Cytotoxic Oleanane-Type Saponins from the Leaves of Albizia anthelmintica Brongn.

Article abstract of DOI:10.1002/cbdv.201600102

Two new oleanane-type saponins: β-d-xylopyranosyl-(1 → 4)-6-deoxy-α-l-mannopyranosyl-(1 → 2)-1-O-{(3β)-28-oxo-3-[(2-O-β-d-xylopyranosyl-β-d-glucopyranosyl)oxy]olean-12-en-28-yl}-β-d-glucopyranose (1) and 1-O-[(3β)-28-oxo-3-{[β-d-xylopyranosyl-(1 → 2)-α-l-

Full text of DOI:10.1002/cbdv.201600102

Received Date: 08-Apr-2016
Revised Date: 17-Jun-2016
Accepted Date: 27-Jun-2016
Article Type: Full Paper
Cytotoxic Oleanane-Type Saponins from the Leaves of Albizia
anthelmintica Brongn.
by Eman Al-Sayed, Omayma A. Eldahshan, Dina M. Bahgat, Abdel Nasser B.
Singab*
Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, 11566,
Egypt (Tel: +2 02 2405 1120; Fax: +2 02 2405 1107; e-mail:
Nasser.Singab.PharmASU@gmail.com; Abdelnasser.sengab@pharma.asu.edu.eg)
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/cbdv.201600102
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Abstract. Two new oleanane-type saponins: 3-O-β-D-xylopyranosyl-(1→2)-β-D-
glucopyranosyl oleanolic acid 28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-
D-glucopyranosyl ester (1) and 3-O-β-D-xylopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→6)-2-
acetamido-2-deoxy-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (2), along
with two known saponins: 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid
(3) and 3-O-β-D-glucopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranosyl
oleanolic acid (4) were isolated from the acetone-insoluble fraction obtained from the 80%
aqueous methanol extract of Albizia anthelmintica Brongn. leaves. Their structures were
1
13
identified using different NMR experiments including: H NMR, CNMR, HSQC, HMBC and
1H,1H COSY, together with HRESIMS/MS, as well as by acid hydrolysis. The four isolated
saponins and the fractions of the extract exhibited cytotoxic activity against HepG-2 and HCT-
116 cell lines. Compound 2 showed the most potent cytotoxic activity among the other tested
compounds against the HepG2 cell line with an IC50 value of 3.60 µM. Whereas, compound 1
showed the most potent cytotoxic effect with an IC50 value of 4.75 µM on HCT-116 cells.
Key words. Albizia anthelmintica; Fabaceae; oleanane saponins; cytotoxicity; MTT.
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Introduction. - The genus Albizia (Fabaceae) comprises about 150 species that are widely
distributed in Africa and South America. Saponin glycosides with diverse pharmacological
activities were isolated from different Albizia species [1, 2]. Several studies have reported the
strong cytotoxicity of Albizia saponins. For instance, Adianthifolioside A-D isolated from A.
adiantifolia showed a potent cytotoxic activity on Jurkat cells [3]. Coriarioside A from A.
coriaria showed a strong cytotoxic activity on different colon cell lines [4]. Furthermore,
grandibracteoside A-C from A. grandibracteata showed cytotoxicity on KB and MCF7 cell lines
[5]. A. anthelmintica Brongn. is a spiny, medium tree native to Africa. Its bark is used widely by
farmers as an anthelmintic for livestock [6]. Previous phytochemical studies have reported the
isolation of few echinocystic acid saponins from the bark of A. anthelmintica [7], and flavonoids
from the leaves [8]; however, no report has been traced regarding the saponin composition of A.
anthelmintica leaves. To the best of our knowledge, no studies have so far been reported on the
cytotoxic activity of A. anthelmintica saponins. Therefore, this study was undertaken to
determine the cytotoxicity of A. anthelmintica and to isolate the compounds responsible for the
bioactivity. Hepatocellular and colon carcinoma cell lines were chosen for this study because the
colon and liver cancers are among the five most common and lethal cancers worldwide [9].
