molecules
Article
Clerodane Diterpenoids from an Edible Plant Justicia insularis:
Discovery, Cytotoxicity, and Apoptosis Induction in Human
Ovarian Cancer Cells
Idowu E. Fadayomi 1 , Okiemute R. Johnson-Ajinwo 1 , Elisabete Pires 2 , James McCullagh 2 , Tim D. W. Claridge 2 ,
Nicholas R. Forsyth 1 and Wen-Wu Li 1, *
1
2
*
Citation: Fadayomi, I.E.;
Johnson-Ajinwo, O.R.; Pires, E.;
McCullagh, J.; W. Claridge, T.D.;
Forsyth, N.R.; Li, W.-W. Clerodane
Diterpenoids from an Edible Plant
Justicia insularis: Discovery,
Cytotoxicity, and Apoptosis
Induction in Human Ovarian Cancer
Cells. Molecules 2021, 26, 5933.
https://doi.org/10.3390/molecules
26195933
Academic Editor: Gian Cesare Tron
Received: 2 September 2021
Accepted: 26 September 2021
Published: 30 September 2021
Publisher’s Note: MDPI stays neutral
School of Pharmacy and Bioengineering, Keele University, Stoke-on-Trent ST4 7QB, UK;
i.e.fadayomi@keele.ac.uk (I.E.F.); okiemute_2002@yahoo.co.uk (O.R.J.-A.); n.r.forsyth@keele.ac.uk (N.R.F.)
Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK;
elisabete.pires@chem.ox.ac.uk (E.P.); james.mccullagh@chem.ox.ac.uk (J.M.);
tim.claridge@chem.ox.ac.uk (T.D.W.C.)
Correspondence: w.li@keele.ac.uk; Tel.: +44-(0)1782-674382; Fax: +44-(0)1782-747319
Abstract: Objectives: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics.
Drug discovery from edible and medicinal plants represents a promising approach towards finding
safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal
plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J.
insularis and investigate their underlying mechanism of action. Methods: The cytotoxicity of the
plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface
epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using
column chromatography including HPLC, and the isolated natural products were characterized
using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated
using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME
and SwissTargetPrediction web tools were used to predict the molecular properties and possible
protein targets of identified active compounds. Key finding: The two cytotoxic compounds were
identified as clerodane diterpenoids: 16(α/β)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound
1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more
potent than compound 2 with IC50 values in the micromolar range against OVCAR-4 and OVCAR-8
cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced
apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound
1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2
are both drug like, and compound 1 may target various proteins including a kinase. Conclusions:
Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via
the induction of apoptosis, providing an abundant and valuable source of hit compounds for the
treatment of ovarian cancer.
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published maps and institutional affiliations.
Keywords: ovarian cancer; Justicia insularis; diterpenoids; cytotoxicity; induction of apoptosis;
target prediction
Copyright: © 2021 by the authors.
1. Introduction
Licensee MDPI, Basel, Switzerland.
Ovarian cancer is the most severe of the gynaecological malignancy worldwide, associated with the highest level of lethality due to lack of efficient screening method and early
symptoms. Each year, ovarian cancer is diagnosed in about quarter of a million women
worldwide, and it stands as the eighth commonest and seventh leading cause of cancer
mortality among women, with 140,000 estimated casualties on a yearly basis [1]. At present,
the available treatments for ovarian cancer include surgery, radiotherapy, chemotherapy,
This article is an open access article
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Attribution (CC BY) license (https://
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Molecules 2021, 26, 5933. https://doi.org/10.3390/molecules26195933
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Molecules 2021, 26, 5933
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and immunotherapy. The most common chemotherapy drugs used for ovarian cancer
treatment are carboplatin and paclitaxel. Others include cisplatin, gemicitabine, etoposide,
topotecan, liposomal doxorubicin, and cyclophosphamide [2,3]. Targeted PARP inhibitors,
such as Olaparib (Lynparza) and Niraparib (Zejula), and antibody based drugs, such as
bevacizumab (Avastin) and rucaparib (Rubraca), are used with or after other chemotherapy for advanced ovarian cancer treatment [3]. These novel therapies have significantly
improved the management of ovarian cancer. However, they have shortcomings, including
severe side effects, development of resistance, and high cost. Therefore, the search for new
and affordable drugs that could also reduce the adverse effects and overcome the resistant
nature of cancer is highly essential.
Natural products from plants or their derivatives represent an important source of
anticancer drugs, such as vinblastine, vincristine, topotecan and irinotecan, etoposide,
and paclitaxel [2,4]. In particular, phytochemicals from edible and medicinal plants (e.g.,
isothiocyanates from cruciferous vegetables, sulforaphane from broccoli [5], curcumin
from tumeric, genistein from soybean, resveratrol from grapes, and apigenin from various vegetables [6], etc.) are promising sources of anticancer compounds effective in the
chemoprevention and treatment of cancer with lower costs and higher safety profiles with
a plethora of mechanisms of action [7,8]. Plants in Nigeria have not been extensively
explored to discover anti-cancer agents. Previously, we isolated and identified a number of
promising cytotoxic alkaloids from several Nigerian medicinal plants [9–16].
