3.74 mb
9
10
Ha 1.87 dd (14.4, 4.2) a
Hb 1.72 dd (14.4, 4.8) a
3.38 s
11
12
1'
57.6
56.8
3.36 s
57.5
57.0
104.5
75.4
77.9
71.6
77.9
62.8
3.39 s
3.40 s
105.2
75.5
78.0
71.6
77.9
62.8
4.48 d (7.8)
4.47 d (7.8)
2'
3.21 dd (9.0, 7.8)
3.28 m
3.20 dd (9.6, 7.8)
3.28 m
3'
4'
3.26 m
3.27 m
5'
3.35 m
3.35 m
6'
3.86 dd (12.0, 1.2)
3.65 m
3.86 dd (12.0, 1.8)
3.66 m
a Assigned by TOCSY spectrum
b Overlapped
Compound 5 has the same molecular formula as 4, and the NMR data of 5 also resembled those of 4. The structure was
elucidated in the same manner as for 4. A careful analysis of the 1D TOCSY, 1H-1H COSY, HSQC, and HMBC correlations
allowed us to conclude that compounds 4 and 5 possessed the same plane structure (Fig. 2 and Supporting Information, Fig.
S43). The relative configuration of the hexatomic ring was established to be the same as that of compound 4 by an analysis of
these 3JH-H coupling constants, as shown in Fig. 5. A comparison of the JH-8, H-7 and JH-8, H-7 coupling constants and the chemical
10

shift of C-8 of 5 with those of 4, showing a larger coupling constant (JH-8, H-9 = 5.4 Hz) and lower field chemical shift of C-8
(δC78.1) indicated that H-8 was in a pseudoaxial orientation, which was supported by the NOE correlations from H-8 to H-6
and H-10 (Supporting Information, Fig. S46). In the ROESY spectrum, the key correlation from CH3-12 to H-4 suggested that
the spiro carbon C-4 and 7-OCH3 were on the same side of the hexatomic ring, as shown in Fig. 5. Thus, the structure of
compound 5 was determined to be 5R*-(7α,8α,9β)-8-O-β-D-glucopyranosyl-7,9-dimethoxy-1-oxaspiro[4,5]decane, and it was
named spirolancinoside B.
Scheme 1. Possible biosynthesis pathways of compounds 4 and 5.
O
OH
O
O
O
CoAS
HO
O
O
Aldol
condesation
SCoA
SCoA
[ H ]
OH
OH
CoA ligase
Hydrolysis
HO
OH
HO
OH
HO
OH
OH
HO
OH
(-)-shikimic acid
OH
a
OH
OH
OH
b
O
H3CO
O
O
HO
O
HO
[ H ]
O
Methylation
[ O ]
O
O
- H2O
OH
5
HO
HO
OH
4
Glycosylation
H3CO
HO
OH
OH
HO
HO
OH
OH
5
and
OH
OH
It is interesting that compounds 4 and 5, which are epimeric at C-4, feature a 7α/8α/9β-oxygen-bearing substituted 1-
oxaspiro[4,5]decane structural unit, which suggests that they are derived from a common biosynthesis precursor of naturally
occurring (-)-shikimic acid, which was also isolated in large quantities from the fruits of this plant, and their proposed
biosynthetic pathways are shown in Scheme 1. Activation of shikimic acid by CoA ligase and subsequent aldol condensation
could lead to the formation of a β-keto ester a, following by reduction and hydrolysis to give a carboxylic acid b.
Subsequently, b undergo oxidation, hydration, reduction, methylation, and glycosylation could produce a pair of
unprecedented oxaspiro-carbon epimeric glycosides (4 and 5).[19]
In addition to the five new compounds (1–5), six known compounds were also isolated from the H2O-soluble fraction (Fig.
1). They were identified asethyl shikimate (6),[20] methyl syringate 4-O-β-D-glucopyranoside (7),[21] 2-trans-2,6-dimethyl-6-
hydroxyocta-2,7-dienoic acid β-glucopyranosyl ester (8),[22] 10-hydroxy-trans-linalyl oxide 7-O-β-D-glucopyranoside (9),[23]
(1R,2R,4S)-trans-2-hydroxy-1,8-cineole β-D-glucopyranoside (10), and (1S,2S,4R)-trans-2-hydroxy-1,8-cineole β-D-
glucopyranoside (11)[24] by a comparison of their 1H and 13C NMR data with those in the literature. Among them, compounds
8–11 were monoterpene glycosides isolated from I.lanceolatum for the first time.
The neuroprotective, anti-inflammatory, antiviral, and cytotoxic activities of 1–11 were evaluated in vitro. Compounds 4, 5
and 8-10 displayed neuroprotective effects against monosodium glutamate-induced injury in human neuroblastoma SH-SY5Y
cells, with protection rates of 23.0, 19.1, 18.2, 17.7 and 15.8%, respectively at 10 μM (Fig. 6), and resveratrol was used as
positive control with protection rate of 25.8% at 10 μM. However, none of the compounds showed cytotoxic, antiviral, or anti-
inflammatory activities.
11

