Phenyl glycosides from the leaves of Flacourtia indica (Burm. f.) Merr (Salicaceae)

Article abstract of DOI:10.1016/j.phytochem.2021.112891

Thirteen phenolic glycosides, together with fourteen various known compounds, were isolated from the methanolic extract of leaves of Flacourtia indica. Twelve of these were composed of gentisyl or salicyl alcohols, glycosylated on the phenol and acylated on the primary alcohol with various more or less oxidized forms of pyrocatechuic acid. A number of positions on the glucose or on the acid were further acylated by benzoic or cinnamic acid. In addition to these, a glucoside of a phenyl propanoid was also isolated. The gross structures were elucidated by spectroscopic means including 1D and 2D NMR experiments and HR-ESI-MS analyses. Several of these structures, for example, xylosmin, were previously described but it proved extremely difficult to conclude on their exact identity with the absence of clear data on absolute configuration in the literature.

Full text of DOI:10.1016/j.phytochem.2021.112891

Phytochemistry 190 (2021) 112891
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phenyl glycosides from the leaves of Flacourtia indica (Burm. f.)
Merr (Salicaceae)
,
,*
Phuc-Dam Nguyena b, Charlotte Sayagha, Georges Massiota, Catherine Lavauda
a
´
Equipe Chimie des Substances Naturelles, Institut de Chimie Moleculaire de Reims, UMR CNRS 7312, BP 1039, Reims Cedex 2, 51687, France
b Department of Chemistry Education, School of Education, Can Tho University, Can Tho City, 94115, Viet Nam
A R T I C L E I N F O
A B S T R A C T
Thirteen phenolic glycosides, together with fourteen various known compounds, were isolated from the meth-
anolic extract of leaves of Flacourtia indica. Twelve of these were composed of gentisyl or salicyl alcohols, gly-
cosylated on the phenol and acylated on the primary alcohol with various more or less oxidized forms of
pyrocatechuic acid. A number of positions on the glucose or on the acid were further acylated by benzoic or
cinnamic acid. In addition to these, a glucoside of a phenyl propanoid was also isolated. The gross structures
were elucidated by spectroscopic means including 1D and 2D NMR experiments and HR-ESI-MS analyses. Several
of these structures, for example, xylosmin, were previously described but it proved extremely difficult to
conclude on their exact identity with the absence of clear data on absolute configuration in the literature.
This contribution is dedicated to the memory of
the late François (alias Frank) Polidano.
Keywords:
Flacourtia indica
Salicaceae
Structural characterization
Phenolic glucosides
Flacourtiosides
Xylosmin
Xylosmic acid
1. Introduction
cough, bronchitis, pneumonia, and phthisis. The juice of the fresh leaves
is used in fevers and in various infections like dysentery, diarrhoea, and
Flacourtia indica (Burm. f.) Merr. is a branched, deciduous and
dioecious shrub or small tree usually up to 5 m high and belonging to the
Salicaceae family, formerly Flacourtiaceae. This species is endemic to
many countries in Africa and Asia and is also commonly known as “In-
dian plum”, “Governor’s plum”, “Madagascar plum”. The ripe fruits are
consumed or are dried and stored as food. The taxonomy of F. indica is
complex with 23 synonyms, and this species can therefore be considered
in a broad sense (Lim, 2013; POWO "Plants of the World Online, 2021).
As well as their ubiquitous metabolites, Flacourtia species contain
phenolic glucoside esters like flacourside, poliothyroside, flacourtio-
sides A-F and xylosmin (Bhaumik et al., 1987; Sashidhara et al., 2013,
2014; Chai et al., 2009; Alakolanga et al., 2014; Ghavre et al., 2017;
Bourjot et al., 2012; Gibbons et al., 1995). The present report describes
the isolation and structural identification of four flavonoids, three caf-
feoylquinic acids, two megastigmane glycosides, two lignans, and
fourteen glycosides of benzyl alcohol derivatives including nine unde-
scribed compounds (5–13) and four recognized compounds (1–4), from
the methanolic extract of leaves of F. indica. All parts of this plant are
used as traditional medicines worldwide for the treatment of various
health ailments. In Ayurvedic medicine, the leaves are used in asthma,
against intestinal worms. Leaf decoctions are used for gynaecological
disorders and hydrocele. In Bengal, the leaves are said to be effective
against snakebites and are taken to treat schistosomiasis, malaria, and
diarrhoea (Lim, 2013). Other various uses are also reported for the bark,
roots and stems of this plant by the locals of the Madhya Pradesh state
(Tiwari, 2017). These numerous traditional uses seem to be linked to the
high antioxidant activity of the extracts of F. indica (Singh et al., 2017a).
Extracts and phenolic compounds of F. indica show a suppressive or
mediated activity in oxidative stress process like cytotoxicity, cardio-
vascular disease, liver damage and diabetes (Akter et al., 2020; Sarkar
et al., 2020; Perera et al., 2018; Palani et al., 2012). Miscellaneous ac-
tivities include hepatoprotective activity of the leaf extracts (Gran-
aprakash et al., 2010; Idoko and Emmanuel, 2019), antidyslipidemic
(Singh et al., 2016), anticancer (Pachute et al., 2011), and anthelmintic
activity (Sreejith et al., 2013). Compounds from the leaves and aerial
parts exhibited intense and selective antimalarial against Plasmodium
falciparum (Kaou et al., 2010; Sashidhara et al., 2013; Singh et al.,
2017b). As reported by some of the authors of these articles, it is difficult
to rationalize the many traditional activities of F. indica due to the
paucity of pharmacology studies and characterization of effective
* Corresponding author.
E-mail address: catherine.lavaud@univ-reims.fr (C. Lavaud).
https://doi.org/10.1016/j.phytochem.2021.112891
Received 3 May 2021; Received in revised form 22 July 2021; Accepted 23 July 2021
Available online 3 August 2021
0031-9422/© 2021 Elsevier Ltd. All rights reserved.

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
phytomolecules. Into attempt to predict the pharmacological activities,
six compounds isolated from F. indica were analyzed through an in silico
approach to determine their pharmacokinetic profiles, health effects and
prediction of diverse pharmacological activities (Hussain et al., 2016).
Eight of the isolated compounds have a common skeleton composed
of a glucopyranosyl unit linked at position 2 of a gentisyl alcohol and a
more or less oxidized form of pyrocatechuic acid. Although these com-
pounds have been isolated on numerous occasions, the lack of hard
evidence on their stereochemistry, makes their structures questionable
´
(Prange et al., 2021). We have attempted to rationalize these elements of
stereochemistry for the isolated compounds and those newly described.
These phenolic glucosides confirm the classification of F. indica in the
Salicaceae family (Mosaddik et al., 2007; Boeckler et al., 2011), and
support the variety of traditional uses of F. indica. Unfortunately, partial
results on isolation of four known compounds and undescribed com-
pound 13, have been prematurely published in an unreferenced journal
and under the wrong denomination of Flacourtia rukam (Dam et al.,
2016).
2. Results and discussion
2.1. General and structure identification of the known compounds
The leaves of F. indica were successively extracted by percolation
with solvents of increasing polarity: petroleum ether, chloroform, ethyl
acetate, methanol and 80 % aqueous methanol to obtain five extracts.
The methanolic extract was purified by using a combination of chro-
matographic techniques: VLC (vacuum liquid chromatography), flash
chromatography and semi-preparative HPLC to yield fourteen known
compounds and thirteen compounds (1–13) (Fig. 1).
Among the known isolated compounds, luteolin (Kubola et al., 2011),
mururin A (Sashidhara et al., 2013), 3-O-E-caffeoylquinic acid, 4-O-E-
caffeoylquinic acid, 5-O-E-caffeoylquinic acid (Alakolanga et al., 2014),
and scolochinenoside D (Bourjot et al., 2012), were previously isolated
from F. indica or synonyms thereof. 7-O-β-D-glucopyranosyl-lute
olin, 7-O-β-D-glucuronopyranosyl-luteolin, 3-O-β-D-galactopyranosyl-
quercetol, blumenol C, 9-O-(6′-O-
α-L-rhamnopyranosyl)-β-D-glucopy
ranosyl-blumenol C, and 4-O-β-D-glucopyranosyl-syringaresinol were
isolated for the first time and identified by analysis of their NMR and MS
spectra and by comparison with literature data (Dictionary of Natural
Products, 2017; see Supplementary Material). The structures of two
further glucosides were elucidated by comparison with the literature:
itoside
A α-L-rhamnopy
(Chai et al., 2007), and the 2-O-(6′-O-
ranosyl)-β-D-glucopyranosyl-salicylic acid methyl ester (Chassagne et al.,
1997).