Justicia insularis T. Anderson (Acanthaceae family) is an annual to perennial edible
plant with medical use as digestive, weaning agent, laxative [17–19], and nutritional
value [20] in Nigeria and across Africa. The Justicia is the largest genus with around
600 species, few of which have been studied in recent decades, although arylnaphthalide
lignans and triterpenoid glycosides are indicated as the major types of chemical constituents [21]. Aqueous extracts of J. insularis leaves were shown to produce estradiol
in vitro [17], promote ovarian folliculogenesis and fertility in female rats [19], possess
anti-oxidant activity [20], and to benefit the treatment of anaemia [22]. However, the cytotoxic activity and chemical constituents of J. insularis have not been characterized. Here,
we report the extraction, bioassay-guided purification/isolation, structural identification,
cytotoxicity, apoptosis induction evaluation and target prediction in human ovarian cancer
cells of the cytotoxic compounds from J. insularis.
2. Materials and Methods
2.1. Reagents
All the chemicals used were of analytical grade. n-Butanol, dichloromethane (DCM),
ethyl acetate (EA), n-hexane, methanol (MeOH), and trichloroacetic acid (TCA) were products of Fischer Scientific, Loughborough, UK. Cell culture media, Roswell Park Memorial
Institute (RPMI) 1640, 10% fetal bovine serum (FBS), L-glutamine, PENSTREP (50 µg/mL
penicillin/streptomycin), and phosphate buffered saline (PBS) were obtained from Lonza
(Basel, Switzerland). Trizma base, trypsin-EDTA solution, glacial acetic acid, dimethyl
sulfoxide (DMSO), sulforhodamine B (SRB) sodium salt, trypan blue, and carboplatin were
purchased from Sigma Aldrich (St. Louis, MO, USA) and Caspase-Glo 3/7, 8, and 9 assay
kits from Promega, Southampton, UK.
2.2. Plant Samples
Justicia insularis T. Anderson (Acanthaceae) leaves were sourced from Isiokolo in
Kokori Town; Region/Local government area: Ethiope East; State: Delta, Nigeria (Latitude:
5◦ 37′ 52” N; Longitude: 6◦ 02′ 06” E), authenticated by Mr. Alfred Ozioko and deposited at
the International Centre for Ethnomedicine and Drug Development (specimen voucher
number: INTERCEDD/1590). The plants were pulverized after drying under shade for
7–10 days at 25 ◦ C.
Molecules 2021, 26, 5933
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2.3. Extraction Procedure for Justicia insularis
The pulverized J. insularis leaves (1.0 kg) were macerated in 1000 mL of DCM and
1000 mL of MeOH for 72 h. The mixture was filtered to obtain the DCM/MeOH extract.
The residue was further macerated with 1000 mL of methanol for 72 h. The solution of the
extract was collected by filtration and repeated two more times within a 24 h maceration
period. The residue was there after soaked in 1000 mL of deionized water and filtered after
72 h of maceration. This was also repeated two more times within 24 h each to increase the
yield. The DCM/MeOH extract was combined with methanol extract to yield the organic
extract, which was dried using a rotary evaporator at <40 ◦ C. The little remaining solvent
was further removed using a desiccator. The aqueous extract was frozen at −80 ◦ C for 24 h
before being lyophilized to dryness.
2.4. Solvent Partition of Plant Extracts
The organic extract of J. insularis (20 g) was further partitioned with three solvents
(n-hexane, ethyl acetate, and n-butanol) as done previously [9,15].
2.5. Bioassay-Guided Purification of Bioactive Fraction of Justicia insularis
The column was firstly prepared by suspending 50–80 g of silica gel in hexane. The
suspended silica gel was poured into the column and allowed to settle with little solvent
above the gel. The bioactive hexane or ethyl acetate fraction J. insularis was dissolved
in hexane and gently transferred to the surface of the gel in the column using Pasteur
pipette. The fractions were eluted with 200 mL of n-hexane/ethyl acetate in the following
ratios (4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4) consecutively based on the optimum thin layer
chromatography profile obtained from a mobile phase of hexane and ethyl acetate combination. The column was finally washed using 100% methanol to obtain the more polar
fractions. Ten sub-fractions were obtained using rotary evaporator and desiccator and
their ovarian cancer cell growth inhibitory activities were evaluated using a cell growth
assay on the OVCAR-4 cell line (Section 2.12). Each of the sub-fractions from the ethyl
acetate fraction showed significant anti-cancer activity. However, EA4 was the most active
sub-fraction of the ethyl acetate fraction while the least activities were observed in EA9
and EA10 which were eluted with ethyl acetate/methanol and methanol respectively. EA4
was further purified using column chromatography to yield sub-fractions of EA4. The
growth inhibitory activity of the sub-fractions of EA4 from column chromatography was
evaluated. Sub-fractions EA4-4 and EA4-6 were further purified using semi-preparative
high performance liquid chromatography.
