1H NMR spectroscopy-guided isolation of new sucrose esters from Physalis alkekengi var. franchetii and their antibacterial activity

Article abstract of DOI:10.1016/j.fitote.2016.09.007

Ten new sucrose esters, physakengoses A–J (1???10), were isolated from the aerial parts of Physalis alkekengi var. franchetii under the guidance of ¹H NMR spectroscopy. Their structures were determined by spectroscopic analyses (HRESIMS, 1D and 2D NMR, and ESIMS) and chemical methods. These new compounds were tested for antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli. Among them, compounds 2 and 5–8 showed potent inhibitory effects against test strains with MIC values ranging from 3.5 to 14.9?μg/mL. These findings may indicate that the P. alkekengi var. franchetii has potential application as an ingredient in pharmaceuticals.

Full text of DOI:10.1016/j.fitote.2016.09.007

Fitoterapia 114 (2016) 138–143
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
1H NMR spectroscopy-guided isolation of new sucrose esters from
Physalis alkekengi var. franchetii and their antibacterial activity
⁎
⁎
Chuan-Yang Zhang, Jian-Guang Luo , Rui-Huan Liu, Ru Lin, Ming-Hua Yang, Ling-Yi Kong
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2016
Received in revised form 7 September 2016
Accepted 14 September 2016
Available online 15 September 2016
Ten new sucrose esters, physakengoses A–J (1−10), were isolated from the aerial parts of Physalis alkekengi var.
franchetii under the guidance of 1H NMR spectroscopy. Their structures were determined by spectroscopic anal-
yses (HRESIMS, 1D and 2D NMR, and ESIMS) and chemical methods. These new compounds were tested for an-
tibacterial activities against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli.
Among them, compounds 2 and 5–8 showed potent inhibitory effects against test strains with MIC values ranging
from 3.5 to 14.9 μg/mL. These findings may indicate that the P. alkekengi var. franchetii has potential application as
an ingredient in pharmaceuticals.
Keywords:
Physalis alkekengi var. franchetii
Solanaceae
© 2016 Published by Elsevier B.V.
Sucrose ester
Antibacterial activity
1H NMR spectroscopy-guided isolation
Chemical compounds
parts of P. alkekengi var. franchetii. Herein we describe the 1H NMR-guid-
ed isolation and identification of ten new sucrose esters (Fig. 1) and their
in vitro antibacterial activities.
Ethyl ethanoate (PubChem CID: 8857)
Methylene chloride (PubChem CID: 6344)
Methanol (PubChem CID: 887)
2. Experimental
Methyl cyanide (PubChem CID: 6342)
2.1. General
1. Introduction
The optical rotation values were recorded on a Jasco P-1020 polarim-
eter and IR data were detected on a Bruker Tensor 27 spectrometer. 1D
and 2D NMR experiments were carried out in methanol-d4 on a Bruker
Avance III NMR instrument at 500 MHz (1H) and 125 MHz (13C), and
TMS was set as the internal standard. Agilent UPLC-Q-TOF (6520B)
was used to acquire HRESIMS data. HPLC analysis was performed on
an Agilent 1260 Series instrument equipped with a DAD detector and
a Shim-pack VP-ODS column (4.6 × 250 mm, i.d.). Silica gel (200–300
and 100–200 mesh, Qingdao Marine Chemical Co., Ltd.), MCI gel (75–
150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and ODS
(40–63 μm, Fuji) were used for column chromatography (CC). All chem-
ical reagents used were analytical grade (Jiangsu Hanbon Science and
Technology Co., Ltd., Nanjing, China).
Physalis alkekengi var. franchetii (Solanaceae), a medicinal and edible
plant, is widely distributed and cultivated in Europe and Asia [1]. The ae-
rial parts of P. alkekengi var. franchetii have been used as a traditional
Chinese medicine for treatment of sore throat, cough, eczema, hepatitis,
urinary problems and tumors [2]. Previous investigations of P. alkekengi
var. franchetii were focused on physalins, which exhibited anti-inflam-
matory, cytotoxic, antioxidant and antibacterial activities [2–4]. As a
part of continuous chemical studies on the genus Physalis [5,6], we
throw our sight to sucrose esters. The presence of sucrose esters are re-
stricted in their distribution, previously being reported from the
Solanaceae genera Physalis, Nicotiana, Petunia, Solanum, and Lycopersicon,
as well as from the Asteraceae, Cannaceae, and Polygalaceae families [7].
Sucrose esters, structurally featured in sucrose and long fatty acid ester,
have exhibited potent antibacterial and anti-inflammatory activities [8–
10]. Since 1H NMR-guided isolation showed the potential to facilitate
the current efforts on distinct natural product discovery [11,12], it was
applied to help us rapidly target and isolate sucrose esters from the aerial
2.2. Plant material
The aerial parts of P. alkekengi var. franchetii were collected in Octo-
ber 2014 from Fuyang city, Anhui province, People's Republic of China,
and identified by Prof. Mian Zhang. A voucher specimen (No. PAF-
20150422) was deposited in the Department of Natural Medicinal
Chemistry, China Pharmaceutical University.
