Iranian Journal of Pharmaceutical Research (2021), 20 (1): 274-282

DOI: 10.22037/ijpr.2020.112557.13824
Received: October 2019
Accepted: April 2020

Original Article

Bioassay-guided Isolation of Flavonoids from Caesalpinia bonduc

(L.) Roxb. and Evaluation of Their Cytotoxicity

Narges Pournaghia, Farahnaz Khalighi-Sigaroodia*, Elahe Safarib and Reza Hajiaghaeea

Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran.
a

b
Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran,
Iran.

Abstract

Cancer is one of the most important causes of death all around the world. Screening plants
and their secondary metabolites as cytotoxic agents is one of the common methods for identifying
new compounds used in chemotherapy and inhibition cancer process. Caesalpinia bonduc (L.)
Roxb. from the Fabaceae family was used for improving wound, fever, tumor, hydrocele, hernia,
smallpox, toothache, inflammation, and as astringent, anthelmintic, antidiabetic, and antimalarial
agent in traditional medicine. A bioassay-guided study of this species led to the isolation of three
flavonoids. At first, the cytotoxicity of methanol extract of aerial parts (leaves and stems), seeds,
and legumes of this plant was tested against MCF-7 and PC-3 by MTT assay. The methanol
extract of legumes showed better inhibitory activities (IC50 < 500 µg/mL). As a result, this extract
was selected for fractionation. In the next step, the ethyl acetate (EtOAc) fraction was selected
for phytochemical analysis based on the inhibitory activity (IC50 = 170 ± 0.9 µg/mL). In this
way, total phenol content (625 ± 7.2 GAE/g extract) and antioxidant activity (IC50 = 6.1 ± 0.3
µg/mL) was compared by BHT (IC50 = 13.5 ± 0.7 µg/mL). Finally, three compounds including,
quercetin-3-methyl ether (1), kaempferol (2), and kaempferol-3-O-α-L-rhamnopyranosyl-
1→2)-β-D-xylopyranoside (3) were isolated from EtOAc fraction, and all isolated compounds
were tested for their cytotoxicity and compound 1 showed better inhibitory activity than other
two compounds. This study suggests that Caesalpinia bonduc could be considered for further
investigations as a natural source of biological compounds.

Keywords: Caesalpinia bonduc; Bioassay-guided; Fabaceae; Flavonoid; Phenol; Antioxidant

activity; MTT assay.

Introduction chemical drugs due to their low side effects

(7). The choice of these plants is usually based
Cancer is the second leading cause of on ethnobotanical resources or traditional
death all around the world. About 10 million medicine (8). Screening plants and their
people are diagnosed with cancer each year, phytochemicals as natural killer agents for
half of whom die (1, 2). The most common cancer cells is one of the most common
and deadly cancers in women and men are methods for identifying new compounds used
breast cancer and prostate cancer, respectively in chemotherapy and inhibiting the cancer
(3-5). The prevalence of these cancers has process. For example, several species of the
increased significantly (6). Medicinal plants Fabaceae family have been studied for their
are better sources of anti-cancer drugs than cytotoxic effects (9-12). Also, in a review
* Corresponding author: article, we expressed a number of compounds
E-mail: [email protected] and properties of different species of the
Pournaghi N et al. / IJPR (2021), 20 (1): 274-282

