Content uploaded by Elvis K Tiburu
Author content
All content in this area was uploaded by Elvis K Tiburu on Mar 15, 2021
Content may be subject to copyright.
separations
Article
Capturing Dioclea Reflexa Seed Bioactives on Halloysite
Nanotubes and pH Dependent Release of Cargo against Breast
(MCF-7) Cancers In Vitro
Srinivasan Balapangu 1,2, Emmanuel Nyankson 3, Bernard O. Asimeng 1, Richard Asiamah 1,
Patrick K. Arthur 2,4 and Elvis K. Tiburu 1,2,*
!"#!$%&'(!
!"#$%&'
Citation: Balapangu, S.; Nyankson,
E.; Asimeng, B.O.; Asiamah, R.;
Arthur, P.K.; Tiburu, E.K. Capturing
Dioclea Reflexa Seed Bioactives on
Halloysite Nanotubes and pH
Dependent Release of Cargo against
Breast (MCF-7) Cancers In vitro.
Separations 2021,8, 26. https://
doi.org/10.3390/separations8030026
Academic Editor: Marcello Locatelli
Received: 23 December 2020
Accepted: 14 February 2021
Published: 27 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Biomedical Engineering, University of Ghana, Legon LG27, Ghana;
ssbalapangu@ug.edu.gh (S.B.); boasimeng@ug.edu.gh (B.O.A.); rasiamah001@st.ug.edu.gh (R.A.)
2West Africa Center for Cell Biology of Infectious Pathogens (WACCBIP), University of Ghana, Legon LG54,
Ghana; parthur14@gmail.com
3Department of Material Science & Engineering, University of Ghana, Legon LG27, Ghana;
enyankson@ug.edu.gh
4Department of Biochemistry, Cell & Molecular Biology, University of Ghana, Legon LG54, Ghana
*Correspondence: etiburu@ug.edu.gh; Tel.: +233-559-585-194
Abstract:
In this work, optimization parameters were developed to capture plant metabolites from
Dioclea Reflexa (DR) seed ex-tracts onto halloysites nanotubes (HNTs). A one-step pool of the crude
extracts at neutral pH from the HNT lumen failed to elicit a reduction in breast cancer, Michigan
Cancer Foundation-7 (MCF-7) cell viability. However, the pH-dependent elution of metabolites
revealed that the acidic pH samples exhibited profound antiproliferative effects on the cancer
cells compared to the basic pH metabolites using both trypan blue dye exclusion assay and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability test. pH~5.2 samples
demonstrated by half-maximal inhibitory concentration (IC50) of 0.8 mg and a cyclic voltammetry
oxidation peak potential and current of 234 mV and 0.45
µ
A, respectively. This indicates that the
cancer cells death could be attributed to membrane polarization/depolarization effects of the sample.
Fluorescence-activated cell sorting (FACS) studies confirmed that the plant metabolites affected
breast cancer apoptotic signaling pathways of cell death. The studies proved that plant metabolites
could be captured using simplified screening procedures for rapid drug discovery purposes. Such
procedures, however, would require the integration of affordable analytical tools to test and isolate
individual metabolites. Our approach could be an important strategy to create a library and database
of bioactive plant metabolites based on pH values.
Keywords:
halloysite nanotubes; cyclic voltammetry; polarization/depolarization Dioclea Reflexa;
plant metabolites; anticancer metabolites
1. Introduction
Traditional herbal medicine practices continue to be inseparable in the lives of many
people in the world, including the inhabitants of sub-Sahara Africa (sSA), due to the impact
of plant metabolites for treating several diseases [
1
–
3
]. Over the years, the global accep-
tance of herbal medicines in homes, as well as health clinics, has resulted in the growth
of the herbal products market in most countries in the subregion. However, there are
still challenges with understanding mechanisms of action due to the lack of standardized
procedures to rapidly prepare plant metabolites to meet pharmacological criteria [
4
–
6
].