⁎
Corresponding authors.
E-mail addresses: luojg@cpu.edu.cn (J.-G. Luo), cpu_lykong@126.com (L.-Y. Kong).
http://dx.doi.org/10.1016/j.fitote.2016.09.007
0367-326X/© 2016 Published by Elsevier B.V.

C.-Y. Zhang et al. / Fitoterapia 114 (2016) 138–143
139
Fig. 1. Structures of compounds 1–10.
2.3. Extraction and isolation
1H NMR and 13C NMR data (see Tables 1 and 3); HRESIMS m/z 699.3559
[M + Na]+ (calcd for C33H56NaO14, 699.3562).
The plant materials (3.0 kg) were extracted with 95% EtOH
(3 × 10 L) under reflux. After removing the solvents under reduced
pressure, the viscous residue (240.0 g) was obtained. The residue was
dissolved in distilled water (2 L), followed by successive partition with
petroleum ether (PE), dichloromethane (CH2Cl2) and ethyl acetate
(EtOAc). The PE, CH2Cl2, EtOAc and H2O fractions were separately ana-
lyzed by 1H NMR spectroscopy, and the characteristic 1H NMR signals
for sucrose esters were detected from PE fraction. Therefore, the PE frac-
tion (80.0 g) was further chromatographed by using a silica gel column
with a gradient of petroleum ether-acetone (100:1–1:1, v/v) to give
four fractions, Frs. A–D. Subsequently, Frs. A–D were subjected to 1H
NMR spectroscopy, and the signals for sucrose esters were observed
particularly in Fr. D. Thus, Fr. D (25.0 g) was further separated by an
MCI gel column using MeOH-H2O (30:10–90:10, v/v) as eluent to
yield six major subfractions (Frs. D1–D6). Fr. D2 (8.0 g) was applied to
an ODS column eluted with solvent system of MeOH-H2O (70:30–
90:10, v/v) to afford Frs. D2a–D2e. Fr. D2b was purified by preparative
HPLC using MeOH-H2O (80:20, v/v) as the mobile phase to give com-
pounds 1 (8.0 mg, tR = 8.5 min) and 5 (17.0 mg, tR = 16.7 min). Com-
pounds 2 (4.0 mg, tR = 10.9 min) and 7 (20.0 mg, tR = 12.5 min) were
obtained from Fr. D2c by recycling-preparative HPLC eluted with MeCN-
H2O (60:40, v/v). The Fr. D2d was subjected to recycling-preparative
HPLC with MeCN-H2O (65:35, v/v) to furnish compounds 6 (24.0 mg,
tR = 11.9 min) and 8 (4.0 mg, tR = 8.5 min). Compounds 4 (5.0 mg,
tR = 6.3 min) and 3 (11.0 mg, tR = 8.4 min) were obtained by prepara-
tive HPLC with MeOH-H2O (80:20, v/v) from Fr. D2e. Fr D3 (6.0 g) was
chromatographed by an ODS column eluted with MeOH-H2O (70:30–
90:10, v/v) to get four major subfractions (Frs. D3a–D3d). Compounds
9 (10.0 mg, tR = 15.7 min) and 10 (15.0 mg, tR = 12.9 min) were obtain-
ed from Fr. D3b and Fr. D3c by recycling-preparative HPLC with MeOH-
H2O (85:15, v/v), respectively.
Physakengose C (3): amorphous solid; [a]2D5+34.0 (c 0.1, MeOH); IR
(KBr) νmax 3418, 2923, 2852, 1747, 1634, 1468, 1383, 1262, 1162 cm−1
;
1H NMR and 13C NMR data (see Tables 1 and 3); HRESIMS m/z 671.3250
[M + Na]+ (calcd for C31H52NaO14, 671.3249).
Physakengose D (4): amorphous solid; [a]2D5+21.0 (c 0.1, MeOH); IR
(KBr) νmax 3366, 2923, 2852, 1721, 1658, 1634, 1469, 1383, 1264 cm−1
;
1H NMR and 13C NMR data (see Tables 1 and 3); HRESIMS m/z 629.3143
[M + Na]+ (calcd for C29H50NaO13, 629.3144).
Physakengose E (5): amorphous solid; [a]2D5+32.0 (c 0.1, MeOH); IR
(KBr) νmax 3409, 2923, 2853, 1741, 1657, 1468, 1384, 1265, 1155 cm−1
;
1H NMR and 13C NMR data (see Tables 1 and 3); HRESIMS m/z 713.3722
[M + Na]+ (calcd for C34H58NaO14, 713.3719).