2.6. Isolation of Compound 1 and 2 Using High Performance Liquid Chromatography (HPLC)
The various sub-fractions were further assayed for anti-cancer activities and the most
significant active sub-fractions were purified further using semi-preparative HPLC. Briefly,
semi-preparative HPLC was done using Agilent 1220 LC, USA. The mobile phase used two
solvent systems. Solvent A consisted of 100% water and solvent B was 100% methanol. The
mobile phase calibration rose from 50% by 50% (A:B) over a period of 25 min to 100% B and
kept at 100% for 10 min at a flow rate of 4mL/min at 215 nm on semi-preparative HPLC
column (Phenomenex, Cambridgeshire, UK; 5µm particle size: 9.4 × 250 mm). A major
fraction and minor fraction eluted at retention times of 22 and 24 min were collected and
dried using a rotary evaporator to yield compound 1 (80 mg, 97% purity) and compound 2
(2 mg, 85% purity), respectively.
2.7. Quantification of Compound 1 in the Extracts and Plant Materials
The purity of both compounds and the composition of compound 1 in the total plant
organic extracts were determined by using analytical HPLC. Stock solutions of the highly
pure compound 1 with a series of concentrations (0.125, 0.25, 0.5, 1.0 and 1.5 mg/mL),
and organic extracts solutions at 1.0 mg/mL were prepared. Hence, 10 µL of each stock
solution and samples were injected into the analytical HPLC system in duplicates. The
Molecules 2021, 26, 5933
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mobile phase rose from 20% B (A + B) to 100% B over a period of 25 min and kept at
100% for 6 min at 215 nm on an analytical HPLC column (Phenomenex, UK; 5 µm particle
size, 4.6 × 250 mm) at a flow rate of 1 mL/min. For the quantification of compound 1 in
organic extracts, a linear calibration curve was made by plotting the area under the peak
against the different contractions. The percentage of compound 1 in the organic extracts
was calculated based on the calibration curve.
2.8. Gas Chromatography Mass Spectrometry Analysis
Briefly, 1.0–2.0 mg of the bioactive fractions and isolated compounds of J. insularis
were dissolved in 200 µL of ethyl acetate and sonicated at <40 ◦ C for 5 min. 1–2 µL of the
solution was subsequently injected into gas chromatography mass spectrometry (GC-MS)
system consisting of an Agilent 7890 coupled to Agilent MS model 5975C MSD (Agilent
Technologies, Cold Spring, NY, USA). The gas chromatography started at 60 ◦ C for 2 min
and increased to 300 ◦ C at the rate of 10 ◦ C/min, which was held at 300 ◦ C for 4 min at a
constant helium pressure (10 psi). The mass spectra data were acquired in the scan mode
in m/z range 40–1000.
2.9. Liquid Chromatography Mass Spectrometry (LC-MS) Analysis
The purified compounds were analysed by LC-MS to determine their high resolution
molecular mass using a Ultimate U3000 ultra-performance liquid chromatography system
with a HESI II electrospray ion source on a Q-Exactive Orbitrap mass spectrometer system
(Thermo Scientific, Waltham, MA, USA) as described [12].
2.10. NMR Spectroscopy
1D and 2D NMR spectra of compounds 1 and 2 were obtained with a Bruker AVII500
NMR spectrometer (Billerica, MA, USA). 1D NMR spectra of the hexane fraction of
J. insularis were obtained with a Bruker Ascend 400 NMR spectrometer. ACD/Labs 10 Freeware (Advanced Chemistry Development Inc., Toronto, ON, Canada) or Bruker TopSpin
4.1.3 software was used to analyse the NMR Spectra.
2.11. Cell Culture
The human ovarian cancer cell lines and normal human ovarian surface epithelial
(HOE) cells were used in this study. Ovarian cancer cell lines (OVCAR-4 and OVCAR-8)
were products of American tissue culture collection (ATCC). The HOE cells were purchased from Applied Biological Materials (ABM) Inc. (Vancouver, BC, Canada). The
Rosewell Park Memorial Institute (RPMI 1640, Lonza) medium was used in the culturing
of OVCAR-4, OVCAR-8, and HOE cells. The medium was supplemented with 2 mM
glutamine, 10% foetal bovine serum (FBS), and 50 µg/mL penicillin streptomycin. These
cells were incubated in a standard humidified incubator) at 37 ◦ C, 5% carbon dioxide (CO2 )
conditions.