Genus Caesalpinia particularly cassane and Experimental

norcassane compounds that have cytotoxicity
General
effects (13).
Nuclear magnetic resonance (NMR; 500
Caesalpinia bonduc (L.) Roxb. (Fabaceae
MHz) spectra were recorded on Bruker FT-
family), the accepted name instead of
500 spectrometer instrument using DMSO-d6
Caesalpinia bonducella, Guilandina bonduc,
solvent and TMS (tetramethylsilane) as
and Guilandina bonducella, is one of the an internal standard. The mass spectra of
Caesalpinia species represented in the flora of the compounds were obtained by Agilent
Iran (14, 15). This species is grown in tropical Technologies mass spectrometer Model:
and subtropical regions (like India, Pakistan). 5975C VL MSD with Triple-Axis Detector
In Iran, this plant is found in the South and (70 eV). All reagents and chemicals for
Southeast of Iran (16). This species was used phytochemical experiments and MTT assay
for improving wound, fever, tumor, hydrocele, were analytical grades.
hernia, smallpox, toothache, inflammation
and as astringent, anthelmintic, antidiabetic, Plant material
and antimalarial agent in traditional medicine The aerial parts (leaves and stems), seeds,
(17). Many studies have been done on this and legumes of Caesalpinia bonduc (L.) Roxb.
plant based on traditional medicine sources. were collected from the Sarbaz region (Sistan
For example, the antibacterial effect of seed and Baluchestan Province) in the southeast of
methanol extract (18), the antidiabetic effect Iranian July 2014. The plant was identified in
of several extracts of seed (19), antitumor the Institute of Medicinal Plants Herbarium
and antioxidant effect of leaves methanol (MPHI.IR).
extract on mice (20) and also antipsoriasis and Extraction and Isolation
increasing uterine smooth muscle contraction The experiment was accomplished in
effects of leaves extract (21, 22) have been several steps. In the first step, air-dried and
proven. Also, several components were powdered legumes (1500 g), seeds (2000
separated from this plant like caesalls H-M g), and aerial parts (840 g) were extracted
(23), caesalpinolide C-E (24), caesalpinianone, at room temperature with 9000 mL, 10130
6-O-methylcaesalpinianone, stereochenol mL, and 8170 mL methanol by percolation
A, hematoxylol, 4’-O-acetylloganic acid, method, respectively. Then these extracts were
6’-O-acetylloganic acid and 2-O-β-D- concentrated by a rotary evaporator (Heidolph
glucosyloxy-4-methoxybenzenepropanoic Laborota 4000 efficient) and dried. The
acid (25), bonducellipins A-D extraction yields were 4.13% w/w (legume),
(26), 7-hydroxy-4’-methoxyl-3,11- 4.75% w/w (seed) and 13.55% w/w (aerial
dehydrohomoisoflavanone, 4,4’-dihydroxy- parts). Then MTT assay was performed on
2’-methoxy-chalcone, 7,4’-dihydroxy-3,11- these extractions and the legume extraction
was selected for fractionations based on the
dehydrohomoisoflavanone, luteolin and
MTT results.
kaempferol-3-O-β-D-xylopyranoside (27).
In the next step, the methanol extract
In another study, cytotoxicity of methanol
of legume was dispersed in distilled water
extracts of different parts (legume, seed, and and partitioned with n-hexane, chloroform
aerial part) of Caesalpinia bonduc (C. bonduc) (CHCl3), ethyl acetate (EtOAc), and n-butanol
was tested through the brine shrimp lethality (n-BuOH) consecutively based on increasing
assay. The legume extract of C. bonduc showed the polarity of solvents to gain n-hexane,
significant cytotoxicity (28). According to the CHCl3, EtOAc, n-BuOH, and water-soluble
evidence of the antitumor effect of methanol fractions. Based on the results of different
extract of this plant, the aim of this study was evaluations, including total phenol content,
to investigate the anti-cancer effect of extracts DPPH radical scavenging assay and the MTT
and different parts of this plant and to isolate assay, the EtOAc soluble fraction was selected
the compounds responsible for this property. for more fractionations and purification.

275
 Bioassay-guided Cytotoxic Isolation of Caesalpinia bonduc

In the final step, 4 g of the EtOAc soluble humidified atmosphere at 37 °C in a 5% CO2

fraction was fractionated by the Sephadex incubator.
LH-20 column chromatography and MeOH
as eluent. Five sub-fraction (1-5) were gained. MTT assay
Sub-fraction 4 (110 mg) and 5 (40 mg) were Antiproliferative activity of extracts,
purified by silica gel plate (20 × 20 cm) with fractions, and isolated compounds was
chloroform-methanol (90:10) to get compound measured by MTT assay using MCF-7,
1 (12.5 mg) and 2 (7 mg). Compound 3 (14 mg) PC-3, and HepG-2 cancer cell lines (29).
was isolated from the sub-fraction 2 (300 mg) Methotrexate was used as a positive control.
using silica gel column chromatography (70- Ten-thousand cells per well (at the exponential
230 mesh ASTM) with CHCl3-MeOH (97:3 growth phase) were seeded into a flat bottom
to70:30). This compound was more purified 96-well plate. After 24 h incubation in a 5%
on the Sephadex LH-20 column using MeOH humidified CO2 incubator at 37 °C, the cells
as eluent (Figure 1). Compound structures were treated with a fresh medium containing
were identified by 1H-NMR and 13C-NMR and different concentrations of extracts, fractions,
MS spectral analysis, as well as by comparing and pure compounds in triplicates. After
with the data published in the literature. Then 48 and 72 h of incubation at 37 °C, 10 μL/
the cytotoxicity of isolated compounds was well MTT (3-(4,5-dimethyl thiazolyl)-2,5-
determined by MTT assay. diphenyltetrazolium bromide: stock solution
5 mg/mL PBS) was added and the plate was
Cell culture again incubated at 37 °C for 4 h. 100 μL
MCF-7 (human breast cancer), PC-3 DMSO was added to each well and shaking
(human prostate cancer), and HepG-2 (Human for 15 min. The absorbance was recorded at
liver cancer) were purchased from the Pasture 630 nm using a microplate reader and IC50
Institute of Tehran (Tehran, Iran). These cell values for each cell line were measured.
lines were cultured in RPMI (Roswell Park
Memorial Institute) medium containing Total phenol content
10% FBS (Fetal Bovine Serum), 100 IU/mL The total phenol content of the fractions
penicillin, and 100 µg/mL streptomycin under was measured by the Folin-Ciocalteu method