Physakengose F (6): amorphous solid; [a]2D5+35.0 (c 0.1, MeOH); IR
(KBr) νmax 3418, 2923, 2852, 1719, 1656, 1469, 1383, 1272, 1156 cm−1
;
1H NMR and 13C NMR data (see Tables 2 and 3); HRESIMS m/z 711.3560
[M + Na]+ (calcd for C34H56NaO14, 711.3562).
Physakengose G (7): amorphous solid; [a]2D5 +25.0 (c 0.1, MeOH); IR
(KBr) νmax 3416, 2923, 2852, 1746, 1634, 1468, 1382, 1261, 1049 cm−1
;
1H NMR and 13C NMR data (see Tables 2 and 3); HRESIMS m/z 755.3820
[M + Na]+ (calcd for C36H60NaO15, 755.3824).
Physakengose H (8): amorphous solid; [a]2D5+36.0 (c 0.1, MeOH); IR
(KBr) νmax 3366, 2922, 2851, 1743, 1633, 1469, 1384, 1262, 1075 cm−1
;
1H NMR and 13C NMR data (see Tables 2 and 3); HRESIMS m/z 753.3667
[M + Na]+ (calcd for C36H58NaO15, 753.3668).
Physakengose I (9): amorphous solid; [a]2D5+22.0 (c 0.1, MeOH); IR
(KBr) νmax 3396, 2924, 2853,1726, 1650, 1467, 1385, 1266, 1154 cm−1
;
1H NMR and 13C NMR data (see Tables 2 and 3); HRESIMS m/z 741.4031
[M + Na]+ (calcd for C36H62NaO14, 741.4032).
Physakengose J (10): amorphous solid; [a]2D5+37.0 (c 0.1, MeOH);
IR (KBr) νmax 3365, 2923, 2853, 1719, 1657, 1468, 1383, 1273,
1155 cm−1; 1H NMR and 13C NMR data (see Tables 2 and 3); HRESIMS
m/z 739.3886 [M + Na]+ (calcd for C36H60NaO14, 739.3875).
2.4. Data of the isolated compounds
2.5. Alkaline hydrolysis
Physakengose A (1): amorphous solid; [a]2D5+25.0 (c 0.1, MeOH); IR
(KBr) νmax 3364, 2924, 2852, 1721, 1658, 1634, 1469, 1424, 1382 cm−1
;
Compounds 1–10 (2.0 mg each), dissolved in 2 M aqueous NH4OH
(2 mL), were heated at 50 °C for 4 h. The reaction mixtures were neu-
tralized with 2 M formic acid to pH 3 and extracted with EtOAc
(3 × 3 mL). Then, the aqueous phase was repeatedly dried with the
method reported in the literature [8]. Co-TLC analysis (CHCl3: HAc:
1H NMR and 13C NMR data (see Tables 1 and 3); HRESIMS m/z 629.3142
[M + Na]+ (calcd for C29H50NaO13, 629.3144).
Physakengose B (2): amorphous solid; [a]2D5+42.0 (c 0.1, MeOH); IR
(KBr) νmax 3417, 2923, 2852, 1742, 1634, 1469, 1384, 1265, 1160 cm−1
;

140
C.-Y. Zhang et al. / Fitoterapia 114 (2016) 138–143
Table 1
Table 2
1H NMR data (500 MHz, CD3OD) of Compounds 1–5.
1H NMR data (500 MHz, CD3OD) of compounds 6–10.
Position
1
2
3
4
5
Position
6
7
8
9
10
δH (multi, J in
δ
H (multi, J
δH (multi, J
δ
H (multi, J in
δH (multi, J
δH (multi, J
δH (multi, J
δ
H (multi, J in
δ
H (multi, J in
δH (multi, J
Hz)
in Hz)
in Hz)
Hz)
in Hz)
in Hz)
in Hz)
Hz)
Hz)
in Hz)
1
2
5.54, d (3.7)
5.57, d (3.5) 5.59, d (3.5) 5.45, d (3.6)
5.57, d
(3.4)
1
2
5.60, d (3.5) 5.57, d (3.1) 5.63, d (3.5)
5.57, d (3.5)
4.80a
5.59, d (3.5)
4.78a
4.78a
4.86, dd
(10.3, 3.4)
5.47, t (9.7) 5.41, t (9.7) 5.47, t (9.7)
3.72, t (9.7) 3.69, t (9.7) 3.72, t (9.7)
4.83a
4.62, dd
(10.1, 3.7)
3.83a
3.46, t (9.5)
3.86, m
3.83a
4.76, dd
4.66, dd
3.63, m
4.83a
(10.4, 3.5)
(10.1, 3.5)
3
4
5
6a
5.42, t (9.7)
3.68, t (9.7)
3.95, m
3.84, dd
(12.6, 1.8)
3.79, m
5.47, t (9.7)
3.71, t (9.7)
3.94, m