In the present study, bioactivity guided fractionation of the acetone-insoluble fraction
obtained from the 80% aqueous methanol extract of A. anthelmintica leaves led to the isolation
of two new oleanane-type saponins: 3-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl
oleanolic
acid
28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-
glucopyranosyl ester (1) and 3-O-β-D-xylopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→6)-2-
acetamido-2-deoxy-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (2),
together with two known ones: 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic
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acid (3) and 3-O-β-D-glucopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranosyl
oleanolic acid (4), for the first time from A. anthelmintica leaves. The cytotoxic activity of the
four isolated saponins and all the fractions of the extract were tested against HepG-2
(hepatocellular carcinoma) and HCT-116 (colon carcinoma) cell lines.
Results and Discussion. - The 80% aqueous methanol extract of A. anthelmintica leaves was
treated with acetone to precipitate saponins. The acetone insoluble fraction was fractionated
using Diaion HP-20 column chromatography to yield four main sub-fractions I-IV. Two new
saponins 1 and 2, together with two known ones 3 and 4 (Fig. 1) were isolated from the sub-
1
fractions III and IV. The structures of the compounds were elucidated on the basis of H NMR,
13C NMR (Table 1), 2D NMR (1H,1H COSY, HSQC and HMBC), HRESIMS/MS
(Supplementary data), as well as acid hydrolysis. The monosaccharides obtained by acid
hydrolysis of 1 and 2 were identified by comparison with authentic sugar samples on TLC.
The HRESIMS of compound (1) displayed a molecular ion peak at m/z 1189.5955 [M−H]−,
1
which is consistent with a molecular formula of C58H93O25. The H-NMR spectrum (CD3OD,
400 MHz) showed characteristic signals of oleanolic acid and five sugar moieties. Seven methyl
singlets and a broad singlet of the olefinic proton H-C(12) at δ(H) 5.25 (1H, brs) suggested that 1
is a triterpenoid saponin with an aglycone of the oleanene skeleton (Table 1). The presence of
five sugar moieties was evidenced by the presence of five anomeric protons at δ(H) 5.48 (1H, d,
J = 7.0 Hz), 5.31 (1H, brs), 4.67 (1H, d, J = 7.7 Hz), and two overlapping doublet signals at δ(H)
4.44 and 4.46. The 13C-NMR spectrum of 1 (CD3OD, 100 MHz) showed a pattern of signals
characteristic for the oleanolic acid aglycone, including seven sharp signals for the methyl
carbons at δ(C) 28.5, 16.9, 16.0, 17.7, 26.1, 33.4 and 24.0 corresponding to C(23), C(24), C(25),
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C(26), C(27), C(29) and C(30), respectively. Two signals at δ(C) 123.8 and 144.9 were typical
for the olefinic carbons C(12) and C(13), respectively (Table 1). Furthermore, an extra methyl
signal at δ(C) 18.2 (which has HMBC correlations with the sugar protons) was assigned to a
methyl group of a rhamnose sugar. The signal of C(3) at δ(C) 91.5 exhibited a glycosylation shift
of about +10 ppm compared to the free un-glycosylated oleanolic acid, which suggested that the
oleanolic acid is glycosylated at C(3). Five anomeric carbon signals at δ(C) 95.3, 101.1, 104.5,
105.4, and 107.1 were detected. The up-field value of 95.3 suggested that one of the sugar
moieties formed an ester linkage with the carboxylic group at C(28). Based on these findings, 1
was suggested to be a bisdesmosidic saponin. Comparing the proton and carbon chemical shift
values of the sugars with those in literature [10-13], together with tracing the COSY, HSQC and
HMBC correlations indicated that the sugar moieties were two glucopyranose moieties, a
rhamnopyranose and two xylopyranose moieties (Fig. 2). Glycosylation shifts were observed for
C(4) of the rhamnopyranose unit at δ(C) 84.4, and C(2) of the glucopyranose unit at C(3) at δ(C)
81.3 indicated the substitution at these positions with terminal sugars. Acid hydrolysis of 1
yielded glucose, rhamnose and xylose as sugar components (detected by co-TLC with authentic
sugars). Extensive HMBC analysis (Fig. 2) confirmed that the sugar sequence at C(28) is β-D-
xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside. While the sugar
sequence at C(3) was determined as β-D-xylopyranosyl-(1→2)-β-D-glucopyranoside. The
structure of 1 was confirmed by HRESIMS/MS (Supplementary data), where a fragment ion at
m/z 1057.5581 [(M−H)−132]− was detected, which accounts for the loss of a terminal xylose unit.