2.2. Structures of compounds 1–4
The known compounds were accompanied by a set of phenolic gly-
cosides (1–12) among which compounds (1–4) were related to xylosmin
(Gibbons et al., 1995) (Fig. 1). The X-ray data of xylosmin was reex-
´
aminated to propose a definitive structure (Prange et al., 2021) (Fig. 2).
Fig. 1. Compounds 1–13 isolated from Flacourtia indica; for 13: tentative ab-
Compound 3 had the spectral characteristics of xylosmin (MS, UV and
NMR). The 1H NMR spectrum was recorded in deuteriated acetone as in
the original publication and a perfect match with an average 0.03 ppm
discrepancy for the glucose due to the small percentage of H2O con-
tained in the acetone. In the absence of a possible direct comparison of
samples, it could be said that there was no noticeable difference between
3 and xylosmin. However, the optical rotation of xylosmin was reported
as ꢀ 4 but our compound gave a value of +14. Xylosmin, independently
isolated from Flacourtia ramontchi (syn. F. indica), had a rotation of ꢀ 6
(Bourjot et al., 2012, Marc Litaudon, personal communication). There
was also a perfect match between the experimental NMR of this com-
pound and ours.
solute configuration.
benzoate at position 6′ of the glucose and therefore resembled 2-(2′-O-
benzoyl-β-D-glucopyranosyl)-7-(1′′α,2′′α,6′′α-trihydroxy-3′′-oxocyclohex-
4′′-ene-1′′-carbonyl)-5-hydroxybenzyl alcohol previously isolated from
F. indica (Sashidhara et al., 2013). Compound 2 was an isomer of 1,
benzoylated at position 6′ of the glucose instead of 2′ and the best match
was obtained with 2-(6′-O-benzoyl-β-D-glucopyranosyl)-7-(1′′α,2′′α
,
6
′′α-trihydroxy-3′′-oxocyclohex-4′′-ene-1′′-carbonyl)-5-hydroxybenzyl
alcohol (Shaari and Waterman, 1995). Compound 4 benzoylated at posi-
tion 6′ of the glucose but also at position 2′′ of the cyclohexene moiety, was
2-(6′-O-benzoyl-β-D-glucopyranosyl)-7-(2′′α-benzoyloxy-1′′α
,
Compounds 1, 2 and 4 were closely related to xylosmin according to
6
′′α-dihydroxy-3′′-oxocyclohex-4′′-ene-1′′-carbonyl)-5-hydroxybenzyl
mass and 13C NMR spectrometry (Table 1). Compound 1 lacked the
2

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
(2) [
α
]
D = +13 (literature = ꢀ 7) and (3) [
α
]
D = +14.7 (xylosmin = ꢀ 4).
No optical rotation was given for compound 4. It was thus impossible to
conclude on the novelty or not of these compounds and to ascertain their
relative and absolute configurations.
It is worth noting at this stage that if the structures of our compounds
1–4 remain uncertain, as far as the absolute configuration of the
cyclohexenone (named xylosmic acid) is concerned, those of related
compounds 1, 2 and 4 from the literature, also need to be secured. The
results of the hydrolysis of 1, which ought to have resolved the problem,
have been questioned (Ghavre et al., 2017) and failed to be reproduced
here on a similar compound.
Fig. 2. The structure of xylosmin.
2.3. Structures of other compounds related to xylosmin
alcohol. (Shaari and Waterman, 1995; Singh et al., 2017b).
To the best of our knowledge, compounds (5–12) have never been
described (Fig. 1). They all possessed one sugar unit in their structures,
which was identified as D-glucose by acid hydrolysis of the methanolic
extract, separation by semi-preparative HPLC, and chiral HPLC analysis
in comparison with authentic enantiomers. Their gross structure was
related to itoside A and xylosmin and differed by the nature and number
In agreement with Sashidhara et al. (2013), the sugar was identified
as D-glucose after hydrolysis and chiral HPLC comparison with an
authentic sample. Comparison of the optical rotations of our isolates
with those of the literature did not allow definitive settling of the
identity of the pairs of compounds: (1) [α]D = +15 (literature = ꢀ 34.6),
Table 1
NMR spectroscopic data (600 MHz, CD3OD) for 1, 2, 3 and 4.
position
1
2
3
4
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
1
2
3
4
5
6
7
127.4
149.0
118.4
116.3
153.7
116.3
63.4
127.9
149.5
119.5
116.3
153.7
116.7
64.2
128.1
149.2
119.3
116.6
154.2
116.8
63.8
127.8
150.0
120.0
117.0
154.0
117.5
64.5
7.07 d (8.8)
7.06 d (8.8)
6.90 d (8.9)
6.79 d (8.8)
6.69 dd (8.8, 3.0)
6.54 dd 8.8, 3.0)
6.38 dd (8.9, 3.0)
6.25 dd (8.8, 3.0)
6.81 d (3.0)
5.19 d (12.9)
5.06 d (12.9)
6.84 d (3.0)
5.38 d (12.5)
5.32 d (12.5)
6.67 d (3.0)
4.90 d (12.3)
4.83 d (12.3)
6.59 d (3.0)
5.16 s
127.8
β-D-glucopyranose
1’
2’
3’
4’
5’
6’
5.16 d (8.0)
101.5
75.4
75.6
71.2
78.0
62.0
4.80 d (7.5)
103.7
74.6
77.5
71.7
75.1
65.0
5.07 d (8.0)
102.0
75.6
76.0
72.3
75.7
65.3
4.54 d-like (7.5)
3.37 m
104.4
74.9
77.9
72.0
75.5
65.4
5.27 dd (9.7, 8.0)
3.83 dd (9.7, 8.7)
3.57 dd (9.7, 8.7)
3.53 ddd (9.7, 5.3, 2.0)
3.96 dd (12.0, 2.0)
3.79 dd (12.0, 5.3)
3.52 dd (8.7, 7.5)
3.51 dd (9.0, 8.7)
3.45 dd (9.7, 9.0)
3.73 ddd (9.7, 7.6, 2.1)
4.72 dd (11.7, 2.1)
4.45 dd (11.7, 7.6)
5.17 dd (9.2, 8.0)
3.76 t (9.2)
3.37 m
3.50 brt (9.4)
3.30 m
3.77 ddd (9.8, 7.6, 2.1)
4.67 dd (11.8, 2.1)
4.39 dd (11.8, 7.6)
3.55 ddd (9.6, 7.6, 2.1)
4.59 dd (11.7, 2.1)
4.54 dd (11.7, 7.6)
7-O-cyclohexenone
1"
85.6
85.8
85.9
83.1
2"
4.45 s
76.9
4.57 s
77.0
4.27 s
77.3
5.85, s
76.7
3"
198.1
126.8
150.6
71.4
198.0
126.8
150.5
71.5
198.5
127.2
151.0
71.7
191.2
126.0,
149.8,
70.7
4"
6.02 dd (10.4, 2.6)
6.74 dd (10.4, 2.0)
4.92 brt (2.3)
6.04 dd (10.4, 2.6)
6.75 dd (10.4, 2.0)
4.99 brt (2.3)
5.99 dd (10.4, 2.6)
6.58 dd (10.4, 2.0)
4.75 brt (2.3)
6.10, dd (10.4, 2.6)
6.83, dd (10.4, 2.0)
5.07, brt (2.3)
5"
6"
7"
171.7
172.0
172.1
170.2
2′-O-benzoyl
1
130.8
130.5
129.3
134.1
129.3
130.5
167.0
131.3
130.9
129.7
134.5
129.7
130.9
167.4
2
8.12 dd (8.5, 1.3)
8.01 dd (8.4, 1.2)
7.41 brt (8.0)
3
7.50 ddd (8.5, 7.5, 1.3)
7.61 dt (7.5, 1.3)
4
7.53 brt (7.8)
5
7.50 ddd (8.5, 7.5, 1.3)
8.12 dd (8.5, 1.3)
7.41 brt (8.0)
6
8.01 dd (8.4, 1.2)
7
6′-O-benzoyl
1
130.9
130.3
129.2
134.0
129.2
130.3
167.4
131.2
130.7
129.7
134.4
129.7
130.7
167.8
131.3
130.7
129.7
134.4
129.7
130.7
167.8
2
8.02 dd (8.3, 1.3)
7.52 brt (8.0)
7.93 dd (8.3, 1.3)
7.38 brt (8.0)
7.91 d (7.5)
7.40 t (7.5)
7.53 t (7.5)
7.40 t (7.5)
7.91 d (7.5)
3
4
7.65 td (7.5, 1.3)
7.52 brt (8.0)
7.49 brt (7.8)
5
7.38 brt (8.0)
6
8.02 dd (8.3, 1.3)
7.93 dd (8.3, 1.3)
7
2′′-O-benzoyl
1
2
3
4
5
6
7
130.3
131.1
129.5
134.5
129.5
131.1
166.5
7.84 d (7.5)
7.29 t (7.5)
7.44 t (7.5)
7.29 t (7.5)
7.84 d (7.5)
3

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
of ester substituents.