This gap, especially in the subregion, has elevated the research community enthusiasm in
pursuing cheap and affordable functional materials to facilitate the development of simple
and effective separation technologies that have similar or even better performance efficien-
cies than the traditional and more expensive technologies. Such easy-to-use technologies
Separations 2021,8, 26. https://doi.org/10.3390/separations8030026 https://www.mdpi.com/journal/separations
Separations 2021,8, 26 2 of 14
will facilitate the capture, release, and compilation of plant metabolites in a library and
database to support further scientific research in the field of plant medicine [7,8].
Recently, solid-phase microextraction (SPME) technology has been developed using
TiO2 nanotube arrays in situ on Ti wires for selective removal of organic compounds [
9
].
However, this technology exhibits some drawbacks, including fragility, which limits
longevity. Halloysites nanotubes (HNTs) are natural tubules of aluminosilicate minerals
composed of different proportions of aluminum, silicon, hydrogen, and oxygen, often with
the chemical formula
Al2Si2O5(OH)4
.
nH2O
[
10
,
11
]. They are empty cylinders with widths
of about 100 nanometers and consist of two structures: the anhydrous structure with an
interlayer dispersing of approximately 7Å and the hydrated structure with an augmented
interlayer dividing of 10 Å, due to the presence of water in the lamellar spaces [
12
–
14
]. In
each layer of the halloysite nanotubes (HNTs), the siloxane (SiOH) groups are found on
the outer surface, while the aluminol (AlOH) groups are situated on the inner surfaces,
making the outer and inner surfaces have different charges. The positive charge of the
internal lumen is a consequence of protonation of the AlOH group at low pH, whereas
the SiOH groups has overall negative charge due to the coordination of the atoms. These
unique properties of HNTs have made it possible for it to be used in various biomedical
applications, such as the development of biohybrid materials for health applications [
15
].
The charge disparity has drawn interest from the research community, whereby overall
negatively charged proteins taken above their isoelectric points were mostly loaded into the
positively charged nanotube lumen [
16
]. Therefore, in a pool of organic compounds, HNTs
can facilitate the formation of a transient bond between selected bioactive compounds and
the AlOH or SiOH as a function of pH conditions and can be very effective as a nano drug
carrier for different applications [
17
–
22
]. The loading efficiency is influenced by the charge
characteristics of the active agents, as well as vacuum pressure [23].
The species Dioclea Reflexa (DR) hook belong to leguminoase plants, which include
legume, pea, and the bean families. There are certain classes of compounds in Dioclea
reflexa (DR) that have clinical usefulness in both temperate and tropical regions [
24
–
26
].
Extract of DR seed has been shown to boost hematological parameters and antioxidant
activities which protect the kidney and blood from oxidative and related injuries under
acute and chronic toxicological challenges [
1
,
2
,
24
,
25
,
27
–
31
]. In addition, the aqueous
extract of the seeds produces 100% mortality in third stage mosquito larvae of Aedes
aegypti. The seed is a potential food source which contains around 14% protein, 8% fats,
and 58% carbohydrates [
26
]. Though these metabolites in the pool continue to show
promise in disease treatment, there is very limited data in the literature of the properties
of single isolates and their medicinal relevance, albeit due to the difficulties in pursuing
systematic separation of the complex mixtures in a single separation method. Thus, the
current work describes the use of a simplified method to systematically isolate bioactive
compounds from extracted complex mixtures from DR and test their inhibitory effects
on breast Michigan Cancer Foundation-7 (MCF-7) cells. The rationale is that the larger
surface area coupled with the differential polarity of the lumen and the surface of the HNTs
will be sufficient to bind selectively with the plant metabolites in the crude extracts of
DR. The authors hypothesized that: (1) the pH dependent elution of the plant metabolites
can identify therapeutic bioactive compounds against cancer cells and that (2) specific
HNT could isolate structurally and functionally related metabolites from complex mixtures
in a single step. The evidence of the entrapped species onto the HNTs was monitored
using X-ray diffractometry (XRD) and Fourier transform infrared spectroscopy (FTIR) to
determine the degree of aluminol (AlOH) and the siloxane (SiOH) groups modification
since these two functional groups will be key sites for bioactive compounds interaction.
pH-dependent eluted samples have been tested on breast (MCF-7) cancer cell lines to
investigate both their inhibitory and the mechanism using cyclic voltammetry and flow
cytometry analyses [
32
–
35
]. The results are reported here and show evidence of differential
inhibitory effects of the bioactive compounds from the various pH conditions.