Another fragment ion appeared at m/z 779.4540 [(M−H)−132−132−146]−, corresponding to the
loss of two terminal xylose units (132 x 2) and a rhamnose (146). A fragment ion at m/z
617.4028 [(M−H)−132−132−146−162]− corresponds to the sequential loss of a glucose unit.
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Finally, a fragment ion at m/z 455.3525 was detected corresponding to the aglycone unit
(oleanolic acid), which results from the loss of all sugar moieties. This fragmentation pattern
confirms the structure of compound (1) suggested by NMR spectral data. Therefore, compound
(1) was identified as 3-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid 28-O-β-
D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester.
The HRESIMS of compound (2) revealed a molecular ion peak at m/z 1084.5526 [M−H]−,
corresponding to a molecular formula of C54H86O21N. The molecular ion peak indicated a
tetrasaccharide saponin containing four sugar units; one acetamido-deoxy hexose, one hexose
and two pentoses. Comparing the NMR data (Table 1) with those of compound (1), compound
(2) was proved to have an oleanolic acid aglycone. The 13C-NMR spectrum (CD3OD, 100 MHz)
showed a pattern of signals characteristic for the oleanolic acid aglycone. Four anomeric carbon
signals were observed at δ(C) 95.4, 101.0, 104.9, and 106.7, that correlated in the HSQC
spectrum with four anomeric proton signals at δ(H) 5.44 (1H, d, J = 7.0 Hz), 5.28 (IH, brs), 4.44
(IH, d, J = 8.2 Hz) and 4.52 (IH, d, J = 7.6 Hz), respectively. Comparing the NMR data with
those of related compounds in literature [11, 14-16], the sugars were confirmed to be one 2-
acetamido-2-deoxy-β-D-glucopyranose, one glucopyranose, one α-L-arabinopyranose and one β-
D-xylopyranose units. The anomeric proton (H-(C1) of the arabinose sugar appeared as broad
singlet which indicated the equatorial-equatorial coupling between H(C-1) and H (C-2) of
arabinose (3J H (C-1),H (C-2) < 3 Hz for equatorial orientation). The arabinose moiety was
1
therefore confirmed to be in the C4 conformation [17]. Acid hydrolysis of 2 confirmed the
presence of these sugars. The up-field value of C(1) of a glucopyranose unit at δ(C) 95.4
suggested that the glucopyranose moiety formed an ester linkage with the carboxylic group at
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C(28). Hence, 2 was suggested to be a bisdesmosidic saponin. The presence of an N-acetamido
group was suggested by the appearance of a signal at δ(H) 1.96 (3H, s, NHCOCH3), correlating
in HSQC with a carbon at δ(C) 23.2 (NHCOCH3); these values are typical for protons and a
carbon of the methyl group of an N-acetamido unit [14, 16]. Furthermore, the appearance of a
carbonyl amide carbon signal at δ(C) 173.4 (NHCOCH3), along with the HMBC correlation
(Fig. 3) between the proton signal at δ(H) 1.96 (NHCOCH3) and the quaternary carbon at δ(C)
173.4 (NHCOCH3) suggested the presence of an N-acetamido group [14-16]. The attachment of
the N-acetamido group to C(2) of the 2-deoxy-β-D-glucopyranose unit was established from the
presence of a carbon signal at δ(C) 57.6 (typical for C(2) of a 2-deoxy-β-D-glucopyranose unit),
which correlates in the HSQC spectrum with a proton at δ(H) 3.69 (H-C(2) of the 2-deoxy-β-D-
glucopyranose moiety). The attachment of 2-acetamido-2-deoxy-β-D-glucopyranosye to C(3) of
the aglycone was evident from the HMBC correlation (Fig. 3) between the anomeric proton (H-
C(1) of the 2-deoxy-β-D-glucopyranose moiety (δ(H) 4.44) and the C(3) of the aglycone at δ(C)
91.1. The sequential assignments of all proton and carbon signals of the individual sugars were