3.84, dd
(12.1, 2.1)
3.78, m
3
4
5
6a
5.41, t (9.7) 3.83a
5.24, t (9.7)
5.41, t (9.7)
3.67, t (9.7)
3.95, m
3.84, d
(11.8)
3.69, t (9.7) 3.48, t (9.3) 3.61,t (9.7)
3.94, m
3.83, dd
(12.2, 1.9)
3.78, m
3.47, d
(11.8)
3.34, d
(11.8)
3.92, m
3.84, m
3.93, m
3.93, m
3.84, d
(11.7)
3.85, m
3.91, m
3.85, dd
(12.5, 2.1)
3.79, m
3.83a
3.82a
6b
1a′
3.78, m
4.09, d
(11.6)
4.01, d
(11.6)
4.04, d (11.6) 3.56, d (11.8) 3.47, d
6b
1a′
3.76, m
3.78, m
3.76, m
4.04, d
(11.5)
3.89, d
(11.5)
3.79, m
3.64, d (12.0) 3.55, d
(11.7)
3.55, d (12.0) 3.44, d
(11.7)
3.78, m
(11.7)
3.94, d (11.6) 3.46, d (11.8) 3.33, d
(11.7)
3.47, d (11.7) 3.46, d
(11.7)
1b′
1b′
3′
3.31a
3.32a
3′
4′
5′
6a′
6b′
3′-O-
2a
5.40, d (8.4) 5.31, d (8.2) 5.27, d (8.4)
4.31, t (8.4) 4.31, t (8.2) 4.30, t (8.4)
5.38, d (8.4)
4.31, t (8.4)
3.88, m
5.40, d (8.4)
4.31, t (8.4)
3.87, m
5.39, d (8.4)
5.39, d (8.4) 5.25, d (8.4) 5.37, d (8.1)
5.39, d
(8.4)
4.31, t (8.4)
3.87, m
3.78, m
3.78, m
3.87, m
3.78, m
3.78, m
Dodecab
2.56, dt
(16.1, 7.8)
2.46, dt
(16.1, 7.8)
1.69, m
1.30, m
3.88, m
3.78, m
3.78, m
Dodecab
2.56, dt
(15.5, 7.5)
2.47, dt
(15.5, 7.5)
1.68, m
1.30, m
3.87, m
3.79, m
3.79, m
Dodecab
3.79, m
3.78, m
4′
5′
6a′
6b′
3′-O-
2a
4.29, t (8.4)
3.85, m
3.78, m
4.30, t (8.4) 4.28, t (8.4) 4.30, t (8.1)
3.79, m
3.78, m
3.88, m
3.78, m
3.78, m
3.84, m
3.78, m
3.78, m
Dodecab
3.90, m
3.79, m
3.79, m
Myrisc
Myrisc
2.56, dt (16.0, 2.55, dt (15.6, 2.56, dt
7.8) 7.5) (15.5, 7.8)
2.46, dt (16.0, 2.47, dt (15.6, 2.46, dt
7.8)
1.69, m
1.30, m
3.78, m
2b
2.43, t (7.4)
2.43, t (7.4)
2.55, m
2.47, m
2.44, t (7.4) 2.50, dt (15.3, 2.55, dt
7.5)
1.70, m
1.30, m
1.30, m
(15.5, 7.8)
1.68, m
1.30, m
1.30, m
1.30, m
0.90, t (6.9)
Tigd
8.0)
(15.6, 7.5)
3
2b
2.44, t (7.4) 2.43, dt (15.3, 2.46, dt
4–11
12
13
14
2-O-
2a
8.0)
(15.6, 7.5)
0.90, t (6.9) 0.91, t (6.9) 0.90, t (6.8)
3
1.66, m
1.33, m
0.90, t (6.8)
Tigc
1.68, m
1.31, m
1.66, m
1.30, m
1.68, m
1.30, m
1.69, m
1.30, m
0.90, t (6.6)
3-MeBud
2.13, d
(7.0)
2.12, d
(7.0)
1.96, m
1.30, m
4–11
12
2-O-
2a
0.91, t (6.8)
3-MeBue
2.13, d (6.9)
2.12, d (6.9)
1.96, m
0.90, t (6.8) 0.90, t (6.8) 0.90, t (6.9)
Tigd
3-MeBue
Tigd
Tigc
Tigc
2.18, d (6.9)
2.17, d (6.9)
1.96, m
2b
3
6.81, m
6.81, dq like
(1.2, 7.0)
6.81, m
2b
3
4
5
3-O-
1.77, d (7.2) 0.87, d (6.6) 1.77, d (7.0)
0.87, d (6.7)
0.87, d (6.7)
Tigd
1.77, d (7.2)
1.75, br s
Tigd
6.94, dq like
(1.1, 7.0)
6.85, m
6.95, q (7.0)
1.75, br s
0.87, d (6.6) 1.75, br s
Tigd
Tigd Tigd
4
1.83, d (7.3)
1.78, d (7.0) 1.82, d (7.0)
0.87, d
(6.7)
0.87, d
(6.7)
Tigc
3
6.81, m
6.87, q (7.1) 6.81, dq like
(1.2, 7.0)
6.87, dq like
(1.1, 7.1)
1.79, d (7.1)
1.82, br s
6.81, m
5
1.85, br s
1.80, br s
1.85, br s
4
5
1.77, d (7.2) 1.79, d (7.1) 1.77, d (7.0)
1.79, br s
1.77, d (7.2)
1.79, br s
1.82, br s
Ac
1.79, br s
Ac
3-O-
i-Bue
Tigc
1′-O-
2
3
2.57, m
1.06, d (7.0)
2
2.09, s
2.06, s
6.93, dq like
(1.3, 7.0)
1.81, d (7.0)
6.87, q
(7.0)
1.79, d
(7.0)
a
Overlapped with other signals.
