Fragranone C: a new dihydrochalcone glucopyranoside from Anneslea fragrans twigs

Article abstract of DOI:10.1080/14786419.2020.1747459

A phytochemical investigation of an ethanolic extract of Anneslea fragrans twigs collected from Thailand resulted in the discovery of a new dihydrochalcone glucopyranoside named fragranone C (1), together with six previously reported compounds. The structural elucidation of the new compound was achieved by HRFABMS, NMR spectroscopic analysis and acid hydrolysis. (Figure presented.).

Full text of DOI:10.1080/14786419.2020.1747459

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Fragranone C: a new dihydrochalcone
glucopyranoside from Anneslea fragrans twigs
Ashraf M. Omar, Dya Fita Dibwe, Sijia Sun, Ahmed M. Tawila, Min Jo Kim,
Ampai Phrutivorapongkul, Naoki Toyooka & Suresh Awale
To cite this article: Ashraf M. Omar, Dya Fita Dibwe, Sijia Sun, Ahmed M. Tawila, Min Jo
Kim, Ampai Phrutivorapongkul, Naoki Toyooka & Suresh Awale (2020): Fragranone C: a new
dihydrochalcone glucopyranoside from Annesleaꢀfragrans twigs, Natural Product Research, DOI:
10.1080/14786419.2020.1747459
To link to this article: https://doi.org/10.1080/14786419.2020.1747459
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1747459
Fragranone C: a new dihydrochalcone glucopyranoside
from Anneslea fragrans twigs
Ashraf M. Omara, Dya Fita Dibwea, Sijia Suna, Ahmed M. Tawilaa, Min Jo Kima,
Ampai Phrutivorapongkulb, Naoki Toyookac,d and Suresh Awalea
aDivision of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, Toyama,
b
c
Japan; Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand; Graduate School of
d
Science and Engineering, University of Toyama, Toyama, Japan; Graduate School of Innovative Life
Science, University of Toyama, Toyama, Japan
ABSTRACT
ARTICLE HISTORY
Received 28 January 2020
Accepted 15 March 2020
A phytochemical investigation of an ethanolic extract of Anneslea
fragrans twigs collected from Thailand resulted in the discovery of
a new dihydrochalcone glucopyranoside named fragranone C (1),
together with six previously reported compounds. The structural
elucidation of the new compound was achieved by HRFABMS,
NMR spectroscopic analysis and acid hydrolysis.
KEYWORDS
Anneslea fragrans;
dihydrochalcone; fragranone
1. Introduction
Anneslea fragrans Wall. (Theaceae) is a tree native to Southeast Asia. Traditionally, the
leaves and bark are used to treat fever, diarrhoea and liver inflammation, and showed
antiplasmodial activity (Nguyen-Pouplin et al. 2007). Our previous study on A. fragrans
leaves collected from Thailand resulted in the discovery of two new chalcones with
unique structures and promising cancer cell migratory inhibitory activity (Omar et al.
2019). As there has been not sufficient investigation on the phytochemical constitu-
ents from the whole plant parts, in the present study, A. fragrans twigs collected from
the Chiang Mai Province of Thailand, was subjected to a phytochemical study which
resulted in the isolation of a new dihydrochalcone glucopyranoside named fragranone
C (1), together with six previously reported compounds. The reported compounds
CONTACT Suresh Awale
suresh@inm.u-toyama.ac.jp
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1747459.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
A. M. OMAR ET AL.
Figure 1. Structure of the compounds isolated from Anneslea fragrans twigs.
were identified as davidioside (2) (Jensen et al. 1977), davidigenin (3) (Chen et al.
2011), betulinic acid (4) (Tala et al. 2013), (epi)catechin (5) (Galotta et al. 2008), isosco-
ꢀ
poletin (6) (Zhao et al. 2014), (þ)-rengyolone (7) (Busque et al. 2003) (Figure 1).
Herein, we describe the isolation, structure elucidation of the new compound by spec-
troscopic techniques.
2. Results and discussion
Fragranone C (1) was isolated as a white amorphous powder. Its molecular formula
was determined by HRFABMS to be C22H26O10 [m/z 451.16484 (M þ H)þ]. The UV
spectrum of 1 showed absorption maxima at 201, 230 and 263 nm. The IR spectrum
of 1 showed absorption bands for hydroxy (3365 cmꢀ1) and a,b-unsaturated ketone
1
(1604 cmꢀ1) groups. The H spectrum of 1 displayed the presence of two ABX-type
phenyl rings [dH 6.78, (d, J ¼ 1.5 Hz, H-2), 6.68 (d, J ¼ 7.9 Hz, H-5), 6.64 (dd, J ¼ 1.5,
7.9 Hz, H-6)] and [dH 6.70 (d, J ¼ 2.1 Hz, H-30), 6.50 (dd, J ¼ 2.1, 9.0 Hz, H-50), 7.59 (d, J
¼ 9.0 Hz, H-60)] along with two mutually coupled methylenes [dH 3.33 (td, J ¼ 1.8, 7.8
Hz, H2-a) and 2.88 (t, J ¼ 7.8 Hz, H2-b)]. These data suggested the presence of a
1
3,4,20,40-tetrahydoxy dihydrochalcone skeleton (de Almeida et al. 1979). H NMR spec-
trum also showed the presence of an anomeric proton [dH 5.00 (d, J ¼ 7.3 Hz, H-100)]
and two diastereotopic protons [dH 3.72 (dd, J ¼ 5.5, 11.9 Hz, H-600b), 3.91 (dd, J ¼
1
1.8, 11.9 Hz, H-600a)] corresponding to a b-glucose moiety. Moreover, H-NMR spectrum
showed the presence of a methoxy group (dH 3.81, s). 13C and DEPT NMR spectra of 1,
also exhibited the signals corresponding to 3,4,20,40-tetrahydoxy dihydrochalcone and
a b-glucose unit, together with a methoxy group (dC 56.73). In the HMBC spectrum of
1 (Figure S1, Supporting Information), the correlation from H-10’ to C-20 (dC 160.62)
confirmed the location of the b-glucose moiety to be attached to C-20 of the dihydro-
chalcone unit. The HMBC correlation observed from the methoxy protons to C-3 sug-
gested the placement of the methoxy group attached to C-3. This was further
confirmed by the presence of the NOESY correlation between the methoxy protons
and H-2 (Figure S2, Supporting Information). The relative configuration of 1 was