2.12. Sulforhodamine B Cell Growth Inhibitory Assay
Sulforhodamine B (SRB) assay was used to determine the inhibition of cell proliferation
by the studied compounds and plant extracts [9,10,15]. Plant extracts/fractions (100
mg/mL) and pure compounds (20 mM) were prepared in DMSO. The 0.2% DMSO in
growth media was added to the cells as vehicle-treated cells (negative control) while
carboplatin was used as positive control. In the SRB assay, OVCAR-4, OVCAR-8, and HOE
cells were seeded in 80 µL growth medium per each well in 96 well plates. OVCAR-4
and HOE were seeded at a density of 5000 cells per well while OVCAR-8 was seeded at
a density of 2000 cells per well. The seeded plates were incubated for 24 h, after which
20 µL of plant extracts and natural compounds 1000 µg/mL and 200 µM (and their serial
two-fold dilutions), respectively, were added at the indicated concentrations. The cell
cultures were incubated at 37 ◦ C under 5% CO2 for 72 h in a humidified atmosphere.
Molecules 2021, 26, 5933
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After 72 h, the medium was decanted, and the cells fixed with 0.1 mL of 10% TCA on
ice for 30 min before drying. The cells were stained with 0.4% SRB, washed with 1% acetic
acid three times and dried. Then, 0.1 mL of Tris-base (10 mM) was added to the plates to
solubilise the protein-bound SRB dye. The absorbance at 570 nm was measured using a
spectroscopic plate reader (Multi-mode microplate reader BioTEK Synergy 2, Winooski,
VT, USA). The data were analysed by non-linear regression to fit a 4-parameter sigmoidal
dose–response curve to determine IC50 values using GraphPad PRISM 6.0 software, Inc.
(San Diego, CA, USA).
2.13. Apoptosis Detection Using Caspase-Glo 3/7, 8 and 9 Activity Assay
Caspase 3/7 activities was measured using assay kits caspase-Glo 3/7 (Promega Corp.,
Madison, WI, USA) on a 96-well microplate. Briefly, the cells (OVCAR-4 and OVCAR-8)
were seeded in 96 well plates at a cell density of 5000 cells/well in 80 µL growth media
and exposed to 10, 20 and/or 30 µM of the natural compounds after 24 h incubation. After
48 h exposure to compound treatments, 25 µL of Caspase 3/7 Glo-reagent was added, and
the cells were incubated in the dark at room temperature for 30 min on a gentle rocker.
The luminescence was measured at 570 nm by a BioTEK Synergy microplate reader (USA).
A similar procedure was followed for caspase 8 and caspase 9 activity.
2.14. Evaluation of Early and Late Apoptosis Using Flow Cytometry
The in vitro method of fluorescence-activated cell sorting (FACS) by Annexin V and
propidium iodide (PI) staining was used to detect the change of cell population as reported [10,23]. OVCAR-8 cells were seeded in 12 well plates at a density of 2 × 105 cells
per well in 1 mL of growth media and incubated for 24 h before treatment with the tested
compound and positive control. After treatment for 48 h, media were decanted into 15 mL
tubes, and cell pellets were collected into the same tubes by trypsinisation, the cells were
centrifuged at 150× g for 3 min and re-suspended into 1 mL growth media, which was
transferred into sterile 2 mL Eppendorf tube, centrifuged at 300× g for 5 min at 4 ◦ C. The
media were aspirated, and the pellet was washed in cold PBS. The cells were centrifuged
under the same conditions and the supernatant aspirated. Cells were washed in 500 µL
annexin-V binding buffer and centrifuged at 300× g for 10 min, annexin-V binding buffer
was aspirated, and the cell pellet was treated with 10 µL of annexin V-FITC in 100 µL of
binding buffer. The cells were thoroughly mixed and incubated in the dark at room temperature for 15 min. After incubation, cells were washed in binding buffer and centrifuged at
300× g for 10 min. The buffer was aspirated, and cells were suspended in 500 µL binding
buffer and subsequently 5 µL of PI was added for flow cytometry analysis.
2.15. Bioinformatic Analysis
The molecular properties of compounds 1 and 2 were determined or predicted through
the SwissADME website tool [24,25], and their molecular targets predicted through SwissTargetPrediction (Swiss Institute of Bioinformatics, University of Lausanne, Lausanne,
Switzerland) web tool [26,27].
2.16. Statistical Analysis
The IC50 s were obtained from at least three repeated experiments. The mean IC50 was
calculated, and the standard error of mean (SEM) was determined. Furthermore, a one
way analysis of variance (ANOVA) and student t test were used to test if the difference
in the mean of control and treatments and mean of treatment at different concentration
were significant. A post hoc Dunnett test was used to determine which of the treatments
was significant to the control while a Tukey test was used to determine which of the
concentrations of a particular treatment were significant using GraphPad prism 6.