Dodeca = dodecanoyl.
Myris = myristyl.
b
c
4
1.11, d (7.0)
d
e
Tig = tigloyl.
5
1.87, br s
1.82, br s
3-MeBu = 3-methylbutanoyl.
1′-O-
Ac
2
2.05, s
a
Overlapped with other signals.
Dodeca = dodecanoyl.
Tig = tigloyl.
3-MeBu = 3-methylbutanoyl.
i-Bu = isobutyryl.
microdilution method was used to measure the minimal inhibition con-
centration (MIC) according to the protocols of the National Committee
for Clinical and Laboratory Standards [14]. Following inoculation of the
test bacteria on nutrient agar for 18–24 h, a colony of approximately
1 mm in diameter was collected by using a sterile loop and was dis-
solved into Mueller Hinton broth. Each bacterial strain was diluted to
105 cfu/mL before antimicrobial assay. Stock solutions were firstly re-
solved in DMSO and diluted to varieties of concentrations (0–50.0 μg/
mL, DMSO b 1%) with broth afterwards. After incubation at 37 °C for
24 h, the optical density was recorded at 578 nm. MIC value was defined
as the minimum concentration of compound at which the growth of mi-
croorganism was half inhibited. Streptomycin and penicillin were used
as positive controls for Gram-positive bacteria and Gram-negative bac-
teria, respectively. All of the antimicrobial assays were performed in
triplicate.
b
c
d
e
H2O = 3: 3.5: 0.5, Rf = 0.41) in comparison with authentic sucrose, in-
dicated the presence of sucrose.
2.6. Antimicrobial assay
Gram-positive Staphylococcus aureus ATCC 25923 (S. aureus), Bacil-
lus subtilis ATCC 6633 (B. subtilis) and Gram-negative Pseudomonas
aeruginosa ATCC 9027 (P. aeruginosa) and Escherichia coli ATCC 25922
(E. coli) were used to test antibacterial activity by disk diffusion assay
[13]. Each paper disk (6 mm diameter) permeating with 10 μL of test
sample (100 μg/mL methanol) was dried and placed on Mueller Hinton
agar plate containing bacterial inoculum. Then the bacterial strains were
incubated at 37 °C for 24 h. For evaluating antibacterial potential, diam-
eter (in mm) of the growth inhibition zone was recorded. The broth
3. Results and discussion
The application of 1H NMR spectroscopy-guided isolation prompted
us to narrow the scope of targeted sucrose esters to the Fr. D of PE layer

C.-Y. Zhang et al. / Fitoterapia 114 (2016) 138–143
141
Table 3
13C NMR data (125 MHz, CD3OD) of compounds 1–10 (δ in ppm).