Figure 1. Schematic procedures for extraction and fractionation.

Figure 1. Schematic procedures for extraction and fractionation.

276
Pournaghi N et al. / IJPR (2021), 20 (1): 274-282

(30). First, 100 µg/mL concentration of the 3, respectively. Therefore, it was selected for
fraction solutions was prepared and oxidized further fractionation (Table 1).
with Folin-Ciocalteu solution followed In the next step, n-hexane, chloroform, ethyl
by neutralization with 7% (w/v) sodium acetate, n-butanol and water soluble fractions
carbonate. After shaking for 2 h at room were fractionated from the methanol extract
temperature, the absorption was measured at of legumes. Total phenol content, antioxidant
765 nm. Gallic acid was used as a standard and activity, and antiproliferative activity of
its solutions (50, 100, 150, 500, and 1000 µg/ fractions were measured to determine effective
mL) were prepared to draw a calibration curve. fraction for phytochemical analysis.
All standard and fractions were performed
in triplicate and total phenol content was Total phenol content of fractions
expressed as Gallic acid equivalent (GAE)/g Total phenol contents of C. bonduc
extract. fractions and methanol extract of legume
were determined and expressed as Gallic acid
DPPH radical scavenging assay equivalent (GAE)/g. Total phenol content
The Free radical-scavenging activity of varied between 175 for water fraction and 625
fractions was evaluated by the 2,2-Diphenyl-1- for ethyl acetate fraction (Table 2). As shown
picrylhydrazyl (DPPH) (31). Briefly, different in Table 2, ethyl acetate fraction has the highest
concentrations of fractions (5, 10, 20, 40, and amount of phenol content (625 GAE/g).
80 µg/mL) and 40 µg/mL solution of DPPH
in methanol were prepared. In each well of DPPH radical scavenging assay
96-well plates, 63 µL of DPPH was added to The antioxidant activity of fractions
a suitable volume of each fraction solution was determined through DPPH free radical
until the final volume became 250 µL. The scavenging activity test. The results revealed
absorbance was measured at 517 nm after 1 that ethyl acetate fraction has higher ability for
h. The test was performed in triplicate. DPPH radical scavenging activity (IC50: 6.1 µg/mL)
solution alone served as control and butylated compared to BHT (IC50: 13.5 µg/mL) (Table
hydroxytoluene (BHT) was used as standard. 2).
According to the results, a comparison of
Results and Discussion total phenol contents of fractions with their
All processes of the experiment contained DPPH radical scavenging activity shows that
phytochemical process followed by biological there is a direct relationship between the total
assays. In the first step, the methanol extracts phenol compound and antioxidant activity of
of aerial parts, seeds and legumes of C. bonduc fractions.
were prepared and tested their cytotoxicity. Antiproliferative activity of fractions
Antiproliferative activity of the legume, The methanol extract of legumes and all
seed, and aerial part extracts fractions were tested for their cytotoxicity
MTT assay was applied to determine which against three human cancer cell lines (MCF-7,
extract had better antiproliferative activity and HepG-2, and PC-3) using the MTT assay. The
fewer IC50. The results showed that the MeOH IC50 values were measured (Table 3). Ethyl
extract of legumes had the least IC50 value of acetate fraction showed better activity against
483 and 337 µg/mL against MCF-7 and PC- all of the tested cell lines. It should be noted

Table 1.
Table 1. Antiproliferative Antiproliferative
activity activityextracts
of the methanol of the methanol extracts cell
on the cancer on the cancer
lines cell ±
(Mean lines
SD,(Mean ± SD, n = 3).
n = 3).

