Two new 3-hexenol glycosides from the calyces of Physalis alkekengi var. franchetii

Article abstract of DOI:10.1080/14786419.2019.1645657

Two new hexenol glycosides, (Z)-hex-3-en-1-ol O-β-d-xylcopyranosyl-(1-6)-β -d-glucopyranosyl-(1-2)-β-d-glucopyranoside (1) and (E)-hex-3-en-1-ol O-β-d-xylcopyranosyl-(1-6)-β-d-glucopyranosyl-(1-2)-β-d-glucopyranoside (2), were isolated from the 50% ethanol elution of macroporous resin of Physalis alkekengi var. franchetii. Their structures were established by detailed spectroscopic analysis, including extensive 2D-NMR data. This is the first time to report the (Z) and (E) 3-hexenol glycosides from Physalis alkekengi var. franchetii.

Full text of DOI:10.1080/14786419.2019.1645657

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Two new 3-hexenol glycosides from the calyces of
Physalis alkekengi var. franchetii
Zunpeng Shu, Yun Tang, Yanni Yang, Zihe Ding, Renxing Zhong, Tianyi Xia,
Xinjia Li, Cuiying Zheng, Zhixiong Wen, Wei Li & Yi Wang
To cite this article: Zunpeng Shu, Yun Tang, Yanni Yang, Zihe Ding, Renxing Zhong, Tianyi
Xia, Xinjia Li, Cuiying Zheng, Zhixiong Wen, Wei Li & Yi Wang (2019): Two new 3-hexenol
glycosides from the calyces of Physalisꢀalkekengi var. franchetii, Natural Product Research, DOI:
10.1080/14786419.2019.1645657
To link to this article: https://doi.org/10.1080/14786419.2019.1645657
View supplementary material
Published online: 25 Jul 2019.
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1645657
Two new 3-hexenol glycosides from the calyces of
Physalis alkekengi var. franchetii
Zunpeng Shua, Yun Tangb, Yanni Yanga, Zihe Dinga, Renxing Zhonga, Tianyi
Xiaa, Xinjia Lia, Cuiying Zhenga, Zhixiong Wena, Wei Lib and Yi Wanga
aResearch Centre for Good Practice in TCM Proessing Technology, Guangdong Pharmaceutical
b
University, Guangzhou, PR China; Key Laboratory of Jiangxi Province Natural Active Pharmaceutical
Ingredients, College of Chemistry and Biology Engineering, Yichun University, Yichun, PR China
ABSTRACT
ARTICLE HISTORY
Received 15 April 2019
Accepted 13 July 2019
Two new hexenol glycosides, (Z)-hex-3-en-1-ol O-b-D-xylcopyrano-
syl-(1-6)-b -D-glucopyranosyl-(1-2)-b-D-glucopyranoside (1) and (E)-
hex-3-en-1-ol O-b-D-xylcopyranosyl-(1-6)-b-D-glucopyranosyl-(1-2)-
b-D-glucopyranoside (2), were isolated from the 50% ethanol elu-
tion of macroporous resin of Physalis alkekengi var. franchetii.
Their structures were established by detailed spectroscopic ana-
lysis, including extensive 2D-NMR data. This is the first time to
report the (Z) and (E) 3-hexenol glycosides from Physalis alkekengi
var. franchetii.
KEYWORDS
Physalis alkekengi var.
franchetii; 3-hexenol
glycosides; Solanaceae
family; activity researches
1. Introduction
Physalis alkekengi L. var. franchetii (Solanaceae) (Chinese name: Jin denglong) are used
as a traditional Chinese herbal medicine for the treatment of sore throat, cough,
eczema, hepatitis, urinary problems, and tumors (Shu et al. 2016). For centuries, the
fruits, roots, calyces and whole plants have been used in traditional Chinese
CONTACT Yi Wang
784789997@qq.com; Wei Li
nobel2025@163.com; Zunpeng Shu
shuzunpeng2010@163.com
Supplementary data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1645657.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Z. SHU ET AL.
prescriptions, particularly its calyces having been considered to be one of the most
important medicines, since compounds isolated from it display a wide spectrum of
biological activities such as anti-trypanosomiasis, immunomodulatory, antitumor, anti-
microbial, cytotoxic, molluscicidal and anti-inflammatory effects (Ge et al. 2009; Kang
et al. 2011; Li et al. 2014; Yang et al. 2015; Li et al. 2018; Wang et al. 2018).