5-hydroxybenzyl alcohol, for which the name flacourtioside J is
proposed.
The positive HR-ESI-MS of compound 5 exhibited a sodium adduct
ion peak [M+Na]+ at m/z 703.1645 (calc. for C34H32O15Na 703.1639)
in agreement with a C34H32O15 composition as in xylosmin. NMR
spectral data showed identical characteristic signals for the 2,5-dihy-
droxybenzyl alcohol moiety, for a β-D-glucopyranosyl unit, for two
benzoic acid esters, and for a 1,2,6-trihydroxy-3-oxocyclohex-4-en-1-
carboxylic acid (Table 2). As in xylosmin, one benzoate was posi-
tioned at C-2′ of glucose, while the second was linked to the cyclo-
hexenone ring as indicated by the HMBC cross-peak observed between
the deshielded H-2′′ (δ 5.77) and the second benzoyl ester carbonyl
carbon at δ 166.5, a feature already encountered in compound 4. The 1H
NMR spectra of compounds 4 and 5 had similar chemical shifts except
for the signals of the xylosmic acid part, which were ca 1 ppm more
deshielded than the corresponding protons in 3. The structure of 5 was
therefore assigned as 2-(2′-O-benzoyl-β-D-glucopyranosyl)-7-(2′′-O-
benzoyl-1′′,6′′-dihydroxy-3′′-oxo-cyclohex-4′′-ene-1′′-carbonyl)-5-
hydroxybenzyl alcohol, for which the name flacourtioside G is proposed.
The molecular formula of compound 6 was established as C43H38O18
with a sodium adduct ion peak [M+Na]+ detected at m/z 865.1951
(calc. for C43H38O18Na 865.1956) in its positive HR-ESI-MS. The 1H and
13C-NMR spectral data of 6 were similar to those of 5 but showed a
higher complexity due to the presence of a supplementary ester group
(Table 2). It was identified as an E-caffeoyl group by the observation of
an ABX aromatic system at δ 7.10 (d, 1.9 Hz, H-2), 6.83 (d, 8.2 Hz, H-5)
and 6.99 (dd, 8.2 and 1.9 Hz, H-6) and of the two protons of a E-
disubstituted double bond at δ 7.64 (d, 15.8 Hz, H-7) and 6.34 (d, 15.8
Hz, H-8) connected to a carbonyl ester at δ 169.0 (C-9). This caffeate
ester was located at position C-6′ of glucose by the HMBC cross-peaks
observed between deshielded glucosidic protons CH2-6′ at δ 4.58 (dd,
11.9 and 1.8 Hz, H-6′a) and 4.44 (dd, 11.9 and 6.6 Hz, H-6′b) and the
caffeate ester carbonyl group. This evidence led to the identification of
compound 6 as 2-(2′-O-benzoyl-6′-O-E-caffeoyl-β -D-glucopyranosyl)-7-
(2′′-O-benzoyl-1′′,6′′-dihydroxy-3′′-oxo-cyclohex-4′′-ene-1′′-carbonyl)-5-
hydroxybenzyl alcohol, named flacourtioside H.
The molecular formula of compound 9 was C27H28O13 according to
the observation of a sodium adduct ion peak [M+Na]+ at m/z 583.1425
(calc. for C27H28O13Na 583.1428) in the positive HR-ESI-MS. The NMR
spectral data of 9 and 2 showed a high degree of similarity and shared
the same 6-benzoyl glucose and xylosmic acid. However, the 2,5-dihy-
droxybenzyl alcohol was here replaced by a 2-hydroxybenzyl alcohol
showing four aromatic protons between δ 6.88–7.29. The structure of 9
was determined as 2-(6′-O-benzoyl-β-D-glucopyranosyl)-7-(1′′,2′′,6′′-
trihydroxy-3′′-oxo-cyclohex-4′′- ene-1′′-carbonyl)-benzyl alcohol, for
which we propose the name flacourtioside K.
Compound 10 had the same molecular formula
C27H28O13
([M+Na]+ at m/z 583.1433 (calc for C27H28O13Na 583.1428)) as com-
pound 9. NMR spectral data of 10 showed the signals of the 6-O-benzoyl-
β-D-glucose and the presence of a 2,5-dihydroxybenzyl alcohol moiety
(instead of 2-hydroxybenzyl alcohol) (Table 3). Differences between the
two compounds were located on the cyclohexenone ring, which in the
1H NMR spectrum exhibited only one singlet at δ 4.69 (H-2′′) and a CH2
at δ 2.65 (dd, 19.3 and 5.5 Hz, H-6′′eq) and 3.09 (dt, 19.3 and 2.7 Hz, H-
6′′ax). All these fitted into a 1,2-dihydroxy-3-oxo-cyclohex-4-enoyl ring
and therefore, compound 10 was identified as 2-(6′-O-benzoyl-β-D-glu-
copyranosyl)-7-(1′′,2′′-dihydroxy-3′′-oxo-cyclohex-4′′-ene-1′′-carbonyl)-
5-hydroxybenzyl alcohol. It may also be seen as 2′-O- desbenzoyl-6-
deoxyxylosmin for which the trivial name flacourtioside L is proposed.
The positive HR-ESI-MS of compound 11 displayed a sodium adduct
ion peak [M+Na]+ at m/z 687.1692 (calc. for C 34H32O14Na 687.1690)
corresponding to the molecular formula C 34H32O14. Comparison of the
NMR spectra of 11 and 10 showed a high degree of similarity, especially
including the high field methylene of the cyclohexenone ring. Two
major differences were signals for the presence of an extra benzoyl group
and a singlet for H-2′′of the cyclohexenone at δ 5.94 instead of 4.69
(Table 3). This latter signal exhibited HMBC correlations with the ben-
zoate carbonyl and with the cyclohexenone ketone, which led to the
proposal of 2-(6′-O-benzoyl-β-D-glucopyranosyl)-7-(2′′-O-benzoyl-1′′-
hydroxy-3′′-oxo-cyclohex-4′′-ene-1′′-carbonyl)-5-hydroxybenzyl alcohol
for compound 11 or 2′′-benzoyl-flacourtioside L. To the best of our
knowledge, the acid appendage has never been described before
The positive HR-ESI-MS of compound 7 displayed a sodium adduct
ion peak [M+Na]+ at m/z 761.1701 (calc. for C36H34O17Na 761.1694)
corresponding to a molecular formula C36H34O17. In comparison with
the preceding compound, 7 lacks a benzoyl group (104 uma) and its 1H
and 13C-NMR spectra showed the absence of a benzoate at position C-2
of the xylosmic acid ring (Table 2). Thus, the structure of 7 was iden-
although derivatives of the similar α-hydroxy cyclohexanone carboxylic
acid were found in Idesia polycarpa (Kim et al., 2014).