Separations 2021,8, 26 3 of 14
2. Materials and Methods
2.1. Materials
N,N-dimethyl sulfonamide, sodium hydroxide (NaOH, >99%), acetic acid (CH
3
COOH,
>99%), sulfuric acid (H
2
SO
4
, >99%), hydrochloric acid (HCl), and propidium iodide were
purchased from Fisher Scientific, Altrincham, UK. Samples of natural halloysite (cat. no.
685 445) were purchased from Sigma Aldrich, St. Louis, USA. All chemicals were analytical
grade and were, therefore, used without further purification. Breast (MCF-7) cancer cells
(HTB-22) were purchased from American Type Culture Collection (ATCC) (Manassas, VA,
USA) and maintained in Dulbecco’s modified eagle medium (DMEM-F12) complete media
supplemented with 10% fetal bovine serum (FBS), minimum essential medium (MEM)
nonessential amino acids, gentamicin, and 10
µ
g/mL insulin in a 5% CO
2
incubator at
37
C. All culturing media were obtained from ATCC (Manassas, VA). RNase A from Sigma
Aldrich, St. Louis, MO, USA.
2.2. Methods
2.2.1. Loading and pH-Dependent Release of DR Metabolites
A 5 g quantity of Dioclea Reflexa (DR) seed powder was suspended in 30 mL of 70%
ethanol (pH 7.4) for 24 h to extract the plant metabolites [
6
]. A volume of 30 mL of the
supernatant was used for the immobilization using 120 to 1320 milligram quantities in
intervals of 120 mg/mL of halloysite nanotubes(HNTs). A UV–Vis spectrophotometer
(Shimadzu UV/Vis 1601 spectrophotometer, Shimadzu Corporation, Tokyo, Japan) was
used to determine the concentration of the crude extracts before and after loading onto the
halloysites nanotubes. A standard curve was then constructed to determine the amount of
entrapped bioactive compounds from the DR seed extracts.
The percentage loading capacity, LC was obtained from Equation (1):
LC =Mm/Mh⇥100 (1)
where
Mm
and
Mh
are the masses of the entrapped metabolites and the amount of hal-
loysites used for the entrapment, respectively.
The loaded HNTs were weighed and stored at 20
C, and the entrapped metabolites
were released using a buffer of pH (4.1–9.6) for 24 h.
2.2.2. Characterization of HNTs and DR Loaded HNTs
FTIR spectra of empty halloysites nanotubes (HNTs) and Dioclea Reflexa (DR) loaded
HNTs were recorded with a Nicolet MAGNA-IR 750 Spectrometer (Nicolet Instrument Co.,
Madison, WI, USA). The spectra were recorded from 500 to 4000 cm
1
wavenumber with
16 scans and spectral resolution of 4 cm1.
XRD (Empyrean, Malvern Panalytical B.V, Almelo, The Netherlands) of the HNTs and
loaded HNTs were performed using a Pan Analytical diffractometer with CuK
↵
radiation.
A2
✓
scan was performed from 5 to 35
in steps of 0.05
, with a tube voltage of 45 kV and a
current of 40 mA.
The Thermogravimetric Analyzer (TGA) (Q600 SDT, TA Instruments, Brussels, Bel-
gium) analysis of the HNT and HNT loaded with the DR extract was conducted using
Pyris 1 TGA equipment. The analysis was conducted in N
2
atmosphere at a heating rate of
20 C/min.
2.2.3. Culturing and Cyclic Voltammetry Analysis of MCF-7 Breast Cancer Cell Lines
MCF-7 cells (HTB-22) were grown and maintained in DMEM-F12 supplemented
media. Media was changed every 2–3 days, and cells were passaged at 65–80% confluence.
