Two New Phenolic Glycosides from Homalium stenophyllum

Article abstract of DOI:10.1007/s10600-021-03445-4

A phytochemical study of the stems of Homalium stenophyllum Merr. & Chun led to the isolation of two new phenolic glycosides (1 and 2) and three known compounds (3–5). The molecular structures of these isolated new phenolic glycosides (1 and 2) were unambiguously established on the basis of comprehensive spectroscopic methods. All the isolated new compounds were evaluated for their cytotoxicities against three human cancer cell lines: HeLa, A-549, and MCF-7 in vitro.

Full text of DOI:10.1007/s10600-021-03445-4

DOI 10.1007/s10600-021-03445-4
Chemistry of Natural Compounds, Vol. 57, No. 4, July, 2021
TWO NEW PHENOLIC GLYCOSIDES FROM Homalium stenophyllum
1
1
2
Shou-Yuan Wu, Zi-Ming Chen, Guang-Ying Chen,
2
*
3*
Xiao-Ping Song, and Chang-Ri Han
A phytochemical study of the stems of Homalium stenophyllum Merr. & Chun led to the isolation of two new
phenolic glycosides (1 and 2) and three known compounds (3–5). The molecular structures of these isolated
new phenolic glycosides (1 and 2) were unambiguously established on the basis of comprehensive
spectroscopic methods. All the isolated new compounds were evaluated for their cytotoxicities against three
human cancer cell lines: HeLa, A-549, and MCF-7 in vitro.
Keywords: Homalium stenophyllum, phenolic glycosides, cytotoxicities.
The genus Homalium (Flacourtiaceae) is comprised of approximately 130 species that are scattered throughout the
subtropical and tropical regions. There are about 12 species of this genus found in China, growing from the southwest to
Taiwan.
Previous chemical investigations on this genus led to the separation and identification of a series of natural products,
containing phenolic glycosides, xanthones, iridoids, triterpenoids, coumarins, and alkaloids, which exhibited a broad range of
biological activities, such as antibacterial, antioxidant, antiviral, antiplasmodial, anti-inflammatory, and antitumor activity
[
1–15]. H. stenophyllum Merr. & Chun has been used in China as a folk medicine for gonorrhea and as an astringent and is
only distributed in China′s Hainan Island [16]. As part of our ongoing research into structurally diverse and bioactive compounds
from this species, two new phenolic glycosides, homastenoside H (1) and homastenoside I (2), along with three known analogues
(
3–5), were separated from the stems of H. stenophyllum. The chemical structures of 1 and 2 were unambiguously established
by means of comprehensive spectral data analyses. The known analogues 3–5 were determined by comparing their experimental
spectral data with those data described in the corresponding literature. In addition, two new phenolic glycosides (1, 2) were
evaluated for their cytotoxicities against three human cancer cell lines in vitro. In this paper, the extraction, separation, structure
elucidation, and cytotoxic activities of these isolated phenolic glycosides will be described in detail.
The 95% EtOH extract of the stems of H. stenophyllum was suspended in water and extracted successively with petroleum
ether and EtOAc. The EtOAc extract fraction was repeatedly subjected to silica gel, Sephadex LH-20, reversed-phase C18
silica gel CC, and semipreparative HPLC to yield five phenolic glycosides, including two new ones.
Compound 1 was obtained as a yellow amorphous powder. Its molecular formula was determined as C H O by
3
2 34 13
+
HR-ESI-MS m/z 627.2075 [M + H] (calcd 627.2072), indicating 16 degrees of unsaturation. The IR spectrum exhibited
characteristic absorption bands at 3440 and 1755 cm , which were indicative of hydroxy and carbonyl functionalities,
respectively. The UV maxima at 288 and 231 nm indicated that 1 possessed aromatic rings. The C NMR and DEPT data
revealed the existence of 32 carbon atoms, including 20 sp carbon atoms, nine sp methines, and three sp methylenes, which
–
1
1
3
2
3
3
were attributable to one benzyl alcohol group, two benzoyl groups, one glucopyranosyl moiety, and one xylofuranosyl moiety.