achieved based on the COSY, HSQC and HMBC spectral data. Glycosylation shifts were
observed for C(6) (δ(C) 67.2) of the 2-deoxy-β-D-glucopyranose unit and C(2) (δ(C) 83.7) of the
α-L-arabinopyranose moiety indicated the substitution at these positions. The HRESIMS/MS of 2
(Supplementary data), revealed a fragment ion at m/z 994.5296 [(M−H)−90]−, which results from
the fragmentation of a glucose unit, where the ring opens and a loss of three carbons with
attached protons and oxygens occurs. A fragment ion also appeared at m/z 952.5217
[(M−H)−132]−, indicating the loss of a terminal xylose unit. Accordingly, compound (2) was
elucidated
as
3-O-β-D-xylopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→6)-2-acetamido-2-
deoxy-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester.
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The acetone insoluble fraction exhibited a potent cytotoxic activity on HepG2 and HCT-116
cell lines, with IC50 values of 1.50 µg/ml and 2.30 µg/ml, respectively. While the acetone soluble
fraction showed a less potent cytotoxic activity, with IC50 values of 5.62 µg/ml and 18.10 µg/ml,
respectively (Table 2). Based on the results of this study, the viability of the hepatocellular
carcinoma cells (HepG2) were reduced in a dose-dependent manner after a 48 h exposure period
by the tested fractions and compounds. The IC50 values of the four sub-fractions I, II, III, IV
were >50.00 µg/ml, 5.71 µg/ml, 1.32 µg/ml and 1.48 µg/ml, respectively. The results showed
that sub-fractions III and IV (rich in saponins) have the most potent cytotoxic effect among the
other sub-fractions, followed by sub-fraction II (containing flavonoids), while sub-fraction I
(containing mainly sugars) showed no cytotoxic activity (Table 2). Regarding the isolated
saponins, compound 2 showed the most potent cytotoxic activity among the other tested
compounds, with an IC50 value of 3.60 µM against the HepG2 cell line, while compounds 1 and 3
showed a moderate cytotoxicity, with IC50 values of 18.47 and 20.50 µM, respectively. Compound
4 showed the least cytotoxic activity with an IC50 value of 33.53 µM. The IC50 value of
doxorubicin (the reference cytotoxic agent used) on HepG2 cells was 0.94 µM (Table 2).
Similarly, the results of the present study demonstrated that the viability of the colon carcinoma
(HCT-116) cells were reduced in a dose-dependent matter after a 48 h exposure period by the
tested samples. The IC50 values were >50.00 µg/ml, 22.40 µg/ml, 10.20 µg/ml and 4.51 µg/ml,
respectively for the four sub-fractions I, II, III, IV, respectively. Whereas, compound 1 showed
the most potent cytotoxic activity among the other tested compounds with an IC50 value of 4.75
µM, while compound 2 showed a moderate cytotoxic activity (IC50 value of 15.11 µM) on HCT-
116 cells. Compounds 3 and 4 showed the least cytotoxic activity with IC50 values of 46.38 and
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41.53 µM, respectively. Doxorubicin produced an IC50 value of 0.85 µM on HCT-116 cells
(Table 2).
3. Conclusions
Bioactivity guided fractionation of the 80% aqueous methanol extract of A. anthelmintica
leaves led to the isolation of four oleanane type saponins, two of them (1 and 2) are new
saponins. Compound 2 showed the most potent cytotoxic activity among the other tested
compounds against the HepG2 cell line with an IC50 value of 3.60 µM. Whereas, compound 1
showed the most potent cytotoxic activity among the other tested compounds with an IC50 value
of 4.75 µM on HCT-116 cells.