In some previous studies, flavonoids and physalins have always been considered as
the main active components of P. alkekengi L. var. Franchetii (Li et al. 2012; Ooi et al.
2013). However, in the recent research, we isolated the two new 3-hexenol glycosides
from the 50% ethanol elution of macroporous resin of 70% ethanol extract of the caly-
ces of P. alkekengi L. var. Franchetii. And we tested the antimicrobial activities of the
two new compounds. Unfortunately, two new compounds showed no antimicrobial
activities. Interestingly, by reviewing the literature, this type of compounds was found
to have anti-insect activity (Sugimoto et al. 2014). Therefore, we will test the anti-
insect activity of these compounds in the future.
2. Results and discussion
Compound 1 was isolated as a white amorphous powder. The molecular formula of 1
was determined as C23H40O15 by the HR-ESI-MS at m/z 579.2279 [M þ Na]þ, (calcd
579.2265), as well as from its NMR spectroscopic data. Two olefinic protons emerged
1
in the high frequency of the H-NMR spectrum at dH 5.35 (1H, dt, J ¼ 6.4, 12.0 Hz) and
5.40 (1H, dt, J ¼ 6.4, 12.0 Hz), correlated with dC 132.9 and 125.5, in the HSQC spec-
trum. Considering the two high frequency tertiary-carbon signals (dC 132.9 and 125.5)
found in 13C-NMR and DEPT spectra, it is believed that the molecule contained one
–CH ¼ CH– fragmen with (Z)-configuration, as indicated by the coupling constant
1
(JH3–H4 ¼ 12.0 Hz). The H-NMR spectrum revealed signals of oxymethylene protons at
dH 3.67 (1H, overlapped) and 3.47 (1H, overlapped), correlated with dc 68.4 in the
HSQC spectrum, revealed the presence of a –CH2–O– fragment. Two methylene pro-
tons at dH 2.26 (2H, m), 2.01 (2H, m), and a methyl proton at dH 0.92 (3H, t, J ¼ 7.6 Hz),
which were correlated with dC 68.4, 27.6, 20.3, and 14.4 in the HSQC spectrum, sug-
gesting that the aglycone was (Z)-3-hexenol (Zheng et al. 2010). Acid hydrolysis of 1
yielded D-glucose and D-xylose in a ratio of 2:1, and their configurations of the mono-
saccharides were determined by GC analysis of the trimethylsilyl ethers derivatives
(Suzuki et al. 1993). Three anomeric protons at dH 4.36 (1H, d, J ¼ 8.0 Hz), dH 4.30 (1H,
1
d, J ¼ 7.6 Hz), and dH 4.19 (1H, d, J ¼ 6.0 Hz) were observed in the H-NMR spectrum,
correlated with dC 104.3, 103.6, and 101.3, respectively, in the HSQC spectrum. On the
basis of 13C-NMR, DEPT, HSQC, HMBC and chemical reactions, one D-glucopyranose (dC
101.3, 82.5, 76.0, 70.7, 77.2, 61.0), one D-glucopyranose (dC 104.3, 69.9, 75.1, 67.5, 76.2,
68.1) and one D-xylcopyranose (dC 103.6, 72.7, 75.5, 69.9, 65.1) were identified. The
anomeric centers of the two glucose units and one xylose were identified as b-config-
3
uration based on large JH-1, H-2 values (8.0, 7.6, and 6.0 Hz). Therefore, compound 1
was considered to be composed of two b-D-glucopyranose, one b-D-xylcopyranose and
an aglycone with (Z)-3-hexenol (Figure 1). The NMR spectra data was shown in
Supplementary material Table S1.

NATURAL PRODUCT RESEARCH
3
Figure 1. The structure of compound 1 and 2.