Molecules 2021, 26, 5933
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3. Results
3.1. Bioassay-Guided Isolation of Diterpenoids from J. insularis
The bioassay-guided fractionation and isolation of two diterpenoids from extracts of
J. insularis are illustrated (Figure S1 in the Supplementary Materials). Both organic (21.0 g,
2.1% yield) and aqueous extracts of J. insularis were obtained. The organic extracts were
partitioned with solvents and the weights and yields of the solid fraction recovered are
4.6 g (25%), 6.0 g (33.3%), 2.5 g (13.9%), and 3.0 g (16.7%) for n-hexane, ethyl acetate (EA),
n-butanol, and aqueous fractions respectively.
The organic extract showed stronger cytotoxicity against OVCAR-4 and OVCAR-8
cell lines (IC50 < 30 µg/mL) than the aqueous extract J. insularis using SRB cell growth
assay. Furthermore, that n-hexane and ethyl acetate fractions derived from the organic
extract are the most active fractions with IC50 less than 20 µg/mL (Figure S2, Table S1).
Analytical HPLC analysis of both n-hexane and ethyl acetate fractions showed similar
patterns of compounds. The more abundant ethyl acetate fraction of J. insularis was focused
on and subjected to silica gel column chromatography. Ten sub-fractions were obtained,
and their in vitro ovarian cancer growth inhibitory activities were evaluated (Table S2).
The most active sub-fraction (EA4) was further purified using reversed-phase HPLC to
yield compounds 1 and 2 with high purity (Figure S3).
3.2. Chemical Identification of the Isolated Bioactive Compounds of J. insularis
The molecular formula of 1 was determined as C20 H30 O3 based on the observed
molar mass of compound 1 (found, 318.2123 Da) by LC-HR-MS (Figure S4). Compound 1
(Figure 1) was identified as 1:1 mixture of 16-hydroxy epimers (α and β) of 16-hydroxycleroda-3,13(14)Z-dien-15,16-olide based on GC-MS (Figure S5), 1 H, 13 C-NMR and HSQC
analysis (Figure S6, Table S3) and a comparison with literature data [28–30]. 13 C-NMR
spectrum of the hexane fraction (Figure S6) showed the presence of epimers of compound
1 before silica gel chromatography and HPLC purification which may cause isomerization,
so the epimers of compound 1 are the natural products in J. inuslaris. Compound 1 showed
a single peak in analytical HPLC chromatogram indicating a purity of 97% (Figure S3).
Surprisingly, three major peaks appeared on the GC-MS chromatogram (Figure S5), the
mass spectrum of a peak at retention time 18.45 min is consistent with the mass data of
compound 1 [24]. The other observed compounds, 1a and 1b (Figure S5), were determined
to be the thermal degradation products of compound 1 due to the presence of a γ–hydroxy
unsaturated 5-membered lactone moiety (Figure 1) under high temperature conditions
(60 ◦ C–300 ◦ C) for 30 min of GC-MS. To support this, the exposure of compound 1 at
200 ◦ C for 0.5 h followed by HPLC analysis indicated the formation of new products
(likely including 1a and 1b), whose structures remain to be determined. Furthermore,
the percentage of compound 1 in organic extracts was determined to be 34%. Thus, the
composition of compound 1 in the plant material is 0.7% (dry weight), a very abundant
secondary metabolite in the leave of J. insularis.
Compound 2 (Figure 1) has the same molecular formula C20 H30 O3 as compound 1 by
LC-HRMS data (Figure S7), which was further identified as 16-oxo-cleroda-3,13(14)E-dien15-oic acid based on 1 H-NMR, 13 C-NMR and HSQC analysis (Figure S8) and a comparison
with literature data (Table S3) [28,29].
Molecules 2021, 26, 5933
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αβ
Figure 1. Chemical structure of compound 1, 16(α/β)-hydroxy-cleroda-3,13
(14)Z-dien-15,16-olide,
and compound 2, 16-oxo-cleroda-3,13(14)E-dien-15-oic acid.
3.3. In Vitro Cytotoxicity of Compounds 1 and 2
Compound 1 (IC50 = 4–6
μ µM) shows greater potency than compound 2 (IC50 =
12–17
µM)
and
a
positive
control
carboplatin (IC50 = μ
8–18 µM) (Table 1) against OVCAR-4
μ
and OVCAR-8 cells (Figure 2). Both compounds 1 and 2 demonstrate less cytotoxic activity
against HOE cells (Table 1).
αβ
Table 1. The growth inhibitory activities of isolated compounds (1 and 2) from J. insularis in OVCAR-4, OVCAR-8 cancer cell
lines and HOE cells. The selectivity index (SI) (the ratio of IC50 against HOE cells to IC50 against OVCAR-8) are indicated.