Acknowledgement
The authors are grateful to the Science and Technology Development Fund in Egypt STDF (drug discovery
and development center) for its grant N-5251 that funded the NMR measurements. The authors are also thankful to
Dr. Maarit Karonen, Department of Chemistry, University of Turku, Finland for carrying out the HRESI-MS/MS
measurements.
The authors report no declarations of interest.
Experimental Part
General. The NMR spectra were measured by a Bruker Ascend 400/R spectrometer (Burker
1
13
Avance III, Fällanden Switzerland) operating at 400 MHz for H and 100 MHz for C (APT).
Spectra were recorded at 25°C using CD3OD-d4 or DMSO-d6 as solvents. Proton and carbon
spectra were referenced internally to TMS signal using a value of 0.00 ppm. The HRESI-MS was
performed on a Bruker micrOTOF-Q quadrupole time-of-flight mass spectrometer (Bremen,
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Germany). The ionization technique was a pneumatically assisted electrospray. The mass
spectrometer was operated in the negative mode with the following settings: capillary voltage,
4000V; end plate offset, –500V. Heated drying gas (N2) flow rate was 12 l/min; the drying gas
o
temperature was 200 C. The gas flow to the nebulizer was set at a pressure of 1.6 bar. For
collision-induced dissociation measurements (MS/MS), the voltage over the collision cell varied
by collision sweeping mode from 4.5 to 85 eV. Argon was used as a collision gas. A melting
point apparatus (Stuart Scientific, Staffordshire, UK) was used to measure the melting point of
the compounds, with a heating rate 1 to 10 oC per min and a temperature range from 50 up to 400
oC. The optical rotation of the compounds was measured using a A.KRÜSS Optronic P8000
GmbH polarimeter (Hamburg, Germany), with a light source (Light Emitting Diode, LED), a
filter and a maximum tube length of 220 mm. TLC was performed on precoated Silica gel 60
F254 plates (Fluka Chemika, Swizerland). Column chromatography was done on Diaion HP-20
polymer gel with particle size 250-850 μm (Mitsubishi Chemical Co., (Tokyo, Japan) and silica
gel 60 of 70–230 mesh (Merck, Darmstadt, Germany). Authentic sugars (glucose, xylose,
arabinose, rhamnose and 2-acetamido-2-deoxy-glucose) were obtained from Sigma-Aldrich
Chemical Co. (St. Louis, MO, USA). All solvents used for extraction and isolation were of
distilled analytical grade.
Plant Material. The leaves of A. anthelmintica Brongn. were collected in January 2013 from
the Zoo, Giza, Egypt. They were kindly authenticated by Mrs. Therease Labib, the taxonomy
specialist in El-Orman botanical garden, Giza, Egypt. A voucher specimen (PHG-P-AA-2013)
was deposited at the herbarium of the Faculty of Pharmacy, Ain shams University, Cairo, Egypt.
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Extraction and Isolation. The air dried leaves (1.5 kg), were extracted by maceration with
80% methanol (3 x 18 l). The extract was evaporated till dryness under reduced pressure then
freeze dried to yield 135 g of the dried extract. The total extract was dissolved in 200 ml MeOH,
then treated with acetone (x 10 volume) to yield the acetone soluble fraction (60 g) and the
acetone insoluble fraction (72 g). The acetone insoluble fraction (50 g) was subjected to Diaion
HP-20 column chromatography (220 g, 5 x120 cm) using H2O-MeOH gradient of decreasing
polarity to give four main sub-fractions: Sub-fraction I (15.6 g, eluted with water), sub-fraction II
(9.2 g, eluted with 50% methanol), sub-fraction III (14.1 g, eluted with 75% methanol), and sub-
fraction IV (2.5 g, eluted with 100% methanol). Sub-fraction III (4 g) was applied to a silica gel
60 column (200 g, 5 x120 cm) (Merck, Germany) using CH2Cl2–MeOH gradient of increasing
polarity (9:1–1:9) to yield four smaller fractions, A (0.04 g), B (0.12 g), C (0.85 g) and D (2.00
g). Sub-fraction B was further purified by preparative TLC using n-BuOH–H2O–AcOH (4:1:5,
upper butanol layer) as a solvent system. The edge of TLC plates was cut then sprayed with
vanillin/ H2SO4 reagent (1% vanillin in 10% H2SO4 in MeOH) to detect the compounds, the
separated bands were scratched, macerated in methanol, filtered then the filtrate was dried under
reduced pressure followed by freeze drying to obtain compounds 1 (65 mg) and 2 (48 mg). Sub-
fraction IV was subjected to preparative TLC using CH2Cl2–MeOH–AcOH–H2O (7:3:1:0.1) a
solvent system to give compounds 3 (35 mg) and 4 (38 mg).