In the HMBC spectrum, key correlation peaks were observed between the proton
signal at dH 4.30 (H-10) and the carbon resonance signal at dC 68.4 (C-1), dH 4.36 (H-100)
and dC 82.5 (C-20), dH 4.19 (H-1000) and dC 68.1 (C-600). Therefore, the sequence and the
attachment of the saccharide chain could be set down (Supplementary material
Figure S2).
Therefore, the structure of compound 1 was determined as (Z)-hex-3-en-1-ol O-b-D-
xylcopyranosyl-(1-6)-b-D-glucopyranosyl-(1-2)-b-D-glucopyranoside, and the NMR spec-
tra data are shown in Supplementary material Table S1.
Compound 2 was obtained as a white amorphous power. The HR-ESI-MS spectrum
of 2 (m/z 579.2273 [M þ Na]þ, calcd for C23H40O15Na, 579.2265) supported a molecular
formula of C23H40O15.
The sugar moieties of 2 were determined to be D-glucose and D-xylose in a ratio of
2:1, as determined by acid hydrolysis described in the part of ‘3.5. Acid hydrolysis’,
and their configurations of the component monosaccharides were determined by GC
1
analysis. The H-NMR spectrum (Supplementary material Table S1) showed a methyl
proton at dH 0.85 (3H, t, J ¼ 7.6 Hz), two methylene protons at dH 2.00 (2H, m) and 2.
13 (2H, m), an oxymethylene protons at dH 3.63 (1H, overlapped) and 3.47 (1H, over-
lapped), two olefin proton signals at dH 5.16 (1H, dt, J ¼ 16.0, 6.0 Hz) and 5.40 (1H, dt,
J ¼ 16.0, 6.0 Hz), in addition to the carbon signal at dC 13.9, 25.7, 31.1, 68.5, 127.0, and
135.0, revealed the aglycone of 2 was also 3-hexenol; signals for three anomeric pro-
tons at dH 4.21 (1H, d, J ¼ 6.0 Hz), 4.32 (1H, d, J ¼ 7.6 Hz), and 4.38 (1H, d, J ¼ 8.0 Hz).
On the basis of 13C-NMR, DEPT, HSQC, HMBC correlations and chemical reactions, two
b-D-glucopyranose (dC 101.1, 82.0, 75.8, 69.5, 76.9, 63.0 and dC 104.0, 70.4, 76.0, 67.4,
75.2, 67.8) and one b-D-xylcopyranose (dC 103.3, 71.9, 75.7, 69.2, 64.7) were identified.
In the HMBC spectrum, key correlation peaks were observed between the proton sig-
nal at dH 4.32 (H-10) and the carbon resonance signal at dC 68.5 (C-1), dH 4.38 (H-100),
and dC 82.0 (C-20), and between the proton signal dH 4.21 (H-1000) and dC 67.8 (C-600).
All in all, compound 2 could be determined as a 3-hexenol glycoside, as well. Analysis
of the 1H- and 13C-NMR spectra established that the NMR spectra of compound 2
were very similar to 1, except for a few signals due to a different double bond config-
uration. Compared with 1, the Z-geometry of the D3-double bond of 1 changed into
E-geometry of 2, which could be deduced from the large coupling constant observed
for H-3 and H-4 (J ¼ 16.0 Hz).
Therefore, the structure of 2 was determined as (E)-hex-3-en-1-ol O-b-D-xylcopyrano-
syl-(1-6)-b-D-glucopyranosyl-(1-2)-b-D-glucopyranoside (Figure 1), and the NMR spectra
data are shown in Supplementary material Table S1.

4
Z. SHU ET AL.
In this activity study, the antibacterial activity of compound 1 and 2 was quantita-
tively assessed by determining the MIC and MBC against seven bacterial strains,
including five Gram-positive bacteria (including Staphylococcus aureus ATCC 26112,
Staphylococcus epidermidis ATCC 27342, Staphylococcus saprophyticus ATCC 24582,
Enterococcus faecium ATCC 35667, and Streptococcus pneumoniae NCTC 7465) and two
Gram-negative bacteria (including Pseudomonas aeruginosa ATCC 27853 and
Escherichia coli ATCC 87394). Unfortunately, we found that two compounds did not
possessed antibacterial potential against each of the tested strains.