1) School of Chemistry and Chemical Engineering, Key laboratory of Clean Energy Materials Chemistry of Guangdong
Higher Education Institutes, Lingnan Normal University, Zhanjiang, P. R. China; 2) Key Laboratory of Tropical Medicinal
Resource Chemistry of Ministry of Education, Hainan Normal University, Haikou, P. R. China, e-mail: sxp628@126.com;
3
) Key Laboratory of Medicinal and Edible Plant Resources of Hainan Province, Hainan Institute of Science and Technology,
Haikou, P. R. China, e-mail: hchr116@126.com. Published in Khimiya Prirodnykh Soedinenii, No. 4, July–August, 2021,
pp. 566–569. Original article submitted September 10, 2020.
©
0
009-3130/21/5704-0663 2021 Springer Science+Business Media, LLC
663

OH
HO
5''''
5''
1
''''
4''''
HO
OH
O
OH
O
1
''
O
3''
6
'''
1
O
O
5
'''
7''
O
6
O
OH
OH
1
'''
2
O
6
''
7'
7
7
1
''
''
O
OH
5'
O
3'''
1'
1
O
7'
O
O
1'''
O
2
6
'''
3''
5'''
3
'
1
'
5
HO
OH
6
'
3'''
3'
OH
1
2
COSY
HMBC
Fig. 1. Selected 2D NMR correlations for compounds 1 and 2.
The above data of 1 showed a partial structure similar to that of chaenomeloidin [17], but with an additional benzoyl group and
a xylofuranose unit. The connectivity between the second benzoyl moiety and the glucose unit was indicated by the HMBC
correlation from H-4′′′ (δ 4.87) to the ester carbonyl carbon (δ 165.2, C-7′′). The HMBC correlation from H -6′′′ (δ 3.06 and
2
2
.90) to C-1′′′′ (δ 104.0) established unambiguously the linkage between the glucose unit and the xylofuranosyl moiety. Thus,
the two sugar units were linked via C-1′′′′ and C-6′′′. In order to further confirm the structure of 1, the acid hydrolysis
reaction of 1 was carried out, and the sugar derivatives were analyzed by HPLC, which indicated the presence of D-xylose and
D-glucose in 1. The relative large coupling constants (J = 7.6 and 7.6 Hz) of the two sugars indicated the β-anomeric orientation
of the D-xylose and D-glucose. Detailed analysis of 2D NMR spectra confirmed the structure of 1 as shown in Fig. 1.
+
The molecular formula of compound 2 was determined as C H O on the basis of HR-ESI-MS [M + Na] at
2
7 30 12
1
13
m/z 569.1636 (calcd 569.1635). Its H and C NMR data exhibited similar signals to those of homaloside D (3) [2], except for
the additional methine (δ 3.25, H-6′′; δ 73.3, C-6′′) group of C-6′′ in 2 and the absence of a keto carbonyl group of C-6′′ in
H
C
homaloside D (3). Elucidation of the 2D NMR of 2 demonstrated that the carbon C-6′′ was connected to a hydroxyl group
because of the HMBC correlations of H-2′′ (δ 5.55) to C-6′′/C-4′′ (δ 22.5), H -4′′ (δ 2.13 and 1.90) to C-2′′ (δ 127.3)/C-6′′,
2
1
1
and H-6′′ to C-2′′/C-4′′. At the same time, the H– H COSY correlations of H-5′′ (δ 1.86) with H-4′′/H-6′′ also supported that
assignment. Furthermore, the coupling constant of the anomeric proton resonating at δ 4.69 (1H, d, J = 7.6 Hz, H-1′′′) suggested
that the glucopyranosyl moiety was β-glucoside. In order to further confirm the structure of 2, the acid hydrolysis
reaction of 2 was carried out, and the monosaccharide derivatives were analyzed by HPLC, which indicated the presence
of D-glucose in 2. However, the stereochemistry at C-1′′ and C-6′′ remained unclear because of the facile aromatization
of the 1,6-dihydroxycyclohex-2-enoyl)carboxylate unit throughout acid and/or base treatment according to the literature [4].