Compound 12 showed a sodium adduct ion peak [M+Na]+ at m/z
727.1626 (calc. for C36H32O15Na 727.1639) in the positive HR-ESI-MS,
in agreement with a molecular formula C 36H32O15. Despite severe
overlap, the 1H-NMR spectrum could be disentangled to reveal the
presence of the usual elements including a benzoate, a caffeate, glucose
and 2,5-dihydroxy-benzyl alcohol (Table 3). The characteristic signals of
the xylosmic acid were missing and were replaced by a C7H3O4 fragment
which was identified as a 2,3-dihydroxybenzoic acid to account for the
observation of three vicinal aromatic protons as two doublets of dou-
blets at 7.15 and 6.98 (8.0, 1.5 Hz) and a sharp triplet at 6.65 (8.0 Hz).
This dihydroxybenzoic acid, also named pyrocatechuic acid is part of
lignin but had also been isolated as a natural product (Sakushima et al.,
1995). Sequencing was performed as in the previous compounds by
assignment of 3J HMBC correlations of the ester carbonyls with the
protons of the adjacent rings. Therefore, compound 12 was identified as
2-(2′-O-benzoyl-6′-E-caffeoyl-β-D-glucopyranosyl)-7-(2′′,3′′-dihydrox-
ybenzoyl)-5-hydroxybenzyl alcohol, named here flacourtioside M.
tified
as
2-(2′-O-benzoyl-6′-O-E-caffeoyl-β-D-glucopyranosyl)-7-
(1′′,2′′,6′′-trihydroxy-3′′-oxo-cyclohex-4′′-ene-1′′-carbonyl)-5-hydrox-
ybenzyl alcohol, for which the name flacourtioside I is proposed.
Compound 8 possessed a C36H34O15 molecular formula yielding an
HR-ESI positive sodium adduct ion peak [M+Na]+ at m/z 729.1791
1
(calc. for C36H34O15Na, 729.1795). Examination of the H NMR spec-
trum of 8 showed the presence of signals for a glucose, a benzoate, 2,4-
dihydroxy benzyl alcohol, xylosmic acid and an E-cinnamate instead of a
benzoate ester (Table 2). The cinnamate was characterized by two sharp
doublets at δ 7.67 (16.0 Hz, H-7) and 6.42 (16.0 Hz, H-8) and a high
order A2B3 system for the five aromatic protons at δ 7.57 (m, H-2 and H-
6) and 7.40 (m, H-3, H-4 and H-5). These building blocks were
sequenced by the observation of 3J HMBC correlations between the ester
carbonyls and deshielded protons such as H-2′′ (δ 5.85, s), CH2-7 (δ 5.30
and 5.36, AB quartet), and the CH2 of glucose (δ 4.69 and 4.38). Thus, as
in xylosmin and compounds 1, 3, 5, 6 and 7, HMBC correlations be-
tween the carbonyl at δ 167.3 and the two protons H-6’ of glucose at δ
4.69 (dd, 11.7 and 2.2 Hz, H-6′a) and 4.38 (dd, 11.7 and 7.3 Hz, H-6′b)
allowed location of a benzoate ester at position C-6′. In an analogous
fashion, a correlation between the cinnamate carbonyl (δ 166.3) and
xylosmic acid H-2′′ (sharp singlet at 5.85) characterized this linkage,
while the third carbonyl of the xylosmic acid (δ 171.2) correlated with
the methylene of the benzyl alcohol (δ 5.36 and 5.30). Thus, the struc-
ture of 8 was elucidated as 2-(6′-O-benzoyl-β-D-glucopyranosyl)-7-(2′′-
O-E-cinnamoyl-1′′,6′′-dihydroxy-3′′-oxo-cyclohex-4′′-ene-1′′-carbonyl)-
2.4. Structure of a phenylpropanoid glycoside
Compound 13, a phenylpropanoid glucoside, was different from all
the others. Its structure was published in preliminary form (Dam et al.,
2016) and the full details are presented here. Its composition was
established as C18H28O11 as shown by a sodium adduct molecular ion
peak [M+Na]+ at m/z 443.1537 (calc. for C18H28O11Na 443.1529) in
the positive HR-ESI-MS mode. A particular feature of the structure was
4

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
Table 2
NMR spectroscopic data (600 MHz, CD3OD) for 5, 6, 7, 8 and 9.
Position
5
6
7
8
9
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
δH mult. (J in
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
Hz)
1
2
3
4
127.2
149.7
117.5
115.4
127.7a
149.8a
120.1
117.0
127.8
148.8
119.0
116.3
127.5
149.5
119.3
116.6
126.5
156.8
131.0
130.7
6.75, d (8.9)
6.76, d (8.8)
6.32, dd (8.8,
2.9)
7.02, d (8.9)
6.62, dd (8.9,
2.9)
6.92, d (8.8)
7.05, brd (8.2)
6.31, dd (8.9, 2.9)
6.39, dd (8.8, 3.0)
6.97, td (8.2, 1.5)
5
6
7
153.8
115.8
64.1
154.0
117.4
64.4
153.9
116.3
63.4
153.7
117.2
63.8
6.88, td (8.2, 1.5)
7.29, dd (8.2, 1.5)
5.31, d (12.5)
123.6
116.9
64.6
6.53, d (2.9)
4.90, d (12.3)
4.83, d (12.3)
6.65, d (2.9)
4.92, d (12.4)
4.96, d (12.4)
6.81, d (3.0)
5.16, d (12.8)
5.11, d (12.8)
6.77, d (3.0)
5.36, d (12.2)
5.30, d (12.2)
5.27, d (12.5)
β-D-glucopyranose
1’
2’
4.93, d (8.0)
102.0
75.8
4.99, d (8.0)
5.32, dd (9.5,
8.0)
102.5
75.7
5.16, d (8.0)
101.7
75.3
4.68, d (7.4)
103.9
74.6
4.88, d (7.8)
102.5
74.8
5.13, dd (9.6, 8.0)
5.30, dd (9.4, 8.0)
3.52, dd (9.0, 7.4)
3.48, dd (9.0, 7.8)
3’
4’
5’
3.68, dd (9.6, 9.1)
3.45, dd (9.6, 9.1)
3.38, ddd (9.6, 5.6,
2.2)
76.0
71.5
78.3
3.84, brt (9.5)
3.62, t (9.5)
3.74, m
76.0
71.9
75.6
3.86, brt (9.4)
3.60, t (9.5)
75.5
71.6
75.3
3.49, brt (9.1)
3.43, dd (9.2, 9.0)
3.61, ddd (9.2, 7.3,
2.2)
77.4
71.5
75.1
3.42, t (9.0)
77.9
71.9
75.6
3.36, dd (9.5, 9.0)
3.71, ddd (9.5, 7.6,
2.1)
3.80, ddd (9.5, 6.7,
2.1)
6’
3.83, dd (12.0, 2.2)
62.5
4.58, dd (11.9,
1.8)
64.6
4.60, dd (11.9, 2.1)
64.1
4.69, dd (11.7, 2.2)
64.9
4.60, dd (11.8, 2.1)
65.4
3.66, dd (12.0, 5.6)
4.44, dd (11.9,
6.6)
4.43, dd (11.8, 6.7)
4.38, dd (11.7, 7.3)
4.34, dd (11.8, 7.6)
7-O-cyclohexenone
1"
84.2
84.2
85.6
84.1
86.2
2"
3"
4"
5.77, s
78.7
5.93, s
78.8
4.43, s
76.9
5.85, s
77.7
4.46, s
77.4
192.4
127.4
192.6
127.5
198.1