3. Experimental
3.1. General
Open column chromatography (Metrailler-Ruchonnet et al. 2010) was carried out using
silica gel (200-300 mesh, Qingdao Marine Chemical Co. (Qingdao, China). TLC
employed precoated silica gel plates (5–7 lm, Qingdao Marine). Preparative HPLC was
carried out on a Waters 600 instrument equipped with a Waters UV-2487 detector. A
Waters Sunfire prep ODS (19 ꢀ 250 mm i.d.) column was used for this purpose. The
1
HR-ESI-MS was determined on IonSpec Ultima 7.0 T FTICR MS. The H-, 13C- and 2D-
NMR (HSQC and HMBC) spectra were recorded on a Bruker AMX-400 spectrometer
using standard pulse sequences. Chemical shifts are reported in ppm (d), and scalar
coupling is reported in Hz. Beef extract, peptone, and agar powder were purchased
from AoboxingBio-techCo. (Beijing, China). Seven bacterial strains, including five Gram-
positive bacteria, Staphylococcus aureus (ATCC 26112), Staphylococcus epidermidis
(ATCC 27342), Staphylococcus saprophyticus (ATCC 24582), and Enterococcus faecium
(ATCC 35667), Streptococcus pneumoniae (NCTC 7465), and two Gram-negative bac-
teria, Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 87394) were
obtained from Beijing ZK Kangtai Biological Co. (Beijing, China). Other reagents were
purchased from various commercial sources.
3.2. Plant material
The calyces of Physalis alkekengi L. var. franchetii. were collected from Shangzhi,
Heilongjiang province in February 2017 and identified by Prof. Wei Ma (Heilongjiang
University of Traditional Chinese Medicine, Heilongjiang, China). The voucher specimen
was authenticated and is deposited in the Heilongjiang University of Chinese Medicine
(No.2017-02-1).
3.3. Extraction and isolation
The air-dried calyces of Physalis alkekengi L. var. franchetii (10 kg) were extracted two
times with 70% EtOH for 2 h at 60 ꢁC. The solvent was evaporated to dryness, and the
dry residue (2.1 kg) was subjected to AB-8 macroporous adsorptive resin, eluted with
water, 50% EtOH, and 95% EtOH respectively to yield three fractions (Fractions 1 ꢂ 3)
(Shu et al. 2016). Fraction 2 (250 g) was separated by column chromatography on silica
gel eluting with CH2Cl2-MeOH (10:1, 5:1, 1:1, and MeOH) to give nine fractions

NATURAL PRODUCT RESEARCH
5
(Fr.1ꢂFr.9). Fr.9 was separated by column chromatography on silica gel eluting with
CH2Cl2-MeOH (8:1, 5:1, and MeOH) to yield three fractions (Fr.9-1ꢂFr.9-3). Fr.9-2 was sep-
arated by column chromatography on silica gel eluting with CH2Cl2-MeOH (9:1 and
MeOH) to yield give twelve fractions (Fr.9-2-1ꢂFr.9-2-12). Fr.9-2-4 was further purified by
HPLC [MeOH-H2O (30:70 v/v)] to give 1 (101 mg, 0.0404%) and 2 (134 mg, 0.0536%).
The thin-layer chromatography of compound 1 and 2 showed a grey-brown spot (Rf
0.58 ꢂ 0.60) with H2SO4/C2H5OH (1:9, v/v) as chromogenic reagent and the upper layer
of n-butyl alcohol/acetic acid/H2O (4:1:5, v/v/v) as developing solvent.
3.4. New compounds information
Compound 1: [a]D : ꢃ56.7 ꢁC (c ¼ 1.00, MeOH); HR-ESI-MS: m/z 579.2279 [M þ Na]þ;
24
1H-NMR (DMSO-d6, 400 MHz) and 13C-NMR data (DMSO-d6, 100 MHz), shown in
Supplementary material Table S1.