3-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-xylopyranosyl-
(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester (1). White amorphous powder.
1
13
m 2 -2 2 C. = −34.6 (c = 0.3, MeOH). H NMR (400 MHz) and C NMR (100 MHz)
data: see Table 1. HRESIMS (neg.): m/z 1189.5955 ([M−H]−, C58H93O25; calc. 1189.5979).
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HRESIMS/MS: m/z 1057.5581 [(M−H)−132]−, 779.4540 [(M−H)−132−132−146]−, 617.4028
[(M−H)−132−132−146−162]−, 455.3525 [(M−H)−132−132−146−162−162]−.
3-O-β-D-xylopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→6)-2-acetamido-2-deoxy-β-D-
glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (2). White amorphous powder. mp
1
187-189 C. = −18.5 (c = 0.3 MeOH). H NMR (400 MHz) and 13C NMR (100 MHz)
spectroscopic data: see Table 1. HRESIMS (neg.): m/z 1084.5526 ([M−H]−, C54H86O21N; calc.
1084.5667). HRESIMS/MS: m/z 994.5296 [(M−H)−90]−, 952.5217 [(M−H)−132]−.
3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid (3). White amorphous
1
powder. H NMR and 13C NMR data: consistent with those reported in literature from A.
inundata [2, 20]. HRESIMS (neg.): m/z 779.4409 ([M−H]−, C42H67O13; calc. 779.4563).
HRESIMS/MS: m/z 617.3883 [(M−H)−162]−, 455.3329 [(M−H)−162−162]−.
3-O-β-D-glucopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranosyl
oleanolic acid (4). White, amorphous powder. 1H NMR and 13C NMR data: consistent with
those reported in literature from A. gummifera [21]. HRESIMS (neg.): m/z 911.4970 ([M−H]−,
C47H75O17; calc. 911.4983). HRESIMS/MS: m/z 749.4363 [(M−H)−162]−, 617.3960
[(M−H)−162-132]−, 455.3356 [(M−H)−162-132-162]−.
Acid Hydrolysis. A solution of 5 mg of saponins 1 or 2 was added to 10 ml 0.02 N H2SO4,
then 1 C for 4 h. The mixture was left to cool, neutralized with 0.02 N KOH and
extracted with CH2Cl2. [22]. The sugars in the aqueous layer were detected by TLC using
authentic sugars and n-BuOH–H2O–AcOH (4:1:5, upper butanol layer) as a solvent system. The
TLC was sprayed by vanillin/ H2SO4 reagent.
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Cytotoxicity MTT Assay. HepG2 (Human Hepatocellular Carcinoma) and HCT-116 (Human
Colon Carcinoma) cell lines were obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA). HepG 2 and HCT-116 cell lines were grown in Roswell Park Memorial
Institute medium (RPMI-1640 medium) supplemented with 10% heat inactivated fetal bovine
serum (FBS), 50 units/ml of penicillin and 50 gm/ml of streptomycin and maintained as a
monolayer culture at 37°C in a humidified atmosphere containing 5% CO2. Cytotoxicity was
monitored by determining the effect of the tested samples on the cell viability using the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay [23]. The cancer cells were
treated for 48 h with the tested samples dissolved in DMSO, such that the DMSO concentration
in the medium did not exceed 0.1%. The plates were read on an ELISA micro-plate reader using
a test wavelength of 570 nm. The IC50 values were calculated according to the equation for
Boltzman sigmoidal concentration–response curve using the nonlinear regression fitting models
(Graph Pad, Prism Version 5). Doxorubicin was used as a standard cytotoxic agent.