Position
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
90.9
74.5
72.4
71.5
74.2
62.3
64.4
105.0
78.6
73.4
84.1
63.0
Dodecaa
174.9
34.9
26.1
23.7–
33.0
14.4
91.1
72.5
73.5
69.0
74.4
61.8
64.1
105.1
78.5
73.3
84.1
63.1
Dodecaa
175.1
34.9
26.1
23.7–
33.1
14.4
91.1
74.4
72.4
71.4
74.4
62.2
66.2
103.1
79.3
73.0
84.1
62.9
Dodecaa
174.8
34.8
26.0
23.7–
33.1
14.4
93.4
71.5
76.9
69.3
74.6
61.9
65.1
104.6
79.7
73.8
84.1
63.0
Dodecaa
175.0
35.1
26.0
23.7–
33.1
14.4
91.1
72.1
73.8
69.4
74.3
61.9
64.4
105.1
78.9
73.5
84.0
63.0
Dodecaa
175.2
34.9
26.1
23.7–
33.0
14.4
91.1
72.7
73.8
69.2
74.5
61.9
64.1
105.1
78.5
73.5
84.1
63.1
Dodecaa
175.1
34.9
26.1
23.7–
33.1
14.5
91.3
71.9
73.7
69.3
74.6
61.9
65.6
103.4
79.1
73.2
84.3
63.0
Dodecaa
175.1
34.8
26.0
23.7–
33.1
14.4
91.4
72.5
73.7
69.1
74.7
61.9
65.9
103.3
79.3
73.2
84.2
63.0
Dodecaa
175.1
34.8
26.0
23.7–
33.1
14.4
91.1
72.1
73.8
69.4
74.4
61.9
64.4
105.1
78.8
91.1
72.7
73.8
69.2
74.5
61.9
64.1
105.1
78.5
73.5
1′
2′
3′
4′
5′
6′
3′-O-
1
73.5
84.0
63.0
84.1
63.1
Myrisb
175.2
34.9
Myrisb
175.1
34.9
2
3
4–11
26.1
23.7–
33.1
23.7–
33.1
23.7–
33.1
26.1
23.7–
33.1
23.7–
33.1
23.7–
33.1
12
13
14
14.4
14.4
2-O-
1
2
3
4
5
3-O-
1
2
3
4
5
Tigc
Tigc
Tigc
3-MeBud
173.7
44.3
26.6
22.7
Tigc
3-MeBud
173.7
44.3
26.7
22.7
Tigc
3-MeBud
173.7
44.3
Tigc
169.1
129.5
139.6
14.5
168.4
129.0
140.4
14.4
169.1
129.3
139.9
14.5
168.5
129.0
140.2
14.4
168.5
128.9
140.4
14.3
12.0
Tigc
168.8
129.6
138.7
14.5
12.2
Ac
168.5
129.0
140.2
14.5
26.7
22.7
12.1
12.0
12.1
22.7
12.0
22.7
22.7
12.2
i-Bue
177.9
35.2
Tigc
Tigc
Tigc
Tigc
Tigc
Tigc
169.7
129.9
138.4
14.4
168.7
129.6
138.9
14.4
168.8
129.6
138.7
14.3
168.6
129.6
138.9
14.3
12.2
Ac
168.7
129.6
138.9
14.4
168.8
129.6
138.7
14.3
19.6
19.1
12.3
12.2
12.1
12.2
12.0
1′-O-
Ac
1
2
171.8
20.6
172.0
20.6
171.8
20.6
a
Dodeca = dodecanoyl.
Myris = myristyl.
Tig = tigloyl.
3-MeBu = 3-methylbutanoyl.
i-Bu = isobutyryl.
b
c
d
e
rapidly. In detail, the 1H NMR spectrum of Fr. D (Fig. S70) showed meth-
ylene groups of fatty acid ester in the range δH 1.2–1.4, terminal methyl
group of fatty acid ester in the range δH 0.8–1.0, sugar moiety signals in
the range δH 3.4–4.5 and anomeric protons of the sugar moiety in the
range δH 5.3–5.8. By comparison of these characteristic 1H signals with
those reported in the literature [15], we assumed that sucrose esters
exist in the Fr. D. As expected, the further isolation of Fr. D led to ten
new sucrose esters (1–10).
174.9) of dodecanoyl group (Fig. 2). Moreover, the ESIMS fragments at
m/z 245 and 345 (Fig. S7) corresponded to tigloylated glucopyranosyl
and dodecanoylated fructofuranosyl units, respectively, thus confirming
physakengose A (1) as 3′-O-dodecanoyl-2-O-tigloylsucrose.
Physakengose B (2) gave the molecular formula C33H56O14 on the
basis of its [M + Na]+ HRESIMS ion peak at m/z 699.3559 (calcd for
C33H56NaO14, 699.3562). The NMR data (Tables 1 and 3) of 2, similar
to those of 1, also showed characteristic signals for sucrose and fatty
acid ester. The main differences were the presence of signals belonging
to an additional isobutyryl unit [δH 2.57 (1H, m), 1.06 (3H, d, J = 7.0 Hz)
Physakengose A (1) was obtained as an amorphous solid. The molec-
ular formula of C29H50O13 was determined by its molecular ion in the
HRESIMS at m/z 629.3142 [M + Na]+ (calcd for C29H50NaO13
,
629.3144). The IR spectrum exhibited strong absorptions at 3364 and
1721 cm−1, which attributed to hydroxyl and ester functions, respec-
tively. The NMR data of 1 revealed that it contained signals for sucrose
and long chain fatty acid ester moieties [15]. The presence of a sucrose
unit was deduced from the analysis of the NMR spectra (Tables 1 and
3), which showed the anomeric CH signals of the glucopyranose (δH
5.54, d, J = 3.7 Hz and δC 90.9; CH-1) and that of the anomeric carbon
of the fructofuranose (δC 105.0, C-2′). Alkaline hydrolysis and compari-
son of the spectroscopic data with those reported in the literature also
confirmed the existence of sucrose [16]. ESIMS, 1H and 13C, TOCSY and
HMBC spectra allowed identification of the acyl groups as dodecanoyl
(m/z 183) and tigloyl (m/z 83). The positions of these groups were
established by the HMBC correlations from H-2 (δH 4.62) to C-1 (δC
169.1) of tigloyl group, as well as that of H-3′ (δH 5.39) to C-1 (δC
Fig. 2. Selected HMBC and TOCSY correlations of compound 1.