Compounds
OVCAR-4
(µM)
1
2
Carboplatin
μ 0.3(1.8 µg/mL)
5.7 ±
16.6 ± 2.8 (5.3 µg/mL)
17.6 ± 4.6
μ
OVCAR-8
(µM)
μ
4.4 ± 0.2 (1.4 µg/mL)
11.8 ± 0.5 (3.8 µg/mL)
8.2 ± 2.2
μ
HOE
(µM)
SI against
OVCAR-8
12.1 ± 0.1μ(3.9 µg/mL)
22.8 ± 0.7 (7.3 µg/mL)
13.0 ± 3.7
3
2
1.6
μ
Figure 2. Mean concentration-response curve of compound 1 (A) and 2 (B) in OVCAR-4 and OVCAR-8 ovarian cancer cells
μ
μ
μ
and HOE. IC50 values determined are listed in Table 1.
μ
μ
μ
3.4. Apoptosis Study
To investigate whether the significant decrease in cell viability by compound 1 and 2
was due to apoptosis, the level of caspase 3/7, or caspase 8 and 9 activation by these compounds were measured. Compound 1 and 2 significantly increased caspase 3/7 activities
in OVCAR-4
and OVCAR-8μcells when compared with
μ
μ control using one-way analysis of
variance (ANOVA) (Figure 3A,B). Compound 1 further increased caspase 8 and 9 activiμ
μ
μ
μ
μ
μ
Molecules 2021, 26, 5933
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ties in OVCAR-8 (Figure 3C,D). These results suggest that the significant decrease in cell
viability induced by these compounds was likely due to apoptosis.
Figure 3. Caspase activities of compound 1 and 2 in ovarian cancer cells. Caspase 3/7 activities of isolated compound 1
and 2 in OVCAR-4 (A) and OVCAR-8 (B) cells; and caspase 8 activity (C) and caspase 9 activity (D) of compound 1 in
OVCAR-8 cells. Carboplatin was used as positive control. The fold increase in caspase activities induced by compound 1
and positive control were compared with the negative control using one-way ANOVA with Dunnett’s multiple comparison
test. Significant difference between treatment and control is denoted with asterisk (*) and student t test was used to test for
concentration dependent activity.
Furthermore, annexin-V and PI assay results also show concentration-dependent and
significant increase of percentage of early and late apoptosis induced by compound 1 (at 5,
10, and 20 µM) after 48 h treatment (Figure 4), similar to the positive control carboplatin
μ
same concentrations.
Molecules 2021, 26, 5933
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Figure 4. Evaluation of apoptotic activities of compound 1 (5, 10 and 20 μµM) and carboplatin (10 and 20 μ
µM) on OVCAR-8
cells using annexin V-FITC and propidium iodide (PI) staining analysed with flow cytometry. (A) Representative flow
cytometry graphs of OVCAR-8 cell line. Lower left (LL), upper left (UL), lower right (LR) and upper right (UR) represent
live cells, necrotic cells, cells in early apoptosis and cells in late apoptosis respectively. (B) Mean percentage of apoptotic cell
populations. The data represent the mean ± SD of three repeats. The significant different between control and treatment is
denoted with asterisk (*), while no significant different is denoted with (ns).
3.5. Bioinformatic Analysis
Compounds 1 and 2 have the same molecular formula, with 1 having a more rigid
structure than 2. Both compounds are druglike, obeying the Lipinski rule (Table S4). The
possible targets of compound 1 are predicted to be mainly kinase (e.g., Ribosomal protein
S6 kinase alpha 5), primary active transporters (e.g., potassium-transporting ATPase alpha
chain 2), and nuclear receptors (e.g., glucocorticoid receptor) (Table S5, Figure S9), while the
possible targets of compound 2 are predicted to be nuclear receptors (e.g., estrogen receptor
beta), oxidoreductases (e.g., steroid 5-alphareductase 2), and phosphatases (e.g., proteintyrosine phosphatase 1B) (Table S6, Figure S10), although with low probability (<25%).
4. Discussion
The ethnopharmacological use and the anti-cancer activity of Justicia species [31–33]
inspired us to investigate the cytotoxic activities of J. insularis from Nigeria against ovarian
cancer cells. In this study, two clerodane diterpenoid compounds (1 and 2) were revealed to
be the cytotoxic compounds in J. insularis for the first time. The very abundant compound 1
in the plant extracts showed higher potency with IC50 values < 6 µM against the two ovarian
μ
cancer cell lines studied and greater SI compared to those of a standard chemotherapeutic
drug carboplatin for ovarian cancer treatment. This makes compound 1 an interesting hit
compound. The cytotoxic activity of compound 1 is likely associated with the more rigid
α,β-unsaturated γ-lactone moiety in the clerodane diterpenoid, whereas compound 2 with
α β rotational bonds
γ (Table S4) demonstrates less cytotoxicity [34].
an open form and more
Compound 1 was found to be thermally unstable at a high temperature, and further
isolation, identification, and testing the cytotoxicity of those degradation compounds
(Figure S5) would be interesting. Compound 1 was found to be a mixture of epimers
of 16α (S) and 16β (R) forms (1:1) based on the analysis of NMR spectra of the isolated
α fraction before
β
purification (Figure S6). The single epimer
compound 1 and the hexane
16α (S) form of compound 1 was previously isolated from P. longifolia [28], P. barnesii [35],
and P. simiarum [36]α(the Annonaceae family). A mixture of 16S and 16R epimers (1:1)
Molecules 2021, 26, 5933
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was also found in P. longifolia [24] and chemically synthesized [30]. Compound 2 was also
present in P. longifolia [28,29]. Previously, phytochemical studies of Justicia species disclosed
the presence of cytotoxic lignans, such as justicidin A [37,38] and 6′ -hydroxy justicidin
A [32] from J. procumbens and triacontanoic ester of 5-hydroxy-justisolin from J. simplex [31].