Finally, compound 2 was established as shown in Fig. 1 and named homastenoside I.
In addition to the two new compounds 1 and 2, three other known phenolic glycosides were identified, homaloside
D (3) [2], 4-hydroxy-2-{[(benzoyl)oxy]methyl}phenyl-β-D-glucopyranoside-6-benzoate (4) [14], and itoside H (5) [18], by
comparing their experimental and reported physical data with those in the literature.
Compounds 1 and 2 were evaluated for their cytotoxicity towards the HeLa, A-549, and MCF-7 cell lines using the
MTT method. They exhibited no cytotoxicity on the growth of the cell lines (IC50 > 40 μM), which indicated that they can be
developed into other agents with low toxicity.
O
O
O
O
O
OH
OH
O
O
R
O
O
O
O
O
O
HO
OH
HO
OH
OH
OH
OH
3
4, 5
4: R = H; 5: R = OH
664

EXPERIMENTAL
General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter.
IR spectra were obtained on a Nicolet 6700 spectrophotometer. UV spectra were recorded on a Beckman DU 640
spectrophotometer. NMR spectra were run on Bruker 400 MHz spectrometers using TMS as an internal standard. HR-ESI-MS
spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer. Semipreparative high-performance liquid
chromatography (HPLC) was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse
XDB-C18 column (250 × 9.4 mm, 5 μm) and (250 × 4.6 mm, 5 μm). Silica gel (Qing Dao Hai Yang Chemical Group Co.;
2
00–300 mesh) and octadecylsilyl silica gel (YMC; 50 μm) were used for column chromatography (CC). Precoated silica gel
plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography (TLC).
Plant Material. The stems of Homalium stenophyllum were collected from JianFengLing Nature Reserve, Hainan
Province of China, inAugust 2015, and identified by Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal University.
Avoucher specimen (No. SONG20150818) has been deposited at the Key Laboratory of Tropical Medicinal Resource Chemistry
of Ministry of Education, Hainan Normal University.
Extraction and Isolation. The air-dried and powdered stems (20 kg) of H. stenophyllum were extracted with 95%
EtOH (3 × 20 L, 5 days each) at room temperature. The solvent was evaporated in vacuum to obtain a crude extract. After
suspension in water, the crude extract was extracted successively with petroleum ether and ethyl acetate. The ethyl acetate
extract (1000.0 g) was subjected to silica gel column chromatograph and eluted with petroleum ether–ethyl acetate (1:0–0:1),
yielding seven fractions (Frs.1–7). Fraction 7 (156.4 g) was subjected to RP-18 eluting with CH OH–H O (from 20% to
3
2
1
00%) to afford eight subfractions (Frs.7A–7H). Subfraction 7E (14.0 g) was purified using Sephadex LH-20 and eluted with
CH OH, then on a silica gel column chromatography and eluted with CHCl –CH OH, 10:1, to yield a new Subfr. 7E-1 (659 mg),
3
3
3
followed by semipreparative HPLC using an Agilent Eclipse XDB column with 30% CH CN–H O to afford 2 (10.2 mg) and
3
2
3
(20.1 mg). Subfraction 7E-3 (96 mg) followed by semipreparative HPLC using a Agilent Eclipse XDB column with 45%
CH OH–H O to obtain 1 (22.3 mg). Subfraction 7H (3.0 g) was purified using Sephadex LH-20 and eluted with CH OH, then
3
2
3
on a silica gel column chromatography and eluted with CHCl –CH OH, 10:1, to yield a new Subfr. 7H-2 (59 mg), followed
3
3
by semipreparative HPLC using aAgilent Eclipse XDB column with 40% CH CN–H O to obtain 4 (10.2 mg) and 5 (5.7 mg).