142
C.-Y. Zhang et al. / Fitoterapia 114 (2016) 138–143
Table 4
and 1.11 (3H, d, J = 7.0 Hz); δC 177.9, 35.2, 19.6, 19.1] and the downfield
shift of H-3 (δH 5.41) relative to that (δH 3.83) in 1. The isobutyryl unit
was located at C-3 determined by the HMBC correlation from H-3 to
its carbonyl carbon (δC 177.9). The 3-isobutyryl-2-tigloyl
glucopyranosyl moiety was further confirmed by the fragment ion at
m/z 315 (Fig. S14). Consequently, the structure of compound 2 was
assigned as 3′-O-dodecanoyl-3-O-isobutyryl-2-O-tigloylsucrose.
The molecular formula of physakengose C (3) was determined to be
C31H52O14 with the [M + Na]+ ion at m/z 671.3250 in the HRESIMS. As
was the case with 2, the 1H and 13C NMR data (Tables 1 and 3) of com-
pound 3 closely resemble that of 1, except for the presence of signals at-
tributable to an acetyl unit [δH 2.05 (3H, s); δC 171.8, 20.6], and the
downfield shift of H2-1′ (δH 3.89, 4.04) relative to those (δH 3.31, 3.47)
in 1. The HMBC correlation from H2-1′ (δH 3.89, 4.04) to acetoxy carbon-
yl (δC 171.8) indicated that the acetyl unit was located at the C-1′.
Therefore, the structure of compound 3 was elucidated as 1′-O-acetyl-
3′-O-dodecanoyl-2-O-tigloylsucrose.
Minimum Inhibitory Concentration (MIC) of compounds 1–10 (μg/mL).a
Compounds
S. aureus
B. subtilis
P. aeruginosa
E. coli
1
2
3
4
5
6
7
8
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
N50.00
9.71 2.83
33.26 6.90
33.71 4.33
9.81 1.48
5.32 1.47
6.57 0.86
5.78 0.96
6.63 0.93
4.70 0.96
0.06 0.03
8.89 1.63
16.78 2.05
N50.00
5.95 0.85
3.50 1.49
8.78 1.67
3.57 1.02
43.55 2.96
18.48 3.69
0.13 0.05
14.91 2.56
N50.00
34.08 2.25
13.12 2.42
5.79 1.15
4.51 3.02
3.21 0.95
15.04 2.24
6.71 1.50
9
10
Penicillinb
Streptomycinb
0.41 0.15
0.45 0.15
a
Values are represented as the means SD based on three independent experiments.
Penicillin and Streptomycin were used as positive controls.
b
Physakengose D (4) owned the same molecular formula C29H50O13
as that of compound 1 with the analysis of its HRESIMS spectrum. A
comparison of the spectroscopic data (Tables 1 and 3) with those of 1
revealed an overall similarity. The difference between them was deter-
mined by the HMBC spectrum; the correlation between H-3 (δH 5.24) of
the glucopyranose to the carbonyl carbon of tigloyl unit (δC 169.7) sug-
gested the tigloyl unit was attached to C-3. Accordingly, the structure of
compound 4 was assigned as 3′-O-dodecanoyl-3-O-tigloylsucrose.
Physakengose E (5) was isolated as an amorphous solid, and its mo-
lecular formula was determined as C34H58O14 by its HRESIMS spectrum.
The NMR data (Tables 1 and 3) showed signals belonging to sucrose,
long fatty acid ester and tigloyl units as in compound 4. In addition, pro-
ton and carbon signals ascribing to 3-methylbutanoyl group [δH 2.13
(1H, d, J = 7.0 Hz), 2.12 (1H, d, J = 7.0 Hz), 1.96 (1H, m) and 0.87
(6H, d, J = 6.7 Hz); δC 173.7, 44.3, 26.6, 22.7, 22.7] were observed in 5
and assigned at C-2 based on HMBC correlation between H-2 (δH
4.83) and its carbonyl carbon (δC 173.7). The peak at m/z 329 (Fig.
S34) also corroborated the difference on the pyranose moiety substitu-
tion and the structure of 5 was established as 3′-O-dodecanoyl-2-O-3-
methylbutanoyl-3-O-tigloylsucrose.
(Figs. S61, S68) corresponding to a myristyl unit. Accordingly, the struc-
tures of compounds 9 and 10 were established as depicted.