Chemical analysis of leave extract of J. insularis indicated the presence of abundant iron [20]
and hemoglobin, which might explain the observed benefit for anaemia [22]. Our study
indicates the clerodane diterpenoids, such as compounds 1 (abundant) and 2, are present
in the family of Acanthaceae, providing a new source of these interesting compounds as
hit compounds for anticancer drug development.
Cancer cells are generally known for their characteristic features of escaping programmed cell death (apoptosis), which is a mechanism that maintains the cell population
and defense against damaged cells [39]. To further investigate the route of anticancer
activity for compound 1 and 2, their roles in the induction of apoptosis were evaluated.
The caspase 3/7 activity of compounds 1 and 2 was evaluated, and the results showed
that the cell death induced by compounds 1 and 2 was via the activation of caspase 3/7
which are apoptosis executioners. Furthermore, compound 1 activated both caspase 8 and
9, which indicates the involvement of both extrinsic and intrinsic pathway [39]. Compound
2 was not further investigated due to the lesser activity and limited quantity isolated. The
pro-apoptotic activity of compound 1 was further verified using annexin V-FITC and PI
staining, which analysed the apoptotic markers, i.e., phosphatidylserine residues, on the
cell surfaces and DNA fragments in the nucleus, respectively [23]. The percentages of both
early and late apoptotic cells caused by compound 1 are similar to those of carboplatin. Further bioinformatic analysis of the potential protein targets of compounds 1 and 2 supports
the observed greater cytotoxicity and apoptotic activity of compound 1 than 2, because
compound 1 may more likely target kinases which are essential for cancer cell initiation
and proliferation [40]. The cytotoxicity and induction of apoptosis of compound 1 were
previously observed in other cancers, such as leukaemia HL-60 [41], CML K562 [42,43],
oral squamous cell carcinoma cancer [44], human renal carcinoma [45], renal cell carcinoma [46], T24 bladder cancer [47], and breast cancer [48] cells. Specifically, compound 1
induces the expression of PRC2 enzyme complex in CML K562 cells [42] and deregulates
phosphoinositide-3 kinase (PI3K) and Aurora kinase B activities [43]. Compound 1 was
also found to be involved with Akt, mTOR, and MEK-ERK pathways in renal carcinoma
cells [45] or the inactivation of EGFR-related pathways in bladder cancer cells [47]. Our
study is the first report showing their cytotoxicity in ovarian cancer cells via the induction of apoptosis via both intrinsic and extrinsic pathways, which is consistent with its
cytotoxicity found in other cancers.
Besides the cytotoxic activity of these diterpenoids, 16α-hydroxy-cleroda-3,13(14)Zdien-15,16-olide (1) was also demonstrated to be an orally active anti-leishmanial and
non-cytotoxic agent [49], a HMG-CoA reductase inhibitor [50], and a dual inhibitor of
COX/5-LOX with potential in the treatment of inflammatory conditions [51,52]. Compound
2 possesses anti-biofilm activity against methicillin resistant Staphylococcus aureus and
Streptococcus mutans [53].
Diterpenoids are a large group of natural products with diverse structures and biological activities including anticancer activity [34,54]. One of the well-known and approved
anticancer diterpenoid drug from plants is paclitaxel, which showed superior potency
against ovarian cancer cells with IC50 s at nanomolar range through stabilizing the tubulin
structure of cancer cells [55]. However, it also caused severe side effects among patients
because of its poor selectivity to cancer cells. So far, more than hundreds of important diterpenoids, including triptolide, oridonin, and andrographolide, have been discovered and
shown in vitro and/or in vivo cytotoxicity with moderate and strong potency [54]. Specifically, clerodane diterpenoids were isolated and showed cytotoxicity to various cancer cell
lines [34]. For example, caseamembrins A–F from Casearia membranacea showed cytotoxicity
to human prostate cancer PC-3 cells with IC50 at the µM range with either intrinsic or
extrinsic apoptotic pathways [56]. Ent-clerodane diterpenoids from Scutellaria barbata D.