126.8
192.2
127.0
198.5
127.2
5.98, dd (10.4, 2.6)
6.72, dd (10.4, 1.9)
4.98, brt (2.2)
6.10, dd (10.4,
2.6)
6.02, dd (10.4, 2.6)
6.72, dd (10.4, 2.0)
4.89, m
6.08, dd (10.4, 2.6)
6.82, dd (10.4, 2.0)
5.13, brt (2.2)
5.91, dd (10.4, 2.6)
6.62, dd (10.4, 2.0)
4.88, m
5"
151.2
6.83, dd (10.4,
2.0)
151.0
150.6
150.8
151.0
6"
7"
72.1
5.07, brt (2.2)
72.1
71.3
71.7
71.9
171.2
171.1
171.7
171.2
172.5
2′-O-benzoyl
1
131.2
130.8
129.7
134.5
129.7
130.8
167.4
131.6
130.8
129.7
134.5
129.7
130.8
167.3
130.8
130.5
129.3
134.1
129.2
130.5
167.0
2
3
4
5
6
7
7.95, d (7.5)
8.10, d (7.5)
7.47, t (7.5)
7.60, t (7.5)
7.47, t (7.5)
8.10, d (7.5)
8.14, dd (8.0, 1.2)
7.51, dd (8.0, 7.7)
7.62, brtd (7.7, 1.2)
7.51, dd (8.0, 7.7)
8.14, dd (8.0, 1.2)
7.33, t (7.5)
7.45, t (7.5)
7.33, t (7.5)
7.95, d (7.5)
6′-O-benzoyl
1
130.9
130.2
129.2
134.0
129.2
130.2
167.3
131.4
130.7
129.6
134.4
129.6
130.7
167.8
2
8.00, dd (8.0, 1.2)
7.49, t (8.0)
7.92, dd (7.5, 1.3)
7.39, t (7.5)
3
4
7.63, brtd (7.5, 1.2)
7.49, t (8.0)
7.52, td (7.5, 1.3)
7.39, t (7.5)
5
6
8.00, dd (8.0, 1.2)
7.92, dd (7.5, 1.3)
7
6′-O-(E)-caffeoyl
1
2
3
4
5
6
127.6a
115.1
146.9
149.7a
116.6
123.2
127.3
114.7
146.4
149.3
116.2
122.8
7.10, d (1.9)
7.10, d (2.1)
6.83, d (8.2)
6.99, dd (8.2,
1.9)
6.82, d (8.2)
6.97, dd (8.2, 2.1)
7
7.64, d (15.8)
6.34, d (15.8)
147.3
114.9
169.0
7.61, d (15.8)
6.33, d (15.8)
146.9
114.4
168.6
8
9
2′′-O-benzoyl
1
2′′-O-(E)-cinnamoyl
130.3
131.0
129.5
134.5
129.5
131.0
166.5
130.4
131.1
129.3
134.3
129.3
131.1
166.3
135.4
129.1
129.6
131.2
129.6
129.1
147.0
117.2
166.3
2
3
4
5
6
7
8
9
7.82, d (7.3)
7.91, d (7.6)
7.36, t (7.6)
7.52, t (7.6)
7.36, t (7.6)
7.91, d (7.6)
7.57, m
7.40, m
7.40, m
7.40, m
7.57, m
7.31, brt (7.4)
7.48, t (7.5)
7.31, brt (7.4)
7.82, d (7.3)
7.67, d (16.0)
6.42, d (16.0)
a,b Interchangeable assignments.
5

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
Table 3
NMR spectroscopic data (600 MHz, CD3OD) for 10, 11, 12 and 13.
Position
10
11
12
13
δH mult. (J in Hz)
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
δC
δC
1
2
3
4
5
6
7
128.5
149.8
119.7
116.8
154.2
117.0
64.3
128.0
150.0
119.8
117.0
154.0
117.4
64.3
127.5
149.2
119.3
116.5
154.0
116.2
62.6
138.4
105.2
154.4
138.5
154.4
105.2
74.8
6.65, s
7.06, d (8.8)
6.80, d (8.8)
7.11, d (8.8)
6.55, dd (8.8, 3.0)
6.29, dd (8.8, 3.0)
6.65, dd (8.8, 2.8)
6.81, d (3.0)
5.29, d (12.5)
5.35, d (12.5)
6.58, d (3.0)
5.16, d (12.2)
5.10, d (12.2)
6.76, d (2.8)
5.18, d (12.4)
5.15, d (12.4)
6.65, s
4.65, d (6.2)
8
9
3.75, m
86.7
63.2
3.50, dd (11.8, 3.7)
3.55, dd (11.8, 5.8)
3.75, s
3-OCH3
4-OCH3
5-OCH3
56.5
61.1
56.5
3.65, s
3.75, s
β-D-glucopyranose
1’
2’
3’
4’
5’
6’
4.81, d like (7.7)
104.2
75.0
78.0
72.1
75.7
65.4
4.56, d (7.3)
104.4
75.0
78.0
72.0
75.5
65.3
5.15, d (8.2)
101.9
75.1
75.5
71.6
75.4
64.1
4.22 d (7.5)
105.1
75.5
77.9
71.5
78.1
62.6
3.51, m
3.35, m
5.31, dd (9.3, 8.2)
3.84, t (9.3)
3.17, dd (9.3, 7.5)
3.23, t (9.3)
3.51, m
3.35, m
3.45, m
3.30, t (9.5)
3.60, t (9.3)
3.18, dd (9.5, 9.3)
3.13, ddd (9.5, 5.9, 2.2)
3.73, dd (12, 2.2)
3.55, dd (12, 5.9)
3.73, ddd (9.7, 7.6, 2.2)
4.72, dd (11.7, 2.2)
4.44, dd (11.7, 7.6)
3.55, ddd (9.5, 7.6, 2.1)
4.58, dd (11.7, 2.1)
4.29, dd (11.7, 7.6)
3.82, ddd (9.3, 6.6, 2.0)
4.62, dd (11.8, 2.0)
4.45, dd (11.8, 6.6)
7-O-dihydroxybenzoyl
7-O-cyclohexenone
1"
81.4
79.8
113.4
150.8
146.6
121.2
119.5
120.9
84.1
2"
3
4.69, s
77.8
5.94, s
79.3
5.85, s
77.7
199.1
127.8
146.7
37.7
193.1
128.2
146.7
38.5
192.2
127.0
150.8
71.7
4"
5"
6"
6.03, dd (10.1, 2.7)
5.98, dd (10.1, 2.5)
6.98, dd (8.0, 1.5)
6.65, t (8.0)
6.08, dd (10.4, 2.6)
6.82, dd (10.4, 2.0)
5.13, brt (2.2)
6.84, ddd (10.1, 5.5, 2.7)
6.81, ddd (10.1, 5.6, 2.5)
2.65, dd (19.3, 5.5)
α-eq
2.68, dd (19.3, 5.6)
α-eq
7.15, dd (8.0, 1.5)
3.09, dt (19.3, 2.7) β-ax
3.13, dt (19.3, 2.5) β-ax
7"
173.7
172.8
170.8
171.2
2′-O-benzoyl
1
130.7
130.3
129.0
133.9
129.0
130.3
166.8
2
8.05, d (7.5)
7.38, t (7.5)
7.50, t (7.5)
7.38, t (7.5)
8.05, d (7.5)
3
4
5
6
7
6′-O-benzoyl
1
2
3
4
5
6
7
131.3
130.7
129.7
134.4
129.7
130.7
167.8
131.3
130.7
129.7
134.4
129.7
130.7
167.8
8.02, dd (8.0, 1.2)
7.52, t (8.0)
7.79, d (7.5)
7.40, t (7.5)
7.53, t (7.5)
7.40, t (7.5)
7.79, d (7.5)
7.65, brt (7.8)
7.52, t (8.0)
8.02, dd (8.0, 1.2)
6′-O-(E)-caffeoyl
1
127.3
114.6
146.4
149.3
116.2
122.8
146.9
114.4
168.5
2
7.09, d (2.0)
3
4
5
6.82, d (8.0)
6
6.98, dd (8.0, 2.0)
7.62, d (15.9)
6.32, d (15.9)
7
8
9
2′′-O-benzoyl
1
2
3
4
5
6
7
130.5
131.1
129.5
134.5
129.5
131.1
166.7
7.79, d (7.5)
7.31, t (7.5)
7.46, t (7.5)
7.31, t (7.5)
7.79, d (7.5)
the presence of three methoxy groups on the aromatic ring as in elemicin
(De Vincenzi et al., 2004) (Table 3). The three-carbon atom chain was
fully substituted by oxygen atoms and a β-D-glucopyranose was linked to
the middle carbon atom (C-8) as demonstrated by the detection of an
HMBC correlation between C-8 δ 86.7 and H-1’ of glucose δ 4.22 (d, 7.5
Hz). Consequently, compound 13 was a diastereoisomer of the alleged
“(7S,8R)-erythro-7,9-dihydroxy-7-(3,4,5-trimethox-
yphenyl)-propan-2-yl β-D-glucopyranoside” isolated from Erica arborea,
the absolute configuration of which, was not determined (Demirkiran
et al., 2010). A threo configuration for (13) was suggested from the
values of JH-7,H-8 (6.2 Hz), and of the ΔδC8-C7 (12 ppm) (Gan et al., 2008).