30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
RSV
Fig. 6. Protection rate of resveratrol (RSV) and compounds 1–11 against monosodium glutamate-induced human
neuroblastoma SH-SY5Y cells injury (at 10 μM).
4. Conclusion
Five new compounds (1–5), including three hexalactone derivatives (1–3) and a pair of new oxaspiro-carbon epimeric
glycosides (4 and 5), and six known compounds (6–11) were isolated from the fruits of I. lanceolatum. The configurations of
the new compounds 1-5 were determined by extensive spectroscopic evidence and CD data. Compounds 4 and 5, a pair of
unusual oxaspiro-carbon epimeric glycosides, possess a 1-oxaspiro[4.5]decane-7α,8α,9β-triol moiety, and plausible biogenetic
pathways for 4 and 5 from the naturally occurring precursor (-)-shikimic acid were proposed. Additionally, the isolated
compounds were evaluated for their neuroprotective, anti-inflammatory, antiviral and cytotoxic activities. Compounds 4, 5,
and 8-10 displayed potential neuroprotective effects at a concentration of 10 μM.
Acknowledgments
The authors are grateful to the Department of Instrumental Analysis of our institute for the acquisition of the IR, NMR, and
MS spectra. This work was supported by grants from the Natural Science Foundation of China (No. 21672261and 21732008 ),
from CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-3-011), and from the National Mega-project for Innovative
Drugs (No. 2018ZX09711001-008-001 and 2018ZX09711001-001-001).
Conflict of interest
The authors declare that there are no conflicts of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version at https://doi.org/10.1016/j.bioorg. 2019.
00.000.
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12

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Graphic abstract
New hexalactone derivatives and a pair of new oxaspiro-carbon epimeric glycosides from the fruits of
Illicium lanceolatum
Yang-Lan Liu†, Xiao-Jing Wang†, Ru-Bing Wang, Mi Li, Wen-Rui Li, Jian-Pei Zhang, Xiu-Qi Bao, Dan Zhang, Shuang-Gang
Ma
13

12
H3CO
HO
7
9
6
O1
O
5
2
O
HO
HO
8
3
4
10
OH
H3CO
11
4
H3CO
HO
7
9
3
O
5
O
HO
HO
8
O1
OH
H3CO
5
Illicium lanceolatum
neuroprotective activities
14

Highlights
Five new compounds (1–5), including three hexalactone derivatives (1–3) and a pair of new oxaspiro-carbon epimeric
glycosides (4 and 5), and six known compounds (6–11) were obtained from the fruits of Illicium lanceolatum.
Compounds 4 and 5, a pair of unusual oxaspiro-carbon epimeric glycosides, possess a 1-oxaspiro[4.5]decane-7α,8α,9β-triol
moiety, and plausible biogenetic pathways for 4 and 5 from the naturally occurring precursor (-)-shikimic acid were proposed.
Compounds 4, 5, and 8-10 displayed potential neuroprotective effects against monosodium glutamate-induced injury in human
neuroblastoma SH-SY5Y cells, with protection rates of 23.0, 19.1, 18.2, 17.7, and 15.8%, respectively.
15