3
2
2
4
Homastenoside H (1). Yellow amorphous powder; [α] –45.4° (c 0.10, CH OH). UV (CH OH, λmax, nm) (log ε):
88 (3.00), 231 (3.10). IR (KBr, ν , cm ): 3440, 2930, 1755, 1602, 1520, 1487, 1136, 1064, 988. HR-ESI-MS m/z 627.2075
D
3
3
–
1
2
[
max
+
1
M + H] (calcd for C H O , 627.2072). H NMR (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.90 (1H, dd, J = 11.6, 6.4,
32 35 13 6
H-6′′′β), 3.06 (1H, dd, J = 11.6, 2.0, H-6′′′α), 3.15 (1H, m, H-2′′′′), 3.31 (1H, m, H-5′′′), 3.66 (1H, m, H-2′′′), 3.71 (1H,
m, H-4′′′′), 3.79 (2H, br.s, H-5′′′′), 3.92 (1H, m, H-3′′′′), 4.38 (1H, d, J = 7.6, H-1′′′′), 4.51 (1H, d, J = 13.2, H-7β), 4.65 (1H,
d, J = 13.2, H-7α), 4.87 (1H, dd, J = 9.2, 8.8, H-4′′′), 5.13 (1H, d, J = 7.6, H-1′′′), 5.30 (1H, dd, J = 9.6, 9.2, H-3′′′), 7.04 (1H,
td, J = 8.0, 2.0, H-5), 7.15 (1H, dd, J = 8.0, 2.0, H-3), 7.23 (1H, td, J = 8.0, 2.0, H-4), 7.40 (1H, dd, J = 8.0, 2.0, H-6), 7.51 (1H,
td, J = 8.0, 2.0, H-3′′), 7.51 (1H, td, J = 8.0, 2.0, H-5′′), 7.56 (1H, td, J = 8.0, 2.0, H-3′), 7.56 (1H, td, J = 8.0, 2.0, H-5′), 7.63
(
8
1H, td, J = 8.0, 2.0, H-4′′), 7.65 (1H, td, J = 8.0, 2.0, H-4′), 7.86 (1H, dd, J = 8.0, 2.0, H-2′′), 7.86 (1H, dd, J = 8.0, 2.0, H-6′′),
1
3
.05 (1H, dd, J = 8.0, 2.0, H-2′), 8.05 (1H, dd, J = 8.0, 2.0, H-6′). C NMR (100 MHz, DMSO-d , δ, ppm): 131.6 (C-1), 154.2
6
(
C-2), 114.8 (C-3), 127.7 (C-4), 122.0 (C-5), 127.2 (C-6), 58.1 (C-7), 130.9 (C-1′), 129.4 (C-2′), 128.5 (C-3′), 133.1 (C-4′),
1
1
7
28.5 (C-5′), 129.4 (C-6′), 165.3 (C-7′), 130.3 (C-1′′), 129.4 (C-2′′), 128.3 (C-3′′), 132.7 (C-4′′), 128.3 (C-5′′), 129.4 (C-6′′),
65.2 (C-7′′), 100.6 (C-1′′′), 71.5 (C-2′′′), 76.1 (C-3′′′), 78.2 (C-4′′′), 67.5 (C-5′′′), 65.3 (C-6′′′), 104.0 (C-1′′′′), 71.3 (C-2′′′′),
6.5 (C-3′′′′), 75.1 (C-4′′′′), 59.4 (C-5′′′′).