An analogous compound, simply lacking the central methyl group on the
6

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
aromatic ring, isolated from Alangium premnifolium, has been described
as threo (7S,8S)-syringoylglycerol 8-O-β-D-glucopyranoside (Kijima
et al., 1997). It is worth noting that this compound and 13 have close
reasonably assumed that these molecules exist in the 1′′-S*, 2′′-S*
configuration (Fig. 3). Both compounds displayed a positive Cotton ef-
fect at 330 nm and a coarse application of the octant rule would suggest
a β orientation for OH-2′′ and therefore 1′′S, 2′′S configuration. This
would require confirmation either by synthesis or by sophisticated
calculations.
values for their optical rotations ([α]D = + 6 for 13 vs + 8). It is however
not possible to use this as an argument to draw a definitive conclusion on
the absolute configuration of compound 13.
2.6. Attempts to solve the absolute configurations of the undescribed
compounds in the xylosmin series
2.5. On the relative configurations of the undescribed compounds in the
xylosmin series
Most of the undescribed compounds were amorphous solids and
despite many attempts, none of them could be crystallized, probably as a
consequence of having too many mobile parts. Therefore, following
Sashidhara et al. (2013), we hydrolyzed flacourtioside I 7 for which the
quantity was sufficiently available. The best results were obtained with
excess sodium methoxide in MeOH at room temperature. After chro-
matography, methyl benzoate, methyl caffeate and the 2-glucoside of
gentisyl alcohol (2,5-dihydroxybenzyl alcohol) were the only products
that could be separated and identified (Fig. 4). Unfortunately, no trace of
an ester which could be related to xylosmic acid could be isolated. It is
likely that, as noted by Ghavre et al. (2017), decomposition had
occurred under reaction conditions. The CD spectra of all the com-
pounds were run, but, although they all showed a weak but definitive
Cotton effect at λ > 350 nm, it seemed difficult to draw a conclusion in
the absence of reference compounds. The absolute configuration of this
part of the molecules thus remains to be determined.
There is no doubt that the configuration of glucose is D- and this
affirmation is based on three arguments: a) the fact that L-glucose has
never been found in plants, b) the hydrolysis of enriched fractions,
which showed no trace of the rare isomer and c) circular dichroism of
compound 3, in agreement with the Nakanishi dibenzoate rule (Liu and
Nakanishi, 1982). As a consequence, the only subject of uncertainty was
the configuration of the xylosmic acid part. In all derivatives, the 1H spin
systems corresponding to the cyclohexenone were very similar, except
for the shift of H-2′′, when this position was esterified (4, 5, 6, 8) and in
compounds 10–12, which had a different deoxyxylosmic acid substitu-
ent. H-2′′ is a sharp singlet, H-6′′ appears in most of the cases as a narrow
triplet and the olefinic protons H-4′′ and H-5′′ are doublets of doublets.
The ROESY experiments showed cross-peaks between H-2′′(s) and H-6′′
(t) confirming that the corresponding OHs were in cis di-equatorial
orientations, as shown by crystallography in xylosmin (Gibbons et al.,
1995). The configuration of C-1′′ remained the only uncertainty in
addition to the question of the absolute configuration. Molecular
modeling of the two possible isomers (1′′S*, 2′′S*, 6′′S* and 1′′S*, 2′′R*,
6′′R*), showed that the first hypothesis was the only one able to favour a
cis di-equatorial relationship for the two alcohols. In the other config-
uration, these hydroxy would be forced into axial positions in order to
accommodate an equatorial position for the ester (Fig. 3). We therefore
conclude that compounds 4–9 all have 1′′S*, 2′′S*, 6′′S* relative con-
figurations, with the three hydroxyls on the same face of the cyclo-
hexene ring.
2.7. Conclusions
The isolation of a large variety of phenol glycosides from Flacourtia
indica is in line with its belonging to the Salicaceae family. What is
striking with this species is the variety of substitutions that occur on the
glucose and on the xylosmic acid parts. This latter acid is a headache for
full structural elucidation, when present in multifunctional molecules
where Mosher derivatization would not apply. It is also not known
whether it is formed from benzoic acid, through protocatechuic acid or
the other way around (Fig. 5). Finally, it may be stated that in this article
and in the literature, all structures must be considered as tentative
except for xylosmin whose structure is now known but which was
published in a wrong configuration. It is worth noting that another false
and different structure was recently published for this compound
(Hussain et al., 2016). This work also illustrates the difficulty in iden-
tifying compounds with the literature when small rotation values are
obtained. All compounds were assayed for antibiotic properties but only
mild activity was observed.
For compounds 10 and 11, the relative configurations of C-1′′ and C-
2′′ of the 1,2-dihydroxy-3-oxocyclohex-4-enoyl moiety were determined
by the observation of ROE effects between H-2′′ (10: δH ppm 4.69 and 11:
δH ppm 5.94) and the axial H-6′′ (10: δH ppm 3.09 (dt, 19.3 and 2.7 Hz) and
11: δH ppm 3.13 (dt, 19.3 and 2.5 Hz)) suggesting that H-2′′ also was in an
axial orientation. Given the propensity for a bulky ester group to prefer
an equatorial configuration in a six-membered ring, it may be
3. Experimental
3.1. General experimental procedures
Optical rotations were determined in MeOH with a PerkinElmer 341
Fig. 3. Relative configurations of flacourtiosides 5–9 (top) and
10–11 (bottom).
Fig. 4. Hydrolysis of flacourtioside I (7).
7

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
Fig. 5. Possible pathways for the formation of the flacourtiosides.
◦
polarimeter at 20 C, λ = 589 nm, and CD spectra were measured in
EtOH on the J-810 Jasco circular dichroism spectrometer. UV–Vis
spectra were measured in MeOH with Shimadzu UV/Vis U-2450 spec-
trophotometer. Fourier transform-infrared (FT-IR) measurements were
carried out by the KBr pellet method using a Nicolet Impact 410 FTIR
spectrometer. HR-ESI-MS experiments were performed using a Micro-
mass Q-TOF micro instrument (Manchester, UK). NMR spectra were
acquired in CD3OD on a Bruker Avance DRX-600 instrument (1H at 600
MHz and 13C at 150 MHz). Standard pulse sequences and parameters
were used to obtain 1D 1H and 13C spectra, and 2D COSY, TOCSY,
ROESY, HSQC and HMBC spectra. TLC was performed on pre-coated
silica gel 60 A: Alugram UV254 Macherey-Nagel in normal phase and
Silicagel 60 RP-18 F254S Merck in reversed phase, and compounds were
visualized by spraying the dried plates with 50% H2SO4, followed by
heating. Vacuum liquid chromatography (VLC) was performed on Kie-
selgel 60 (63–200 mesh) Merck which was packed in a column equipped
with a sintered glass No 4 to a height of 5–7 cm, using 25 times the mass
of the fractionated extract. Flash chromatography (FC) was carried out
on a Grace Reveleris apparatus equipped with an ELSD detector, a UV/
vis detector and Reveleris Flash System software. HPLC was performed
on a Dionex apparatus equipped with a LPG 3400AB pump, an ASI-100
auto-sampler, a diode array detector UVD 340 S, an oven STH 585 and
Chromeleon software. An RP-18 column (Interchrom Uptisphere Strat-
coordinating expert at the Conseil National des Sciences du Laos) in Ban
Namalat (17◦ 19′ 40′′ N, 104◦ 52′ 31′′ E), Laos in September 2003.