IC50 (µg/mL)
Extract MCF-7 PC-3
48 (h) 72 (h) 48 (h) 72 (h)
Legume 546 ± 0.5 483 ± 1.2 456 ± 1.9 337 ± 1.1
Seed >1000 >1000 800 ± 2.2 730 ± 4.2
Aerial part >1000 850 ± 6.1 780 ± 5.3 510 ± 3.1

277
 Bioassay-guided Cytotoxic Isolation of Caesalpinia bonduc

that we used 72 h of incubation at 37 °C in this 5-OH), 7.54 (1H, d, J = 2.2 Hz, H-2′), 7.45
test because it had better response in previous (1H, dd, J = 8.35, 2.2 Hz, H-6′), 6.91 (1H, d,
test. J = 8.35 Hz, H-5′), 6.39 (1H, d, J = 2.00 Hz,
According to the results listed above (total H-8), 6.18 (1H, d, J = 2.00 Hz, H-6), 3.78 (3H,
phenol content, DPPH radical scavenging assay s,3-OCH3);13C-NMR (125 MHz, DMSO-d6);
and antiproliferative activity of fractions), the δ 178.21 (C-4), 165.21 (C-7), 161.65 (C-5),
ethyl acetate fraction was selected for more 156.80 (C-9), 155.93 (C-2), 149.28 (C-4′),
fractionations and purification. 145.73 (C-3′), 138.11 (C-3), 121.13 (C-1′),
In the final step, from the bioactive guided 120.99 (C-5′), 116.18 (C-2′), 115.74 (C-6′),
fractionation of ethyl acetate fraction of C. 104.35 (C-10), 99.14 (C-6), 94.11 (C-8), 60.09
bonduc legume, three flavonoids (compounds (OCH3); EI-MS m/z: [M+H]+ 317 (32,27).
1 to 3) were obtained. Fractionation Compound 2: Kaempferol (C15H10O6);
and purification of ethyl acetate fraction pale yellow powder; 1H-NMR (500 MHz,
were performed by the Sephadex LH- DMSO-d6); δ 12.73 (1H, 5-OH), 8.10 (2H,
20 column chromatography and silica gel d, J = 9.7 Hz, H-2′, 6′), 6.92 (2H, d, J = 9.7
plate chromatography. The structures of the Hz, H-3′, 5′), 6.79 (1H, br s, H-8), 6.25 (1H,
isolated compounds were characterized as br s, H-6); 13C-NMR (125 MHz, DMSO-d6);
Quercetin-3-methyl ether (1), Kaempferol δ 180.36 (C-4), 164.92 (C-7), 162.00 (C-5),
(2), Kaempferol-3-O-α-L-rhamnopyranosyl- 161.63 (C-4′). 157.90 (C-9), 149.50 (C-2),
1→2)-β-D-xylopyranoside (3) using 135.21 (C-3), 130.12 (C-2′, 6′), 121.45 (C-1′),
1
H-NMR, C-NMR, and MS evaluations, as
13 116.31 (C-5′, 3′), 103.52 (C-10), 99.70 (C-6),
well as comparison with those reported in the 94.50 (C-8) (33).
literature (32, 33 and 27). Figure 2 illustrates Compound 3: Kaempferol-3-O-α-L-
the chemical structures of compounds 1-3. rhamnopyranosyl-1→2)-β-D-xylopyranoside
(C26H28O14); pale yellow crystalline solid;
Spectroscopic data of the isolated 1
H-NMR (500 MHz, DMSO-d6); 7.98 (2H,
compounds d, J = 8.85 Hz, H-2′,6′), 6.89 (2H, d, J =8.35
Compound 1: Quercetin-3-methyl ether Hz, H-3′,5′), 6.17 (1H, br s, H-8), 5.97 (1H,
(C16H12O7); pale yellow crystalline solid; br s, H-6), 5.52 (1H, d, J = 7.35 Hz, H-1′′),
1
H-NMR (500 MHz, DMSO-d6); δ 12.70 (1H, 5.09 (1H, s, H-1′′′); 13C-NMR (125 MHz,

Table 2. Results of the total phenol content and DPPH IC50 (Mean ± SD, n = 3).
Table 2. Results of the total phenol content and DPPH IC50 (Mean ± SD, n = 3).

Methanol n-Hexane Chloroform Ethyl acetate n-Butanol Water

BHT
extract fraction fraction fraction fraction fraction
GAE/g 475 ± 3.2 275 ± 2.4 575 ± 5.1 625 ± 7.2 300 ± 3.2 175 ± 1.8 -
DPPH IC50
14.3 ± 0.3 22.5 ± 0.9 7.7 ± 0.5 6.1 ± 0.3 17.2 ± 0.9 27.3 ± 1.1 13.5 ± 0.7
(µg/mL)

Table 3. In-vitro cytotoxic activities of fractions.