The cells were harvested after complete rinsing with 0.25% (w/v) Trypsin and 0.53 mM
Ethylenediamine tetra acetic acid (EDTA) solution to remove all traces of fetal bovine
serum, which contains trypsin inhibitor. A volume of 2.0 to 3.0 mL of Trypsin-EDTA
solution was added to the flask, and the cells were observed under an inverted microscope.
Separations 2021,8, 26 4 of 14
A volume of 6.0 to 8.0 mL of complete growth medium was used to aspirate the cells, and
the suspension was centrifuged at 125 mg for 5 to 10 min. After re-suspension, cell density
of 5.6
⇥
10
6
was obtained. The inhibitory effects (expressed as Percentage Activity, (PA)) of
the metabolites concentration of 2 mg/mL on the cells was determined using Equation (2).
Mo
is the initial concentration of the extracts,
Mph
is the concentration of the extracts at
particular pH.
PA =MoMph
Mo
⇥100 (2)
A stock solution of 2 mg/mL of the metabolites from pH ~5.2 was prepared for elec-
trochemical detection studies using cyclic voltammetry under steady-state conditions. The
electrochemical detection was carried out using A CheapStat potentiostat device (IO Rodeo,
Pasadena, CA, USA) connected to interdigitated gold electrodes (IDEs)/Microelectrodes
(Metrohm, DropSens Llanera, Asturias, Spain). A volume of 5
µ
L of cells of cell density
of 2.3
⇥
10
6
cells/well was suspended in 0.1 mM Phosphate Buffer Saline (PBS), and the
metabolite was also dispensed in 0.002 mM dimethyl sulfoxide (DMSO). The samples
were deposited on the active electrode for cyclic voltammetry measurements. The voltam-
mograms were obtained using a potential range from 690 to 970 mV at a scan rate of
10 mVs
1
. Cell viability studies was conducted using trypan blue assay and confirmed
by MTT assay. The MTT assay protocol is based on the conversion of water soluble MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) compound to an insoluble
formazan product by the viable cells. [36–40].
3. Results
3.1. Characterization of HNT and loaded HNT using XRD, TGA, and FTIR
In an effort to determine the presence of trapped compounds in the HNT, preparations
were subjected to analysis using XRD, TGA, and FTIR techniques. In Figure 1a (red), the
characteristic 2
peak positions of HNT occur at 11.7, 20.5, 24.8, 37.5, and 62.2, representing
(001), (110), (011), (131), and (331) crystallographic planes, respectively. After immobi-
lization of the DR extracts on the nanotubes, there was dramatic reduction of the peak
intensities at the same 2
positions, indicating that the resulting structure of the composite
material (HNT with DR) is more amorphous than that of HNT. This is expected since
the metabolites from the DR are mostly amorphous in nature. The absence of additional
peaks aside the characteristic peaks of HNTs after loading with DR depicts the amount
of DR loaded was less than 5% by weight of the HNTs. Since the metabolite may be
amorphous, it is likely that the peaks of the HNTs overshadowed the amorphous peaks of
the extract, and, as a result, no clearly defined XRD peaks were found. The bioactive con-
stituents were eluted with 70% ethanol and resulted in HNTs signature peaks, as shown in
Figure 1a (blue). The characteristic peak intensities reverted to those observed in the
control, implying that the reduced intensities were due to the plant extract.