Figure legends
Fig. 1. Chemical structures of compounds 1-4
Fig. 2. HMBC correlations of compound 1
Fig. 3. HMBC correlations of compound 2
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Table 1. 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CD3OD-d4) of Compounds 1 and 2.
1
2
aglycone
sugar
δ(H)
aglycone
sugar
δ(H)
δ(C)
δ(H)
δ(C)
δ(C)
δ(H)
δ(C)
Glu at 28
Pos.
Pos.
Pos.
Pos.
39.9
26.1
91.5
40.4
57.0
19.4
29.2
0.98/1.62 (m)
1.36/1.95 (m)
3.19
39.9
26.8
91.1
39.8
57.0
19.4
31.9
1.00/1.63 (m)
1.70/1.93 (m)
3.11
1
2
3
4
5
6
7
Glu I at 28
1
2
3
4
5
6
7
95.3
76.7
77.6
71.9
78.4
62.8
5.48 (d, J = 7.0)
95.4
5.44 (d, J = 7.0)
1
2
3
4
5
6
1
2
3
4
5
6
(m)
3.61
3.28
3.24
3.55
(m)
(m)
(m)
(m)
(m)
76.4
75.9
71.8
77.6
62.7
3.58
3.47
3.34
3.27
(m)
(m)
(m)
(m)
0.77
1.39/ 1.57 (m)
1.55/1.72 (m)
(m)
0.77
1.36/1.54 (m)
1.71/1.91 (m)
(m)
3.65/3.85 (m)
3.70/3.84 (m)
40.7
49.2
37.8
23.9
123.8 5.25
144.9
40.8
48.1
37.8
23.7
123.4 5.32
144.7
8
Rha
8
Glu-NAc at 3
1.57
(m)
101.1
71.8
72.3
84.4
68.8
18.2
5.31
3.96
3.85
3.53
3.76
(brs)
(m)
(m)
(m)
(m)
1.62
(m)
104.9 4.44 (d, J = 8.2)
9
1
2
3
4
5
6
9
1
2
3
4
5
6
57.6
75.9
72.0
76.1
67.2
3.69
3.48
3.34
3.49
(m)
(m)
(m)
(m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
11
12
13
1.90; 1.15 (m)
1.91;1.16 (m)
(brs)
(m)
(brs)
42.9
1.29 (d, J = 5.3)
3.19/3.88 (m)
30.8
24.6
49.6
42.7
48.2
31.5
34.9
34.0
28.5
16.9
16.0
17.7
26.1
177.9
33.4
24.0
1.30
1.89;1.15 (m)
42.7
Xyl I at 4 Rha
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
NHCOCH3
107.1
76.1
78.3
70.8
67.2
4.46
30.7
24.5
50.2
42.2
47.9
31.3
36.5
34.3
28.6
17.1
16.1
17.7
27.1
177.0
33.3
24.9
0.90/1.29 (m)
1.91;1.16 (m)
CO
NH
CH3
173.4
1
2
3
4
5
3.24
3.33
3.53
(m)
(m)
(m)
8.55
1.96
(brs)
(s)
2.83
1.57
(m)
(m)
23.2
2.95
1.05/2.28 (m)
(m)
Ara at 6 Glu-NAc
3.19/3.87 (m)
101.1 5.28
(brs)
(m)
(m)
1
2
3
4
5
1.22/1.36 (m)
83.7
72.2
68.9
63.1
3.57
3.85
3.74
Glu II at 3
1.42
1.09
0.86
0.95
0.77
1.15
(m)
(s)
(s)
( s)
(s)
(s)
105.4
4.44
1.45/1.69 (m)
1.42/1.52 (m)
1
2
3
4
5
6
81.3
77.8
71.5
78.2
63.1
3.59
3.29
3.24
3.21
(m)
(m)
(m)
(m)
(m)
0.97
0.86
0.96
0.77
1.36
(s)
(s)
(s)
(s)
(s)
3.62/3.84(m)
Xyl at 2 Ara
106.7 4.52 (d, J = 7.6)
1
2
3
4
5
3.64/3.85 (m)
76.0
78.0
71.1
66.5
3.26
3.35
3.49
(m)
(m)
(m)
Xyl II at 2 Glu
0.90
0.92
(s)
(s)
104.5
76.3
78.3
71.1
66.5
4.67 (d, J = 7.7)
1
2
3
4
5
3.25
3.34
3.49
(m)
(m)
(m)
0.88
0.95
(s)
(s)
3.32/3.92(m)
3.31/3.91 (m)
Assignments were based on the COSY, HSQC and HMBC spectra
Overlapped signals are reported without multiplicity
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Table 2. Cytotoxicity of the acetone soluble and insoluble fractions, four sub-fractions and the isolated
saponins from A. anthelmintica leaves on HepG 2 and HCT-116 cell lines.