Previous investigations have suggested that sucrose esters from
Physalis species and the ethanol extracts of P. alkekengi var. franchetii
inhibited the growth of Gram-positive and Gram-negative bacteria
[17,18]. Thus, the ten isolates were evaluated for antibacterial activity
against S. aureus, B. subtilis, P. aeruginosa and E. coli. As shown in Table
4, all compounds had no activity against E. coli, but exhibited various de-
grees of antibacterial activity against other three bacteria. The antibacte-
rial data against Gram-positive bacteria of the sucrose esters provided
clear evidence for the first time that the C-2 and C-3 substitution at
the glucopyranose is a subtle but critical parameter, variations of
which would remarkable spoil antibacterial activity. To elaborate, com-
pounds 2 and 5–10, which were disubstituted at C-2 and C-3, showed
antibacterial activity against S. aureus and B. subtilis. Contrastively, com-
pounds 1, 3 and 4, with only one substituted group at C-2 or C-3,
displayed weaker or no antibacterial activity. Notably, compounds 6
and 8 showed potent antibacterial activity to the test bacteria (S. aureus,
B. subtilis and P. aeruginosa), with MIC values below 6 μg/mL. These re-
sults indicated that the di-tigloyl substitution may play an important
role in the antibacterial activity.
Physakengose F (6), designated with the molecular formula of
C34H56O14 in accordance with its HRESIMS spectrum. Although the 1H
and 13C NMR data (Tables 2 and 3) of compound 6 were analogous to
those of 1, the presence of signals attributable to an additional tigloyl
unit [δH 6.81 (1H, m), 1.77 (3H, d, J = 7.2 Hz) and 1.79 (3H, br s); δC
168.8, 129.6, 138.7, 14.3, 12.1] were obviously observed in 6. The posi-
tion of the second tigloyl unit at C-3 was established by the correlation
between its carbonyl carbon (δC 168.8) and H-3 (δH 5.47) of the gluco-
pyranose observed in the HMBC spectrum. Thus, the structure of com-
pound 6 was identified as 3′-O-dodecanoyl-2, 3-di-O-tigloylsucrose.
On the basis of HRESIMS and NMR experiments, the molecular for-
mulas of physakengoses G (7) and H (8) were found to be C36H60O15
and C36H58O15, respectively. Their 1H and 13C NMR spectra (Tables 2
and 3) were similar to those of 5 and 6, except for the presence of signals
belonging to additional acetyl groups (δH 2.09, δC 172.0, 20.6; δH 2.06, δC
171.8, 20.6). The further analysis of their HMBC spectra, together with
ESIMS fragments at m/z 329 and 387 for 7 (Fig. S48) and m/z 327 and
387 for 8 (Fig. S54), allowed us to formulate 7 as 1′-O-acetyl-3′-O-
dodecanoyl-2-O-3-methylbutanoyl-3-O-tigloylsucrose and 8 as 1′-O-
acetyl-3′-O-dodecanoyl-2,3-di-O-tigloylsucrose.
In conclusion, this study reported the 1H NMR Spectroscopy-guided
isolation of ten new sucrose esters and evaluation for their in vitro anti-
bacterial activity. The activity results showed that compounds 2 and 5–8
possessed potent activity which provided a scientific basis for the sup-
plement of the active ingredients in P. alkekengi var. francheti as well
as for the development of novel agents against bacteria.
Conflict of interest
The authors declare no competing financial interests.
Acknowledgements
This research work was supported by Huahai Graduate Innovation
Program (1010020006), the Program for Changjiang Scholars, Innova-
tive Research Team in University (IRT_15R63), the Project Funded by
the Priority Academic Program Development of Jiangsu Higher Educa-
tion Institutions (PAPD), and the Program for New Century Excellent
Talents in University (NCET-12-0977).
The molecular ions at m/z 741.4031 and 739.3886 ([M + Na]+) in
the HRESIMS spectra of compounds 9 and 10 confirmed their molecular
formulas as C36H62O14 and C36H60O14, respectively, and indicated they
both contained an additional C2H4 than compounds 5 and 6. The 1H
and 13C NMR data (Tables 2 and 3) of 9 and 10 were almost superimpos-
able with those of 5 and 6. A comprehensive study on the 1D and 2D
NMR spectra of compounds 9 and 10 indicated that these two com-
pounds each had two more methylene groups in its fatty acid chain,
which were further confirmed by the ESIMS fragment ion at m/z 211
Appendix A. Supplementary data
HRESIMS, 1H NMR, 13C NMR, HSQC, HMBC, TOCSY and ESIMS spectra
of 1-10 and the 1H NMR spectra of PE, Fr. D, CH2Cl2, EtOAc and H2O frac-
tions of Physalis alkekengi var. franchetii are available in Supporting in-
formation. Supplementary data associated with this article can be
found in the online version, at http://dx.doi.org/10.1016/j.fitote.2016.
09.007.

C.-Y. Zhang et al. / Fitoterapia 114 (2016) 138–143
143
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