The changes in the signatures peak intensities of the halloysite nanotubes (HNTs) was
monitored using FTIR spectroscopy, as shown in Figure 1b. The FTIR spectra revealed all
the functional groups present in the empty HNTs (black). Six major peaks were identified
in the HNT. The inner Al-OH and outer Si-OH groups have characteristic stretching peaks
at 3622 and 3694 cm
1
, respectively. Bending vibrations of Al-OH and Si-OH revealed
absorption peaks at 902 cm
1
. In addition, the uneven stretching vibrations of the Si-OH
bond gave a strong absorption peak at 995 and 1118 cm
1
. In addition, the deformation
vibration of the interlayer water molecules of the HNT was observed at 1647 cm
1
. There
was a significant increase of the transmission peak intensities after immobilization of the
DR extracts on the HNTs (blue). Four distinct additional peaks were observed in the FTIR
spectra of the halloysite nanotubes loaded with the DR extract, as can be seen in Figure 1b.
These peaks were also present in the FTIR spectra of the DR extract. The peak observed at
1744 cm
1
is due to the C = O stretching, while the peak observed at 1460 cm
1
represents
the symmetric C–H vibration. The symmetric and asymmetric vibrations of CH
2
were
represented by the peaks observed at 2923 and 2854 cm
1
[
41
]. Since the peaks present
Separations 2021,8, 26 5 of 14
in the HNT and DR extract were observed in the HNT loaded with DR extract sample, it
depicts that the HNT was indeed loaded with the DR extract.
To further confirm that the DR extract was loaded into the HNT, TGA analysis of the
raw HNTs and HNT loaded with the DR extract was conducted. This analysis also helped in
estimating the amount of DR extract loaded into the HNTs. The TGA results is presented in
Figure 1c. Two distinct decompositions were observed at approximately 60
C and 450
C.
The weight losses observed at 60
C and 450
C can be attributed to the decomposition
of the water molecules absorbed onto the HNT surface and the dehydroxylation of the
HNTs, respectively. Considering the HNT loaded with the DR extract, a significant mass
loss was observed between 25–85
C. This mass loss can be attributed to the decomposition
of the 70% ethanol solution used in the loading process. The HNT loaded with the
DR extract contained some moisture when the TGA analysis was being conducted. In
addition to this observed decomposition, another gradual decomposition was observed
from approximately 240–370
C. This gradual decomposition can be attributed to the
extract. This implies that the HNT loaded with the DR extract indeed contained the DR
extract. The amount of the loaded DR extract was estimated to be approximately 5.18 wt.%.
Separations 2021, 8, x FOR PEER REVIEW 5 of 14
Si-OH bond gave a strong absorption peak at 995 and 1118 cm−1. In addition, the defor-
mation vibration of the interlayer water molecules of the HNT was observed at 1647 cm−1.
There was a significant increase of the transmission peak intensities after immobilization
of the DR extracts on the HNTs (blue). Four distinct additional peaks were observed in
the FTIR spectra of the halloysite nanotubes loaded with the DR extract, as can be seen in
Figure 1b. These peaks were also present in the FTIR spectra of the DR extract. The peak
observed at 1744 cm−1 is due to the C = O stretching, while the peak observed at 1460 cm−1
represents the symmetric C–H vibration. The symmetric and asymmetric vibrations of
CH2 were represented by the peaks observed at 2923 and 2854 cm−1 [41]. Since the peaks
present in the HNT and DR extract were observed in the HNT loaded with DR extract
sample, it depicts that the HNT was indeed loaded with the DR extract.
To further confirm that the DR extract was loaded into the HNT, TGA analysis of the
raw HNTs and HNT loaded with the DR extract was conducted. This analysis also helped
in estimating the amount of DR extract loaded into the HNTs. The TGA results is pre-
sented in Figure 1c. Two distinct decompositions were observed at approximately 60 °C
and 450 °C. The weight losses observed at 60 °C and 450 °C can be attributed to the de-
composition of the water molecules absorbed onto the HNT surface and the dehydroxyla-
tion of the HNTs, respectively. Considering the HNT loaded with the DR extract, a signif-
icant mass loss was observed between 25–85 °C. This mass loss can be attributed to the
decomposition of the 70% ethanol solution used in the loading process. The HNT loaded
with the DR extract contained some moisture when the TGA analysis was being con-
ducted. In addition to this observed decomposition, another gradual decomposition was
observed from approximately 240–370 °C. This gradual decomposition can be attributed
to the extract. This implies that the HNT loaded with the DR extract indeed contained the
DR extract. The amount of the loaded DR extract was estimated to be approximately 5.18
wt.%.