Compound 2: [a]D : 57.8 ꢁC (c ¼ 1.00, MeOH); HR-ESI-MS: m/z 579.2273 [M þ H]þ;
24
1H-NMR (DMSO-d6, 400 MHz) and 13C-NMR data (DMSO-d6, 100 MHz), shown in
Supplementary material Table S1.
3.5. Acid hydrolysis
Solution of compound 1 (8 mg) and 2 (8 mg) in MeOH-HCl (1:1) was heated in a water
bath at 90 ꢁC for 2 h. After cooling, the solution was diluted to 20 mL with water and
then extracted with CH2Cl2 (20 mL ꢀ 2) (Yang et al. 2014). Then, the water layer was
neutralized with 1 M aqueous KOH. After concentration, each layer was subjected to
silica gel thin layer chromatography (TLC), together with the standard samples. N-
BuOH-AcOH-H2O and CHCl3-MeOH-H2O were used as the developing solvents and O-
phthalic acid-aniline was used as the detection reagent. Glucose and xylose were
detected. The reduced sugars were dissolved in 0.5 mL pyridine, and then 0.5 mL hex-
amethyl disilazane and 0.2 mL trimethylchlorosilane were added and the mixture was
heated at 20 ꢁC for 15 min. After that the sample was extracted with H2O (2 mL). Each
aqueous layer was examined by GC, H2 flame ionization detector. The column tem-
perature was programmed from 60 to 260 ꢁC at a rate of 10 ꢁC/min. Nitrogen was
used as carrier gas at 1.5 mL/min. The injector and detector temperature were at
280 ꢁC and the injection volume was 1 lL with the split ratio being 10:
1. The standard monosaccharides were subjected to the same reaction and GC analysis
under the same condition. The derivatives of D-glucose and D-xylose were detected by
comparison of its retention times with those of the standard sample.
3.6. Antibacterial activity
The antibacterial activities of compound 1 and 2 were evaluated by determining the
minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)
in vitro. The MIC of compound 1 and 2 for the isolated bacterial strains were deter-
mined by 96 well-plates dilution method as previously described with as light modifi-
cation (Shu et al. 2016). Briefly, bacterial strains were grown on Mueller-Hinton (MH)

6
Z. SHU ET AL.
agar plates and suspended in MH broth. The inoculum suspensions were prepared
from 6 h broth cultures incubation and adjusted to obtain a 0.5 McFarland standard
turbidity and were then diluted 1000-fold with the respective medium to the concen-
tration of 1.5 ꢀ 105 CFU/Ml (Lu et al. 2011). Two folds serial diluted concentrations of
compound 1 and 2 were added in MH broth ranging from 0.01 to 5 mg/mL. To adjust
the interference by plant pigments, a parallel series of mixtures with uninoculated
broth was prepared. The bacterial suspensions were aerobically incubated for 18 h at
37 ꢁC. Triplicate samples were performed for each test concentration. MIC was defined
as the lowest concentration inhibiting visible growth. The MBC determination was car-
ried out by spreading 0.1 mL of the cultures in each well without visible growth onto
sample free MH agar and incubated for 18 h. MBC was considered as the highest dilu-
tion at which almost bacterial inoculum was killed. The experiments were performed
in triplicate.
Disclosure statement
The authors declare that there are no conflicts of interest.
Funding
This study was supported by the National Natural Science Foundation of China (Grant Nos.
81603366), the Natural Science Foundation of Heilongjiang (Grant Nos. H2015041), the
Foundation of China Postdoctoral Science (Grant Nos.2017M610816), and the Foundation of
Postdoctoral Science of Chinese Academy of Medical Sciences & Peking Union Medical
College (2016).
References
Ge Y, Duan Y, Fang G, Zhang Y, Wang S. 2009. Study on biological activities ofPhysalis alkeken-
givar.franchetipolysaccharide. J Sci Food Agric. 89(9):1593–1598.