2
4
Homastenoside I (2). White amorphous powder; [α] –72.1° (c 0.10, CH OH). UV (CH OH, λmax, nm) (log ε): 272
D
3
3
–
1
(
[
3.68), 218 (4.16). IR (KBr, ν , cm ): 3356, 2889, 1721, 1598, 1156, 1473,1277, 1065, 714. HR-ESI-MS m/z 569.1636
M + Na] (calcd for C H O Na, 569.1635). H NMR (400 MHz, DMSO-d , δ, ppm, J/Hz): 1.86 (2H, m, H-5′′), 1.90 (1H,
max
+
1
2
7
30 12
6
m, H-4′′β), 2.13 (1H, m, H-4′′α), 3.25 (1H, overlapped, H-6′′), 3.26 (1H, overlapped, H-2′′′), 3.30 (1H, m, H-3′′′), 3.66 (1H,
m, H-5′′′), 3.74 (1H, m, H-4′′′), 4.27 (1H, dd, J = 11.6, 6.4, H-6′′′β), 4.62 (1H, dd, J = 11.6, 2.0, H-6′′′α), 4.69 (1H, d,
J = 7.6, H-1′′′), 5.12 (2H, br.s, H-7), 5.55 (1H, d, J = 10.4, H-2′′), 5.79 (1H, d, J = 10.4, H-3′′), 6.46 (1H, dd, J = 8.8, 2.4, H-4),
6
.78 (1H, d, J = 2.4, H-6), 6.94 (1H, d, J = 8.8, H-3), 7.56 (1H, t, J = 8.0, H-3′), 7.56 (1H, t, J = 8.0, H-5′), 7.69 (1H, t, J = 8.0,
1
3
H-4′), 7.97 (1H, d, J = 8.0, H-2′), 7.97 (1H, d, J = 8.0, 2.0, H-6′). C NMR (100 MHz, DMSO-d , δ, ppm): 126.8 (C-1), 147.0
6
(
1
(
C-2), 117.1 (C-3), 114.4 (C-4), 152.6 (C-5), 114.6 (C-6), 61.0 (C-7), 129.7 (C-1′), 129.2 (C-2′), 128.7 (C-3′), 133.4 (C-4′),
28.7 (C-5′), 129.2 (C-6′), 165.5 (C-7′), 75.8 (C-1′′), 127.3 (C-2′′), 129.4 (C-3′′), 22.5 (C-4′′), 26.1 (C-5′′), 73.3 (C-6′′), 172.9
C-7′′), 102.2 (C-1′′′), 70.3 (C-2′′′), 76.3 (C-3′′′), 72.2 (C-4′′′), 73.7 (C-5′′′), 64.3 (C-6′′′).
665

Acid Hydrolysis of the New Compounds. A solution of each compound (3 mg) in 2 N HCl (10 mL) was refluxed at
00°C for 4 h. The reaction mixture was evaporated to obtain a residue and extracted with an equal volume of chloroform and
1
water.After decompression drying of the water layer, the residue was dissolved with anhydrous pyridine (1 mL). After addition
of L-cysteine methyl ester hydrochloride (3 mg), the solution was heated at 60°C for 1 h. After the reaction, o-tolyl isothiocyanate
(
30 μL) was added and the whole heated at 60°C for another 1 h. The reaction mixture was finally analyzed by HPLC under the
following conditions: chromatographic column ZORBAX Eclipse XDB-C18 (250 × 4.6 mm, 5 μm); test wavelength 250 nm;
column temperature 30°C; mobile phase CH CN–0.05% HCOOH–H O (25:75,); flow rate 0.8 mL/min. The standard sugars
3
2
were treated in the same manner and gave the t (min): D-glucose (18.824), L-glucose (17.247), D-xylose (21.866), L-xylose
R
(
20.313). The absolute configurations of the sugars obtained from the acid hydrolysate of each compound were determined by
comparing the retention time of monosaccharide derivatives to the derivatives of the standard sugars in HPLC analysis.
Cytotoxicity Bioassays. The following human tumor cell lines were used: HeLa, A-549, and MCF-7. All cells were
cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone)
in 5% CO at 37.8°C. The cytotoxicity assay was performed using the MTT method in 96-well microplates [19]. Briefly,
2
adherent cells (100 μL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug
5
addition, and suspended cells were seeded just before drug addition with an initial density of 1 × 10 cells/mL. Each tumor cell
line was exposed to the tested compound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM in triplicate for 48 h. Cisplatin
(
Sigma, St. Louis, MO, USA) was used as positive control. After treatment, the cell viability was measured and the cell growth
curve plotted. IC50 values were calculated by the Reed and Muench method [20].
ACKNOWLEDGMENT
This work was financially supported by the National Natural Science Foundation of China (Nos. 21362009, 81360478,
and 21302181), International S&T cooperation Program of China (ISTCP) (No. 2014DFA40850), Hainan special
Project for TCM Modernization (No. 2015ZY19), Science and Technology Planning Project of Guangdong Province
(
Nos. 2016A030307021 and 2018A0303070005), and Special program for Natural Science Talents of Lingnan Normal
University (No. 1170919617).
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