Table 3. In-vitro cytotoxic activities of fractions.
MCF-7 HepG-2 PC-3
fractions
IC50 ± SDa (µg/mL) IC50 ± SDa (µg/mL) IC50 ± SDa (µg/mL)
Methanol extract 483 ± 1.2 800 ± 3.2 337 ± 1.1
n-Hexane fraction >1000 >1000 >1000
Chloroform fraction 333 ± 2.1 560 ± 0.9 300 ± 1.4
Ethyl acetate fraction 280 ± 1.3 191 ± 3.1 170 ± 0.9
n-Butanol fraction 700 ± 5.2 850 ± 3.4 540 ± 2.1
Water fraction >1000 >1000 >1000

a
Values presented mean ± SD of triplicate experiments.
a
Values presented mean ± SD of triplicate experiments.

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Pournaghi N et al. / IJPR (2021), 20 (1): 274-282

DMSO-d6); δ 176.84 (C-4), 167.05 (C-7), antiproliferative activity with IC50 values of
161.40 (C-5), 160.83 (C-4′). 157.08 (C-9), 45 µg/mL against PC-3, but it showed low
155.27 (C-2), 132.59 (C-3), 130.77 (C-2′,6′), antiproliferative activity with IC50 values
121.04 (C-1′), 115.74 (C-5′,3′), 107.01 (C-10), of 78 and 99 µg/mL against MCF-7 and
102.30 (C-1′′′), 101.10 (C-1′′), 99.69 (C-6), HepG-2, respectively. Previous investigation
88.70 (C-8), 77.60 (C-2′′), 77.35 (C-3′′), 72.31 has proven the cytotoxicity of compound 1
(C-4′′), 70.99 (C-2′′′), 70.18 (C-5′′′), 68.83 (C- against Hela cell line and mouse epidermal
5′′), 17.89 (C-6′′′); EI-MS m/z: [M+H]+565 JB6 P1 cells (27, 34). Compounds 2 and 3
(32, 27). did not show any significant antiproliferative
activity, and both had IC50 values greater than
Antiproliferative activity of isolated 100 µg/mL. The lack of cytotoxic activity of
compounds compound 3 might be due to the additional
The isolated compounds were tested for sugar component attached at position 3 of the
antiproliferative activity against MCF-7 C-ring so that increasing their polarity and
(human breast cancer), HepG-2 (human liver limiting their cellular permeability and also
cancer) and PC-3 (human prostate cancer). increased molecular weight of compound
The results of the in-vitro antiproliferative 3 might limit its cellular permeability (35).
activity of the compounds isolated from C. Due to the structure of the compounds and its
bonduc are summarized in Table 4. relationship with antiproliferative activity, the
The results show that compound 1 presence of a hydroxyl group at 3’ of the B-ring
(Quercetin 3- methyl ether) has moderate and methylation of the 3 hydroxyl group at

R2

3'
OH
2'
4'
8 1
HO 7 9 O 2 5'
1'
6'

6 3
10 R1
5 4

OH O

Compound 1: R1= OCH3, R2= OH Compound 3

Compound 2: R1= OH, R2= H

Figure 2. Chemical structures of compounds 1-3 isolated from Caesalpinia bonduc.

Figure 2. Chemical structures of compounds 1-3 isolated from Caesalpinia bonduc.

Table 4. In-vitro cytotoxic activities of isolatedTable 4. In-vitro cytotoxic activities of isolated compounds.
compounds.

MCF-7 HepG-2 PC-3

Isolated compounds
IC50 ± SDa (µg/mL) IC50 ± SDa (µg/mL) IC50 ± SDa (µg/mL)
1 78 ± 0.8 99 ± 1.0 45 ± 0.5
2 > 100 > 100 > 100
3 > 100 > 100 > 100
methotrexateb 25.3 ± 1.2 8.5 ± 0.8 12.5 ± 1.0

Values present mean ± SD of triplicate experiments.

a
a
Positive control.
b Values present mean ± SD of triplicate experiments.
Positive control.
b

279
 Bioassay-guided Cytotoxic Isolation of Caesalpinia bonduc

the C-ring are the factor for increasing this from the Medicinal Plants Research Center,
property. Institute of Medicinal Plants, ACECR. This
It is necessary to mention that article is a result of PhD dissertation of Narges
different flavonoid compounds were Pournaghi. The authors wish to thank Mrs
separated from this plant, for example Mona Ghiasi-Yekta, Dr. Saeedeh Momtaz and
caesalpinianone, 6-O- methylcaesalpinianone, Dr. Saeed Tavakoli for their assistance.
7-hydroxy-4’-methoxyl-3,11-
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