Kang H, Kwon SR, Choi HY. 2011. Inhibitory effect of Physalis alkekengi L. var. franchetii extract
and its chloroform fraction on LPS or LPS/IFN-c-stimulated inflammatory response in periton-
eal macrophages. J Ethnopharmacol. 135(1):95–101.
Li AL, Chen BJ, Li GH, Zhou MX, Li YR, Ren DM, Lou HX, Wang XN, Shen T. 2018. Physalis alke-
kengi L. var. franchetii (Mast.) Makino: An ethnomedical, phytochemical and pharmacological
review. J Ethnopharmacol. 210:260–274.
Li X, Zhang C, Wu D, Tang L, Cao X, Xin Y. 2012. In vitro effects on intestinal bacterium of phys-
alins from Physalis alkekengi var. Francheti. Fitoterapia. 83(8):1460–1465.
Li X, Zhao J, Yang M, Liu Y, Li Z, Li R, Li X, Li N, Xu Q, Khan IA, et al. 2014. Physalins and witha-
nolides from the fruits of Physalis alkekengi L. var. franchetii (Mast.) Makino and the inhibitory
activities against human tumor cells. Phytochem Lett. 10:95–100.
Lu Y, Joerger R, Wu C. 2011. Study of the chemical composition and antimicrobial activities of
ethanolic extracts from roots of Scutellaria baicalensis Georgi. J Agric Food Chem. 59(20):
10934–10942.
Metrailler-Ruchonnet I, Pagano A, Carnesecchi S, Khatib K, Herrera P, Donati Y, Bron C,
Barazzone C. 2010. Bcl-2 overexpression in type II epithelial cells does not prevent hyperoxia-
induced acute lung injury in mice. Am J Physiol Lung Cell Mol Physiol. 299(3):L312–322.
Ooi KL, Muhammad TS, Sulaiman SF. 2013. Physalin F from Physalis minima L. triggers apop-
tosis-based cytotoxic mechanism in T-47D cells through the activation caspase-3- and c-myc-
dependent pathways. J Ethnopharmacol. 150(1):382–388.

NATURAL PRODUCT RESEARCH
7
Shu Z, Xing N, Wang Q, Li X, Xu B, Li Z, Kuang H. 2016. Antibacterial and anti-inflammatory
activities of Physalis Alkekengi var. franchetii and its main constituents. Evid Based
Complement Alternat Med. 2016:4359394–4359403.
Sugimoto K, Matsui K, Iijima Y, Akakabe Y, Muramoto S, Ozawa R, Uefune M, Sasaki R, Alamgir
KM, Akitake S, et al. 2014. Intake and transformation to a glycoside of (Z)-3-hexenol from
infested neighbors reveals a mode of plant odor reception and defense. Proc Natl Acad Sci
USA. 111(19):7144–7149.
Suzuki N, Miyase T, Ueno A. 1993. Phenylethanoid glycosides of Sesamum indicum.
Phytochemistry. 34(3):729–732.
Wang A, Wang S, Zhou F, Li P, Wang Y, Gan L, Lin L. 2018. Physalin B induces cell cycle arrest
and triggers apoptosis in breast cancer cells through modulating p53-dependent apoptotic
pathway. Biomed Pharmacother. 101:334–341.
Yang BY, Guo R, Li T, Liu Y, Wang CF, Shu ZP, Wang ZB, Zhang J, Xia YG, Jiang H, et al. 2014.
Five withanolides from the leaves of Datura metel L. and their inhibitory effects on nitric
oxide production. Molecules. 19(4):4548–4559.
Yang J, Yang F, Yang H, Wang G. 2015. Water-soluble polysaccharide isolated with alkali from
the stem of Physalis alkekengi L.: structural characterization and immunologic enhancement in
DNA vaccine. Carbohydr Polym. 121:248–253.
Zheng X, Kuo G, De-Qiang D, Gui-Rong C, Ting-Guo K, Yu-Yuan S, Xing-Tai L, Feng D. 2010. A
new hexenol glycoside from leaves of Smallanthus sonchifolius. Nat Prod Res. 24(17):
1592–1597.