Molecules 2021, 26, 5933
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Don. (Labiatae) showed cytotoxicity against KB oral epidermoid carcinoma, HONE-1
nasopharyngeal, and HT29 colorectal carcinoma cells with IC50 values in the range of
3.1–7.2 µM [57]. Kurzipene B from the leaves of Casearia kurzii showed an IC50 value of
5.3 µM against Hela cells, induced apoptosis, and arrested the cell cycle at the G0 /G1
stage [58]. The activity of the two compounds found in J. insularis is comparable to clerodane diterpenoids from other plants.
Further work on the experimental identification of molecular targets, structure–activity
relationships, efficacy, and safety of diterpenoids in J. insularis remain under investigation
in in vitro and animal xenograft models, which may provide a safer, more sustainable, and
affordable anticancer drug.
5. Conclusions
This study shows that the extract of the edible J. insularis leaves demonstrate significant cytotoxic activities against ovarian cancer cell lines, providing a new and abundant
potential source of anticancer agents. Two diterpenoids, 16α/β-hydroxy-cleroda-3,13(14)Zdien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2), were isolated and
identified as the cytotoxic compounds through bioassay-guided fractionation from the
Acanthaceae family. Compound 1 showed greater selectivity towards in vitro cancer cells
over normal cells. Compound 1 was established to induce apoptosis through both intrinsic
and extrinsic pathways, which warrants the further investigation of compound 1 as an
anticancer agent from an edible plant as with the potential of lower toxicity.
Supplementary Materials: The following are available online. Figure S1. Scheme showing the extraction, bioassay-guided purification, and identification of cytotoxic compound 1 and 2 from J. insularis.
Figure S2: Mean concentration-response curve of the active extract of J. insularis (DCM/MeOH)
in OVCAR 4 (A) and OVCAR 8 (B) ovarian cancer cell lines, showing potent cytotoxic activity of
the organic extracts. Figure S3. Analytical HPLC chromatograms of purified active compounds of
J.insularis. Figure S4: LC-HRMS chromatogram (A) and negative ESI-MS spectrum (B) of isolated
compound 1 showing the HRMS of the major peak at a retention time of 12.87 min. Figure S5:
GC-MS chromatogram and mass spectra of the isolated compound 1 at Rt 18.45 min and two thermal
degradation products of 1a and 1b under high temperature in the oven of GC-MS. Figure S6: NMR
data analysis of J. insularis of the purified compound 1 and hexane fraction in CDCl3 : (A) 1 H-NMR
(500 MHz), (B) 13 C NMR (125 MHz) spectrum, (C) 13 C-NMR (100 MHz) spectrum (range of 110-180
ppm) of hexane fraction containing 1 before silica gel chromatography and HPLC. Figure S7: LC-MS
chromatogram (A) and negative ESI-MS spectrum (B) of isolated compound 2 at retention time
of 13.45 min. Figure S8: NMR spectra of J. insularis purified compound 2 in CDCl3 : (A) 1 H-NMR
and (B) 13 C-NMR spectra. Figure S9. Distribution of predicted targets molecules of compound 1
by the SwissADME web tool (Table S5). Figure S10. Distribution of predicted targets molecules of
compound 2 by the SwissADME web tool (Table S6). Table S1: The results of the growth inhibitory
activities of J. insularis extracts and fractions on ovarian cancer OVCAR-4 and OVCAR-8 cell lines.
Table S2: The growth inhibitory activities of EA fractions and EA4 sub-fractions of J. insularis on
OVCAR-4 ovarian cancer cell line. Table S3: 1 H (500 MHz) and 13 C-NMR (125 MHz) assignments
of isolated compound 1 and 2 (CDCl3 ). Table S4. Molecular descriptors and drug-likeness of compounds 1 and 2 in J. insularis calculated by SwissADME web tool. Table S5. Predicted protein targets
of compound 1 by SwissTargetPrediction web tool. Table S6. Predicted protein targets of compound
2 by SwissTargetPrediction web tool.
Author Contributions: I.E.F. performed the experimental work; O.R.J.-A. collected the plant materials and performed the initial plant extraction; E.P. and J.M. performed LC-MS analysis; T.D.W.C.
performed NMR measurement; N.R.F. provided resources and advice; W.-W.L. and I.E.F. designed
and conceived the studies and carried out structural determination and bioinformatic analysis; I.E.F.
drafted, and all authors contributed to writing the article. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by the Tertiary Education Trust Fund, Nigeria.
Institutional Review Board Statement: Not applicable.
Molecules 2021, 26, 5933
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Informed Consent Statement: Not applicable.
Data Availability Statement: Data available within the article or Supplementary Materials.
Acknowledgments: We thank Sian Woodfine at Keele University for recording NMR spectra of the
hexane fraction of J. insularis. We also thank Alexander Kagansky, Ted Hupp and Alan Richardson
for advice and an anonymous reviewer for suggesting to use SwissADME web tools.
Conflicts of Interest: All authors do not have conflict of interest to disclose.
Sample Availability: Samples of the compounds are not available from the authors.
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