Voucher specimens (7FR-UR) are deposited in Laos (Centre Inter-
disciplinaire Herbier National du Laos (CIHNL); Vientiane) and Pierre
Fabre laboratories (Toulouse).
3.3. Extraction and isolation of undescribed compounds
Dried and powdered leaves of Flacourtia indica (500 g) were suc-
cessively macerated for 15 h with petroleum ether, CHCl3, EtOAc, MeOH
and finally 80 % aqueous MeOH (5 l each time). The methanolic extract
(27.23 g; yield 5.45%) was purified using a silica gel VLC (2 × 10 g of
extract; column 7 × 9.5 cm) eluted with a gradient of solvent CHCl3
–
MeOH – H2O (100:0:0–0:70:3) to give 20 fractions. All phenolic glyco-
sides were isolated from fraction 7 (3 g) eluted with CHCl3 – MeOH
(80:20 and 75:25), which was separated by C-18 FC using a gradient of
MeCN – H2O (10:90–100:0) to have 28 sub-fractions. Compound 13 (11
mg) was purified from the sub-fraction 6 (50 mg) by semi-prep. HPLC
eluted with isocratic solvent of 10% MeCN (column 1 at 4 ml/min). Sub-
fraction 13 (148 mg, eluted with MeCN – H2O (24:76) was purified by
semi-preparative HPLC (column 1 at 4 ml/min) using an isocratic sol-
vent of 20% MeCN (pH = 2.4) during 25 min to yield 1 (20 mg, tR
=
13.52 min). Sub-fraction 17 (148 mg, eluted with MeCN – H2O (24:76))
was purified in the same way using an isocratic solvent of 22 % MeCN to
furnish 2 (30 mg, tR = 19.07 min). Compound 10 (3.5 mg, tR = 26.24
min) was obtained from the sub-fraction 19 (76 mg, eluted with MeCN –
H2O (26:74)) was purified by semi-preparative HPLC (column 1 at 4 ml/
min) using an isocratic solvent of 22% MeCN. Sub-fraction 22 (243 mg,
eluted with MeCN – H2O (75:25)) was chromatographed by silica gel C-
18 FC using a gradient of MeCN – H2O (20:80–0:100) to obtain 9 frac-
tions, among them the fraction eluted with MeCN – H2O (32:68) (83 mg)
was purified by semi-preparative HPLC (column 1 at 4 ml/min) during
30 min using an isocratic mode of 27% MeCN (pH = 2.4) to give 7 (39
mg, tR = 16.68 min) and 9 (15 mg, tR = 24.83 min). The same silica gel
C-18 FC was used for sub-fraction 23 (206 mg, eluted with MeCN – H2O
(78:22)) and then the major fraction (64 mg, eluted with MeCN – H2O
(29:71)) was purified by semi-preparative HPLC using an isocratic mode
of 28% MeCN (pH = 2.4) to yield 5 (20 mg, tR = 24.874 min). The sub-
fraction 24 (317 mg, eluted with MeCN – H2O (80:20)) was separated by
silica gel FC using a gradient of CHCl3 – MeOH (100:0–0:100) to have 12
fractions; the purification of fraction eluted with CHCl3 – MeOH (92:8)
(62 mg) was performed by semi-preparative HPLC using an isocratic
mode of 32% MeCN (pH = 2.4) to give 3 (28 mg, tR = 16.41 min) and 11
(4.5 mg, tR = 23.79 min). The fraction eluted with CHCl3 – MeOH
(90:10) (96 mg) was purified by semi-preparative HPLC using an iso-
cratic mode of 30 % MeCN (pH = 2.4) to give 4 (50 mg, tR = 25.88 min).
A silica gel FC was carried out on the sub-fraction 25 (171 mg, eluted
with MeCN – H2O (85:15)) and then the major fraction eluted with
CHCl3 – MeOH (94:6) (96 mg) was purified by semi-preparative HPLC
egy C18, 2–5
μm, 250 × 10 mm; column 1) was used for semi-
preparative HPLC with binary gradient eluent (Solvent A, H2O (pH
2.4, 0.025% TFA); solvent B, MeCN or MeOH) and a flow rate 4 ml/min;
the chromatogram was monitored at 205, 230, 254 and 256 nm. Waters
chromatographic chain was controlled by the Empower software,
comprising a 600 E pump, a 717 plus auto-sampler, and a refractive
index (RI) detector (Waters 410); two types of column were used for this
analysis: Rezex ROA Column, 250 × 21.2 mm (Phenomenex; column 2)
for the purification of sugars by semi-preparative HPLC (flow rate = 3.5
ml/min, pressure = 1400 psi, solvent = H2SO4 2.5 μM), and Chiralpak IC
Column, 5 μm, 250 × 4.6 mm (Chiraltech; column 3) for identification of
sugars (flow rate = 0.5 ml/min, pressure = 300 psi, solvent = n-hexane/
EtOH/TFA (80/20/1)).
3.1.1. Molecular calculations
The two possible diastereoisomers (1′′S*, 2′′S*, (6′′S*) and 1′′R*,
2′′S*, (6′′S*)) of compounds 7 and 10 were constructed using the
Maestro software and subjected to force field MM2 calculations. The
enthalpies were found for 7 at ꢀ 122.47 kJ/mol for isomer 1′′S* and
ꢀ 144.29 kJ/mol for isomer 1′′R*, and for 10 at ꢀ 90.8 kJ/mol for 1′′S*
and ꢀ 95.8 kJ/mol for the 1′′R*.
3.2. Plant and biological material
The leaves of Flacourtia indica (Burm.f.) Merr. (Salicaceae) were
collected and identified by François Polidano (formerly principal
8

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
using an isocratic mode of 32% MeCN (pH = 2.4) to give 6 (28 mg, tR
=
982, 714 cmꢀ 1; CD [θ] nm (c 0.001, EtOH): +14,000 (223); +3000
(232), +9000 (241), ꢀ 2000 (280); 1H-NMR (CD3OD, 600 MHz) and 13C-
NMR (CD3OD, 125 MHz), see Table 2; HRESIMS m/z 761.1701
13.73 min), 8 (8.5 mg, tR = 21.60 min) and 12 (7 mg, tR = 27.19 min).
The HPLC chromatograms and the purification of known compounds
are described in the supplementary material section.
[M+Na]+ (calcd for C36H34O17Na, 761.1694).
25
Flacourtioside J (8): amorphous powder; [
α
]
ꢀ 17 (c 0.091,
MeOH); UV (MeOH) λ
(log
ε
) 280 (3.35) nm; IRD(KBr) νmax 3423,
3.4. Acid hydrolysis of the methanolic extract
max
2925, 1702, 1634, 1500, 1452, 1382, 1281, 1206, 1139, 1068, 715
cmꢀ 1; CD [θ] nm (c 0.001, EtOH): +21,000 (236), ꢀ 5000 (285), +5000
The MeOH extract (1 g) was refluxed in 2 N HCl (25 ml) for 4 h. After
extraction with EtOAc (3 × 25 ml), the aqueous layer was neutralized
with 0.5 M NaOH and freeze-dried. The sugar mixture was identified as
glucose by comparison with authentic sugar samples by TLC using
MeCOEt – i-PrOH – MeCOMe – H2O (20/10/7/6). Glucose was separated
(340); H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125 MHz),
1
see Table 2; HRESIMS m/z 729.1791 [M+Na]+ (calcd for C36H34O15Na,
729.1795), 559.4258 [M-cinnamoyl]+.
25
Flacourtioside K (9): amorphous powder; [
α
]
D +17 (c 0.18, MeOH);
UV (MeOH) λmax (log ε) 272 (3.30) nm; CD [θ] nm (c 0.001, EtOH):
by semi-preparative HPLC (column 2) using an isocratic of H2SO4 2.5 μM
+18,000 (234); 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125
MHz), see Table 2; HRESIMS m/z 583.1425 [M + Na]+ (calcd for
35%, then identified by chiral analytical HPLC (column 3) using an
isocratic of n-hexane/EtOH/TFA (80/20/1) in comparison with
authentic L-glucose and D-glucose.