NATURAL PRODUCT RESEARCH
3
assigned by analysing the coupling constants and NOESY NMR spectrum (Figure S2,
0
Supporting Information). The coupling constant J1’’,2 ’ (7.3 Hz) was in agreement with
the trans-diaxial disposition of H-10’ (a) and H-20’ (b). The presence of the NOESY corre-
lations H-10’/H-300, H-100/H-500 and H-500/H-600a suggested their a-axial orientation, while
correlations between H-400/H-600b indicated their b-axial orientation. Finally, acid
hydrolysis of 1 was carried out to confirm the absolute configuration of the glucose
moiety, which yielded a sugar with [a]25 þ48.5 (c 0.04, H2O) (Figure S11, Supporting
D
Information), similar to those reported for D-glucose having [a]25 þ52 (c 0.02, H2O)
D
(Kim et al. 2015). Thus, the structure of 1 was assigned as 3-methoxy-4,20,40-trihydroxy
dihydrochalcone-20-O-b-D-glucopyranoside. Natural dihydrochalcones, with methoxyla-
tion and glucosylation in the A-ring, have been previously reported (Fuendjiep et al.
2002; Yao et al. 2005). To the best of our knowledge, this is the first report in the
nature of a B-ring methoxylated 20-glucosylated dihydrochalcone.
All isolated compounds were tested for preferential cytotoxicity against the PANC-1
human pancreatic cancer cell line utilising the antiausterity strategy (Awale et al.
2006). The isolated compounds were not active at the maximum tested concentration
of 100 mM.
3. Experimental
3.1. General experimental procedures
Optical rotations and IR spectra were measured using a JASCO P-2100 digital polarim-
eter and a JASCO FT/IR-460 Plus spectrophotometer, respectively. An U-5100 UV ꢀ
Visible ratio-beam spectrophotometer (Hitachi) was used to record the UV/Vis spec-
trum. A JEOL ECX400 Delta spectrometer was used to record the NMR spectra. A JEOL
€
JMS-AX505HAD mass spectrometer was used for the HRFABMS measurement. A Buchi
MPLC C-605 binary gradient pump system using normal-phase silica gel (silica gel 60
N, Kanto Chemical, Japan) was used to perform medium-pressure liquid chromatog-
raphy (MPLC). Analytical and preparative TLC was carried out on pre-coated silica gel
60 F254 and RP-18F254 plates (0.25 or 0.50 mm, Merck KGaA, Darmstadt, Germany) and
pre-coated silica gel 70 PF254 plates (0.75 mm, Wako Pure Chemical Industries,
Ltd., Japan).
3.2. Plant material
Twigs of A. fragrans were collected in Omoli district, Chiang Mai province, Thailand, in
November 2017. The plant material was identified by Mr. Wittaya Pongamornkul, and
a voucher specimen (NDD2017-AFT) has been deposited in Natural Drug Discovery
Laboratory, University of Toyama, Japan.
3.3. Extraction and isolation procedures
Shade-dried twigs (257 g) of A. fragrans were ground to a fine powder then extracted
ꢂ
with 95% EtOH in three cycles (three days/cycle) at room temperature (ꢁ26 C). The
extracts were combined and concentrated under reduced pressure to yield an