(a)
10 20 30 40 50 60 70 80
0
50
100
150
200
250
300
Intensity
2θ / degree
HNT loaded DR
HNT
Eluted HNT
11.7
20.5
24.8
37.5 62.2
Figure 1. Cont.
Separations 2021,8, 26 6 of 14
Separations 2021, 8, x FOR PEER REVIEW 6 of 14
(b)
(c)
Figure 1. (a) X-ray diffractometry (XRD) spectra of halloysites nanotubes (HNT) and HNT loaded
with the Dioclea Reflexa (DR) extract. (b) Fourier transform infrared (FTIR) spectra of HNTs and
loaded HNTs with plant metabolites. (c) TGA of HNT and HNT loaded with DR extract.
050010001500200025003000350040004500
1460 1744
2854
2923
902
995
1118
1647 3622
3694
Transmittance (%)
Wavenumber (cm
-1
)
HNT
DR Extract
HNT loaded with DR Extract
100 200 300 400 500 600 700 800
40
60
80
100
Weight %
Temperature (Degree C)
HNT
HNT with DR Extract
DR Extract
Figure 1.
(
a
) X-ray diffractometry (XRD) spectra of halloysites nanotubes (HNT) and HNT loaded
with the Dioclea Reflexa (DR) extract. (
b
) Fourier transform infrared (FTIR) spectra of HNTs and
loaded HNTs with plant metabolites. (c) TGA of HNT and HNT loaded with DR extract.
3.1.1. Optimization of Parameters to Increase Metabolites Immobilization onto HNTs
Concentration dependent analysis of the bioactive compounds entrapped on the HNT
using varied concentrations of the nanotubes is displayed in Figure 2a. It was observed that
the maximum extractable bioactive compounds at the loading range from 720 to 1080 mg
Separations 2021,8, 26 7 of 14
of HNTs using DR concentration of 0.2 g/mL in a total volume of 30 mL of crude extract
solution was about 14–19%.
Separations 2021, 8, x FOR PEER REVIEW 7 of 14
3.1.1. Optimization of Parameters to Increase Metabolites Immobilization onto HNTs
Concentration dependent analysis of the bioactive compounds entrapped on the
HNT using varied concentrations of the nanotubes is displayed in Figure 2a. It was ob-
served that the maximum extractable bioactive compounds at the loading range from 720
to 1080 mg of HNTs using DR concentration of 0.2 g/mL in a total volume of 30 mL of
crude extract solution was about 14–19%.
Figure 2 showed the inhibitory effects of 2 mg/mL of the entrapped metabolites on
breast (MCF-7) cancer cell lines at cell density 6.3 × 106. It was observed that the crude
extracts from the HNTs lumen did not show significant increase in percent antiprolifera-
tive effects on the MCF-7 cells, as indicative by the statistically insignificant variations in
cell viability.
Figure 2. Optimization of the conditions for DR entrapment using HNTs. (a) The relative amount
in percentage of entrapped bioactive compounds (mg/mL) using 5 grams bed volume of HNTs.
The release was conducted for 2 days to ensure all the bound molecules were completely released
from the lumen of the HNT. (b)MTT assay for MCF-7 cell viability analysis of the entrapped bioac-
tive compounds from the various crude extracts using 2 mg/mL. (c) The effects of buffer pH on the
release of the immobilized bioactive compounds. (d) The relative activity of pH dependent bioac-
tive compounds on MCF-7 cell viability using 2 mg/mL concentrations.