C
27H28O13Na, 583.1428), 463.3469 [M + Na-benzoyl]+, 413.1277 [M
+ Na-cyclohexenoyl]+.
25
Flacourtioside L (10): amorphous powder; [α] D ꢀ 6 (c 0.11, MeOH);
3.5. Undescribed isolated compounds
UV (MeOH) λmax (log ε) 280 (3.36) nm; CD [θ] nm (c 0.001, EtOH):
+2000 (330); 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125
MHz), see Table 3; HRESIMS m/z 583.1433 [M+Na]+ (calcd for
25
Compound (1): amorphous powder; [
α
]
+7 (c 0.35, MeOH); UV
(MeOH) λ
(log
ε
) 282 (3.4) nm; IR (KBrD) νmax 3413, 2928, 1701,
1601, 150m1a,x1455, 1379, 1276, 1209, 1074, 715 cmꢀ 1; CD [θ] nm (c
0.01, EtOH): +30,000 (224); +19,500 (241), +5000 (330); 1H-NMR
(CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125 MHz) spectral data, see
Table 1; HRESMS m/z 599.1375 [M+Na]+ (calcd for C27H28O14Na
C
27H28O13Na, 583.1428), 429.1264 [M + Na-cyclohexenoyl]+.
Compound (11; 2′′-benzoyl-flacourtioside L): amorphous powder;
25
[
α]
D ꢀ 36 (c 0.24, MeOH); UV (MeOH) λmax (log
ε
) 282 (3.50) nm; CD
[θ] nm (c 0,001, EtOH): +4000 (220), ꢀ 26000 (231), +5000 (330); 1H-
NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125 MHz), see Table 3;
HRESIMS m/z 687.1692 [M+Na]+ (calcd for C34H32O14Na, 687.1690),
599.1377), 473.1676 [M-benzoyl]+, 407.1434 [M-cyclohexenyl]+.
25
Compound (2): amorphous powder; [
α
]
D +39 (c 0.23, MeOH); UV
559.4116 [M-benzoyl]+.
(MeOH) λmax (log ε) 282 (3.6) nm; IR (KBr) νmax 3421, 2923, 1702,
25
1502, 1454, 1382, 1283, 1209, 1139, 1069, 964, 715 cmꢀ 1; CD [θ] nm (c
0.01, EtOH): +29,000 (235), +3000 (330); 1H-NMR (CD3OD, 600 MHz)
and 13C-NMR (CD3OD, 125 MHz) spectral data, see Table 1; HRESMS m/
z 599.1365 [M+Na]+ (calcd for C27H28O14Na 599.1377), 429.0976 [M-
Flacourtioside M (12): amorphous powder; [
α
]
ꢀ 20 (c 0.045,
D
MeOH); UV (MeOH) λ
(log ε) 328 (3.38), 294 (3.33) nm; IR (KBr)
max
νmax cmꢀ 1: 3422, 1682, 1602, 1499, 1453, 1385, 1273, 1206, 1139,
1074, 981, 714; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD,
125 MHz), see Table 3; HRESIMS m/z 727.1626 [M+Na]+ (calcd for
benzoyl + Na]+.
C
36H32O15Na, 727.1639), 705.5147 [M+H]+.
25
Compound (3): amorphous powder; [
α
]
D +14 (c 0.25, MeOH); UV
25
Compound (13): amorphous powder; [
α]
+ 6.5 (c 0.79, MeOH);
D
(MeOH) λ
(log ε) 282 (3.7) nm; IR (KBr) νmax 3422, 2962, 1708,
1601, 150m1a,x1454, 1379, 1267, 1209, 1125, 1074, 987, 714 cmꢀ 1; CD
[θ] nm (c 0.01, EtOH): +31,000 (223); +11,500 (246), +3000 (330); 1H-
NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125 MHz) spectral
data, see Table 1; HRESMS m/z 703.1647 [M+Na]+ (calcd for
UV (MeOH) λmax (log
ε
) 270 (3.19) nm; IR (KBr) νmax cmꢀ 1: 3424, 2938,
1681, 1599, 1514, 1463, 1417, 1331, 1234, 1204, 1128, 1078, 1038,
720, 658; 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125
MHz), see Table 3; HRESIMS m/z 443.1537 [M+Na]+ (calcd for
34H32O15Na 703.1639), 533.1472 [M + Na-cyclohexenyl]+.
C
18H28O11Na, 443.1529), 389.1987 [M-OCH3]+, 241.1222 [M-
C
glucose]+.
25
Compound (4): amorphous powder; [
α
]
D ꢀ 16 (c 0.10, MeOH); UV
(MeOH) λ
(log ε) 282 (3.8) nm; IR (KBr) νmax 3423, 2922, 1708,
max
1601, 1502, 1453, 1380, 1281, 1209, 1067, 960, 713 cmꢀ 1; CD [θ] nm (c
0.01, EtOH): 1000 (220); ꢀ 10000 (230), +10,000 (245), +2000 (330);
1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD, 125 MHz) spectral
data, see Table 1; HRESMS m/z 703.1630 [M+Na]+ (calcd for
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
C
34H32O15Na 703.1639), 414.3108 [M-benzoyl-glucose]+.
25
Flacourtioside G (5): amorphous powder; [
α
]
D ꢀ 16 (c 0.22, MeOH);
Acknowledgments
UV (MeOH) λmax (log ε) 282 (3.40) nm; IR (KBr) νmax 3448, 2931, 1700,
1602, 1499, 1452, 1384, 1318, 1273, 1207, 1138, 1072, 1026, 712
cmꢀ 1; CD [θ] nm (c 0.001, EtOH): +15,000 (221), ꢀ 6000 (232),
+17,000 (241), ꢀ 1000 (280), +5000 (330); 1H-NMR (CD3OD, 600
MHz) and 13C-NMR (CD3OD, 125 MHz) spectral data, see Table 2;
HRESMS m/z 703.1645 [M+Na]+ (calcd for C34H32O15Na 703.1639),
The Ministry of Education in Vietnam (VIED) is acknowledged for
the 4-year doctoral fellowship for N.P.D. (grant 671/QD-BGDDT). D.
Harakat (ICMR) is thanked for measuring mass spectra, and A. Martinez
(ICMR) for molecular modeling and calculations. We are greatly
indebted to professor J. D. Connolly for fruitful discussions and careful
reading of the manuscript.
559.4249 [M-benzoyl]+, 397.2863 [M-benzoyl-glucose]+.
25
Flacourtioside H (6): amorphous powder; [
α
]
D ꢀ 29 (c 0.20, MeOH);
UV (MeOH) λmax (log ε) 328 (3.45), 294 (3.45) nm; IR (KBr) νmax 3421,
1706, 1603, 1501, 1452, 1379, 1273, 1178, 1117, 1071, 980, 712 cmꢀ 1
;
Appendix A. Supplementary data
CD [θ] nm (c 0.001, EtOH): +12,000 (220), ꢀ 5000 (232), +18,000
(241), ꢀ 1000 (280); 1H-NMR (CD3OD, 600 MHz) and 13C-NMR (CD3OD,
125 MHz) spectral data, see Table 2; HRESIMS m/z 865.1951 [M+Na]+
(calcd for C43H38O18Na, 865.1956), 703.1672 [M + Na-caffeoyl]+,
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2021.112891. Protocol of purification for
known compounds and their 1H and 13C-NMR Spectroscopic Data. NMR
spectra of undescribed compounds (5–13).
413.2957 [M-glucose-benzoyl-caffeoyl]+.
25
Flacourtioside I (7): amorphous powder; [
α
]
D +15 (c 0.19, MeOH);
UV (MeOH) λmax (log
ε) 328 (3.47), 292 (3.44) nm; IR (KBr) νmax 3419,
2966, 1700, 1602, 1499, 1451, 1375, 1275, 1207, 1178, 1118, 1074,
9

P.-D. Nguyen et al.
Phytochemistry 190 (2021) 112891
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Conflicts of interest
The authors declare no conflicts of interest.
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