Sample
Main fractions
Sub-fractions
Compounds
Acetone Acetone
sol. fr. insol. fr.
Cell line
HepG 2
HCT-116
I
II
III
IV
1
18.47
4.75
2
3
4
5.62
1.50
2.30
>50.00 5.71
1.32
1.48
3.60
20.50
33.53
41.53
18.10
>50.00 22.40 10.20 4.51
15.11 46.38
a) Cells were treated with the tested samples for 48 h, and the cell viability was determined by the MTT assay. IC50 values
(µg/mL for fractions or µM for compounds) was calculated from the % cell viability on the basis of triplicate
measurements.
b) IC50 values of doxorubicin were 0.94 µM and 0.85 µM on HepG 2 and HCT-116, respectively.
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29
30
29
30
20
17
20
17
19
18
21
22
19
18
21
22
12
26
12
26
11
11
13
25
13
25
H
16
14
9
H
14
28 O
O
16
9
15
COOH
Glu II
1
4
15
8
1
4
10
28
2
2
3
8
7
10
27
HO
HO
5
3
H
27
7
HO
HO
HO
HO
H
5
O
6
O
O
O
6
O
O
HO
HO
H
HO
H
24
HO
HO
HO
23
24
23
Glu I
O
O
O
O
HO
HO
O
O
HO
HO
O
OH
OH
OH
29
30
OH
Rha
Xyl II
Xyl I
OH
29
30
20
19
18
21
22
12
26
20
17
13
17
11
19
18
21
22
OH
25
Ara
12
26
H
15
11
16
28 O
O
9
14
13
1
4
25
OH
8
7
10
5
2
H 16
27
OH
14
9
O
15
H
COOH
28
3
1
4
O
2
3
10 H
6
8
O
HO
Ara
OH
O
27
HO
HO
O
O
O
7
5
H
24
O
6
HO
HO
Glu OH
23
HO
HO
O
H
HO
NH
24
HO
23
O
H3C
O
O
O
HO
HO
HO
O
HO
OH
OH
Xyl
Fig. 1. Chemical structures of compounds 1-4
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29
30
20
17
19
18
21
22
12
26
11
13
25
H
15
14
9
16
Glu II
HO
HO
28
O
O
1
4
2
8
10
27
H
HO
HO
3
7
O
O
O
6
5
O
Glu I
HO
HO
H
24
23
O
O
HO
HO
O
O
HO
HO
OH
O
OH
OH
Xyl II
OH
Xyl I
Rha
Fig. 2. HMBC correlations of compound 1
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29
30
20
17
19
18
21
22
12
13
11
26
25
H 16
15
28 O
O
14
1
4
8
10
5
2
OH
27
H
Ara
3
7
O
O
OH
O HO
HO
HO
O
O
6
H
OH
HO
24
23
HO
Xyl
HO
NH
Glu
O
H3C
O
O
HO
OH
Fig. 3. HMBC correlations of compound 2
This article is protected by copyright. All rights reserved.