S. Perveen, et al.
Fitoterapia139(2019)104377
chromatography (1 × 100 cm) with CHCl3/methanol (9:1 → 6:4, v/v;
total 1 l) and fractions were monitored by TLC (CHCl3/methanol, 3:1)
to afford two major sub-fractions C1 and C2 (October 2017), which
were then stored as solutions at 5 °C in the dark. Sub-fraction C1
(150 mg) was re-run (November 2017) on the same silica-gel column
with CHCl3/methanol (8:2, v/v) to afford montbresides C (15 mg) and D
(16 mg). Sub-fraction C2 (250 mg) was also re-run (December 2017) on
the same silica gel column, eluting with CHCl3/methanol (9:1 → 7:3, v/
v) to yield montbresides A (15 mg) and B (19 mg). Fraction D (0.5 g),
which eluted in 30% methanol, was re-run (December 2017) on the
silica gel column in CHCl3/methanol (4:1, v/v), yielding cinnamic acid
glucosyl ester (5; 28 mg). Fraction E (500 mg), which eluted in 40%
methanol, was sub-fractionated by HPLC (on ODS gel (Inertsil ODS-3,
GL-science, 10 × 250 mm) in 50% methanol at 1.0 ml/min; January
2018), to afford trans-p-coumaric acid (6; 18 mg) and trans-cinnamic
acid (7; 25 mg).
2.4. Spectroscopic data of compounds
Selected NMR and mass spectra of montbresides A–D are in the
supplementary data.
Montbreside A: White amorphous powder; [α]25D + 37 (c 0.0010,
methanol); 1H NMR (800 MHz, methanol‑d4) data, see Table 1; 13C
NMR (125 MHz, methanol‑d4) data, see Table 2; HRESIMS m/z
1133.3297 [M.Na]+ (calcd for C50H62O28·Na, 1133.33198).
Montbreside B: White amorphous powder; [α]25D + 48 (c 0.0015,
methanol); NMR as above; HRESIMS m/z 1133.3299 [M.Na]+ (calcd
for C50H62O28·Na, 1133.33198).
Montbreside C: White amorphous powder; [α]25D + 44 (c 0.0012,
methanol); NMR as above; HRESIMS m/z 1091.3209 [M.Na]+ (calcd
for C48H60O27·Na, 1091.3214).
Fig. 1. Montbresides A and C. Montbresides B and D are identical to these but
the coumarate residues are in the cis-configuration.
and products were analysed by TLC. Enzymic digestion of the tetra-
saccharide was performed with a 1% (w/v) solution of Driselase
(Sigma–Aldrich; purified as in [4]) in pyridine/HOAc/H2O (1:1:98,
containing 0.5% chlorobutanol) at 25 °C for 0–99 h; products were
analysed by TLC on Merck silica-gel plates in butan-1-ol/acetic acid/
water (4:1:1) or ethyl acetate/pyridine/acetic acid/water (6:3:1:1) with
1–3 ascents. Staining was with iodine vapour or thymol/H2SO4 [5].
Montbreside D: White amorphous powder; [α]25D + 52 (c 0.0012,
methanol); NMR as above; HRESIMS m/z 1091.3210 [M.Na]+ (calcd
for C48H60O27·Na, 1091.3214).
2.2. Plant material
Montbretia [Crocosmia
× crocosmiiflora (Lemoine) N.E. Br.
2.5. Cytotoxicity assay
(monocot: Asparagales: Iridaceae)] flowers (500 g) were collected from
gardens in Edinburgh, in July 2017, and identified by SCF. Voucher
specimen: Royal Botanic Garden Edinburgh, herbarium barcode
E00873192.
MCF-7 cells (human breast cancer cell line), HCT-116 (colon car-
cinoma), HepG-2 cells (human hepatocellular carcinoma), HeLa (cer-
vical carcinoma), A-549 (lung carcinoma) and WI-38 (human lung fi-
broblast normal cells) were obtained from Vacsera (Giza, Egypt). The
cells were cultured in Dulbecco's modified Eagle's medium with 10%
heat-inactivated foetal bovine serum, HEPES buffer (0.1 M, pH 7.0), 1%
L-glutamine and 50 μg/ml gentamycin. The cells were kept at 35 °C
under humidified condition with 5% CO2 and were sub-cultured twice a
week. Montbresides (up to 50 μg/ml) were evaluated for cytotoxicity
against these six cell-lines by the MTT method (reduction of a tetra-
2.3. Extraction and isolation
Montbretia flowers (collected in July 2017) were air-dried (40 g dry
weight), powdered, stored dry at room temperature in the dark, and
percolated with 2 × 1.5 l of 80% (v/v) ethanol at 25 °C in the dark
(August 2017). The extract was concentrated in vacuo to a residue
(15 g), which was stored dry at room temperature in the dark then
suspended in water (150 ml; September 2017) and partitioned against
first chloroform (2.0 l) then butan-1-ol (2.5 l). The butanol fraction was
dried (8 g), redissolved in 25 ml water and passed through Sephadex
LH-20 (3 × 70 cm) with a 0 → 100% methanol gradient (4 l total) to
afford five fractions (A–E; September 2017), which were monitored by
TLC on silica-gel and RP-18 F254 (Merck, Darmstadt, Germany) in me-
thanol/water/acetic acid (10:10:1; detection was under 254-nm UV and
by spraying with Ce(SO4)2 followed by heating at 150 °C which black-
ened the UV spots. The fractions were stored as solutions at 5 °C in the
dark. The chloroform and aqueous fractions and the butanolic fractions
A and B, which between them are expected to contain most of the
quantitatively major phytochemicals such as sucrose, glucose, fructose,
citrate, malate, glutamate etc., did not show up under UV and were not
characterised further.
2.6. Antibacterial assay
Antibacterial activities were assayed by the methods of [7]. Pa-
thogenic bacteria, including Gram-negatives [Pseudomonas aeruginosa
(NR-117678.1), Escherichia fergusonii (CU928158.2), Enterobacter
xiangfangensis (CP017183.1)] and Gram-positives [Bacillus licheniformis
(KX785171.1), Staphylococcus aureus (CP011526.1)] were suspended
for 24 h in nutrient broth. After spreading on Müller–Hinton agar
plates, wells were filled with 10 μl of various concentrations of each
sample (up to 25 μg/ml). Amikacin was the positive control. The dia-
meters of clear (inhibited) zones were measured. The experiment was
conducted three times; the mean value of minimal inhibitory con-
centration (MIC [8]) is reported.
Fraction C (1 g), which eluted from Sephadex LH-20 in 20% me-
thanol, was further resolved by silica-gel (Merck) column
2