3.1.2. PH-Dependent Elution of the Metabolites from HNTs
The pH-dependent release of the bioactive compounds is displayed in Figure 2. The
results revealed pH effect on the release of the bioactive compounds from HNTs was the
same based on the profile exhibited in Figure 2. To investigate the inhibitory effects of the
pH eluted samples, a 2 mg/mL was prepared from all the samples and tested on the cancer
cells, as shown in Figure 2d. All the samples tested demonstrated some level of percent
inhibition with the extract from pH(~5.2 and ~9.6), revealing the highest and the lowest
degree of percent antiproliferative effects of 74 and 36, respectively. The IC in Table 1
of the water and ethanol extracts, as well as those of the pH dependent eluted samples,
10
20
30
40
50
60
35
40
45
50
55
Loading capacity (%)
120 240 360 480 600 720 840 960 1080 1200 1320
0
5
10
15
20
25
30
35
40
Antiproliferate effects (%)
Concentration
(
mg/mL
)
Entrapment efficiency (%)
(c)
(d)
(b)
(a)
4.1 5.2 6.4 7.5 8.6 9.7
0
10
20
30
40
50
60
70
Antiproliferate effects (%)
p
H
Figure 2.
Optimization of the conditions for DR entrapment using HNTs. (
a
) The relative amount in percentage of entrapped
bioactive compounds (mg/mL) using 5 grams bed volume of HNTs. The release was conducted for 2 days to ensure all the
bound molecules were completely released from the lumen of the HNT. (
b
)MTT assay for MCF-7 cell viability analysis of
the entrapped bioactive compounds from the various crude extracts using 2 mg/mL. (
c
) The effects of buffer pH on the
release of the immobilized bioactive compounds. (
d
) The relative activity of pH dependent bioactive compounds on MCF-7
cell viability using 2 mg/mL concentrations.
Figure 2showed the inhibitory effects of 2 mg/mL of the entrapped metabolites on
breast (MCF-7) cancer cell lines at cell density 6.3
⇥
106. It was observed that the crude
extracts from the HNTs lumen did not show significant increase in percent antiproliferative
effects on the MCF-7 cells, as indicative by the statistically insignificant variations in
cell viability.
3.1.2. PH-Dependent Elution of the Metabolites from HNTs
The pH-dependent release of the bioactive compounds is displayed in Figure 2. The
results revealed pH effect on the release of the bioactive compounds from HNTs was the
same based on the profile exhibited in Figure 2. To investigate the inhibitory effects of the
pH eluted samples, a 2 mg/mL was prepared from all the samples and tested on the cancer
cells, as shown in Figure 2d. All the samples tested demonstrated some level of percent
inhibition with the extract from pH(~5.2 and ~9.6), revealing the highest and the lowest
degree of percent antiproliferative effects of 74 and 36, respectively. The
IC50
in Table 1of
the water and ethanol extracts, as well as those of the pH dependent eluted samples, were
determined to confirm the trend of inhibition demonstrated in Figure 2. The inhibitory
effects of the bioactive compounds eluted at acidic pH had much lower IC
50
of 0.8–1.6,
with pH~5.2 revealing the lowest
IC50
, which indicated the metabolites from that fraction
are more potent to the cells, as shown in Table 1.
Separations 2021,8, 26 8 of 14
Table 1.
IC
50
values of the extracts eluted at different pH conditions, all measured in milligram, and
quantities of the seed extract.
Treatment Normalized
Sample/Dry Extract IC50 (mg) R Squared Value
Water 33.3 0.959
Ethanol 1.6 0.991
pH 4.1 1.4 0.967
pH 5.2 0.8 0.998
pH 6.4 1.6 0.975
pH7.4 1.9 0.983
pH 8.1 2.3 0.948
pH 9.6 3.1 0.994
3.1.3. CV Response of the MC-7 Cells in the Presence of the Metabolites
The influence of the metabolites from pH~ 5.2 on breast (MCF–7) cancer cells viability
was monitored with cyclic voltammetry (CV). Figure 3showed the oxidation peak potential
and current fluctuations as the response variables when the cells were treated with the
metabolites at different pH. In Figure 3A, the empty electrode did not exhibit electrochemi-
cal response, as indicated by a horizontal line, whereas the treated cells samples showed
a quasi-reversible CV profile. The voltage–pH at the same concentration of metabolites
described an inverse correlation between metabolite concentration and oxidation peak
potential, revealing a Pearson correlation coefficient (R2) 98.64, as in Figure 3B. However,
the peak current correlation exhibited a triangular wave pattern as a function of metabolite
concentration, as shown in Figure 3C.