4
A. M. OMAR ET AL.
ethanolic extract (15.5 g). The extract (15.4 g) was suspended in H2O (200 mL), and
partitioned successively with EtOAc (4 ꢃ 200 mL) to give EtOAc (4.1 g) and H2O (11 g)
soluble fractions, respectively. The EtOAc-soluble fraction was chromatographed on sil-
ica gel with MPLC using a CH2Cl2–EtOAc gradient mixture (0 ꢀ 100%) then
EtOAc–MeOH gradient system (0 ꢀ 100%) to give ten fractions (Fr.1, 457 mg; Fr.2, 174
mg; Fr.3, 31 mg; Fr.4, 282 mg; Fr.5, 50 mg; Fr.6, 420 mg; Fr.7, 117 mg; Fr.8, 61 mg;
Fr.9, 1.8 g; Fr.10, 594 mg). Fraction 8 was obtained as pure (þ)-rengyolone (7, 61 mg).
Fraction 2 (174 mg) was recrystallized from a CH2Cl2–EtOAc solvent mixture to give
betulinic acid (4, 25 mg). The supernatant of fraction 2 (140 mg) was purified by pre-
parative TLC with a CH2Cl2–EtOAc solvent mixture (9:1) to give isoscopoletin (6, 2.5
mg). Fraction 3 (31 mg) was also purified by preparative TLC with an n-hexane–EtOAc
solvent mixture (1:1) to give davidigenin (3, 6 mg). Fraction 6 (420 mg) was rechroma-
tographed on silica gel by MPLC using a CH2Cl2–MeOH gradient system (0 ꢀ 100%) to
give three subfractions (Fr.6-1, 125 mg; Fr.6-2, 72 mg; Fr.6-3, 204 mg). Subfraction 6-2
(72 mg) was further purified by preparative TLC using a CH2Cl2–MeOH solvent mixture
(9:1) to give (epi)catechin (5, 14 mg). Fraction 9 (1.8 g) was rechromatographed on RP-
silica gel by MPLC using an eluent system consisted of solvent A [H2O] and solvent B
[CH3CN-Ac (1:1)], with a gradient (0-100% B) to give four subfractions (Fr.9-1, 73 mg;
Fr.9-2, 950 mg; Fr.9-3, 68 mg; Fr.9-4, 647 mg). Subfraction 9-2 (950 mg) was further
chromatographed on silica gel by MPLC using an EtOAc–MeOH gradient system
(0–100%) to give three subfractions (Fr.9-2-1, 790 mg; Fr.9-2-2, 71 mg; Fr.9-2-3, 39 mg).
Subfraction 9-2-2 (71 mg) was purified by preparative TLC with a CH2Cl2/MeOH solvent
mixture (88:12) to give davidioside (2, 6 mg). Subfraction 9-3 (68 mg) was purified by
preparative TLC with an EtOAc–MeOH solvent mixture (94:6) to give fragranone C (1,
5 mg).
Fragranone C (1): white amorphous powder; [a]25 –45.5 (c 0.5, MeOH); UV (MeOH)
D
kmax (log e) 201 (5.31), 230 (4.96), 263 (4.73) nm; IR ꢀmax (KBr) 1604, 3365 cmꢀ1; H
1
NMR (400 MHz, CD3OD): dH 7.59 (1H, d, J ¼ 9.0 Hz H-60), 6.78 (1H, d, J ¼ 1.5 Hz, H-2),
6.70 (1H, d, J ¼ 2.1 Hz, H-30), 6.68 (1H, d, J ¼ 7.9 Hz, H-5), 6.64 (1H, dd, J ¼ 1.5, 7.9, H-
6), 6.50 (1H, dd, J ¼ 2.1, 9.0, H-50), 5.00 (1H, d, J ¼ 7.3 Hz, H-100), 3.91 (1H, dd, J ¼ 1.8,
11.9 Hz, H-600a), 3.81 (3H, s, OCH3), 3.72 (1H, dd, J ¼ 5.5, 11.9 Hz, H-600b), 3.50-3.53 (1H,
m, H-200), 3.46–3.50 (1H, m, H-500), 3.44–3.60 (1H, m, H-400), 3.39–3.42 (1H, m, H-300), 3.33
(td, J ¼ 1.8, 7.8 Hz, H2-a) 2.88 (2H, t, J ¼ 7.8 Hz, H2-b). 13C NMR (100 MHz, CD3OD): dC
203.14 (CO), 164.81 (C-40), 160.62 (C-20), 149.08 (C-3), 145.84 (C-4), 134.86 (C-1), 133.59
(C-60), 122.09 (C-10), 121.99 (C-6), 116.35 (C-5), 113.44 (C-2), 111.13 (C-50), 104.11 (C-30),
102.83 (C-100), 78.71 (C-300), 78.56 (C-500), 75.22 (C-200), 71.48 (C-400), 62.78 (C-600), 56.73
(OCH3), 46.51 (C-a), 31.76 (C-b). HRFABMS m/z 451.16484 [M þ H]þ (calcd for
C22H27O10, 451.16038).
3.4. Acid hydrolysis of 1
ꢂ
Fragranone C (1, 1.7 mg) was heated with 6% HCl (1 mL) for 5 h at 80 C. The reaction
mixture was extracted with CH2Cl2 (4 ꢃ 3 mL), and the aqueous phase was dried by
using the Genevac EZ-2 vacuum centrifuge. The resulting residue was purified by pre-
parative TLC with CH2Cl2/MeOH/H2O solvent mixture (8:5:1) to yield glucose (0.5 mg),

NATURAL PRODUCT RESEARCH
1
5
[a]25 þ48.5 (c 0.04, H2O). The identification of glucose was made through H NMR
D
spectroscopic analysis (Gurst 1991).
3.5. Preferential cytotoxicity assay against PANC-1 cells
Preferential cytotoxicity of A. vulgaris extract and the isolated compounds were deter-
mined by a procedure described previously (Awale et al. 2006).
4. Conclusions
In conclusion, the phytochemical investigation of A. fragrans twigs resulted in the dis-
covery of a new dihydrochalcone glucopyranoside (1) along with six previously
reported compounds. The structural elucidation of the new compound was achieved
by HRFABMS, NMR spectroscopic analysis and acid hydrolysis. From the screening of
the isolated compounds for preferential cytotoxicity against PANC-1 cells, none of the
compounds were found active at the maximum tested concentration of 100 mM.
Acknowledgments
Authors thank the Egyptian-Japanese Education partnership (EJEP) fellowship for supporting
AMO and AMT.
Disclosure statement
The authors reported no potential conflict of interest.
Funding
This work was supported by the Japanese Society for the Promotion of Science (JSPS), Japan,
Kakenhi (16K08319), and Kobayashi International Scholarship to SA.
ORCID
Suresh Awale
http://orcid.org/0000-0002-5299-193X
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