Separations 2021, 8, x FOR PEER REVIEW 8 of 14
were determined to confirm the trend of inhibition demonstrated in Figure 2. The inhibi-
tory effects of the bioactive compounds eluted at acidic pH had much lower IC50 of 0.8–
1.6, with pH~5.2 revealing the lowest IC, which indicated the metabolites from that
fraction are more potent to the cells, as shown in Table 1.
Table 1. IC50 values of the extracts eluted at different pH conditions, all measured in milligram,
and quantities of the seed extract.
Treatment Normalized
Sample/Dry Extract IC50 (mg) R squared Value
Water
Ethanol
pH 4.1
pH 5.2
pH 6.4
pH7.4
pH 8.1
pH 9.6
33.3
1.6
1.4
0.8
1.6
1.9
2.3
3.1
0.959
0.991
0.967
0.998
0.975
0.983
0.948
0.994
3.1.3. CV Response of the MC-7 Cells in the Presence of the Metabolites
The influence of the metabolites from pH~ 5.2 on breast (MCF–7) cancer cells viability
was monitored with cyclic voltammetry (CV). Figure 3 showed the oxidation peak poten-
tial and current fluctuations as the response variables when the cells were treated with the
metabolites at different pH. In Figure 3A, the empty electrode did not exhibit electrochem-
ical response, as indicated by a horizontal line, whereas the treated cells samples showed
a quasi-reversible CV profile. The voltage–pH at the same concentration of metabolites
described an inverse correlation between metabolite concentration and oxidation peak
potential, revealing a Pearson correlation coefficient (R2) 98.64, as in Figure 3B. However,
the peak current correlation exhibited a triangular wave pattern as a function of metabo-
lite concentration, as shown in Figure 3C.
-600 -400 -200 0 200 400 600 800 1000
-400
-200
0
200
400
I/µA
V/mV
MCF-7 cells with plant extracts
Empty electrodes
(A)
Figure 3. Cont.
Separations 2021,8, 26 9 of 14
Separations 2021, 8, x FOR PEER REVIEW 9 of 14
Figure 3. (A) Effects of the bioactive compounds from the pH ~5.2 on the depolarization potential
of the MCF-7 cells. (B,C) The influence of the voltage on current of the MCF-7 cells as a function of
pH. The cyclic voltammogram measurements conditions were: Scanning from 690 mV to 970 mV
at a scan rate of 10 mV s−1. MCF-7 cancer cell viability studies of the bioactive compounds ex-
tracted at pH 5.2 at cell concentration of 1 × 106 cells/well.
3.1.4. Flow Cytometry Analysis of the Active Metabolites on Cell Behavior
Flow cytometry analysis (BD LSRFortessa X-20, BD Biosciences, Le Pont de Claix,
France) was conducted to investigate the mechanism of inhibition of the breast (MC-7)
cancer cells by the metabolites obtained from pH~5.2 and the results compared with a
commercially available drug (Curcumin), as shown in Figure 4. The quadrants referred to
the cell condition, as displayed in the flow cytometry results, after a 48 h incubation time
frame. The symbols Q1, Q2, Q3, and Q4 on the graph were used to represent debris, dead
cells, live cells, and apoptotic cells, respectively. The conditions of the cells before and
after treatment with the drug, as well as the metabolites, and the results compared with
the control indicated that the curcumin showed dramatic cell death at concentration of
0.02 mg/mL without going through significant apoptosis. Conversely, the metabolites
showed some level of cell death but with significant apoptotic effects.
-0.2
-0.1
0.0
0.1
45678910
80
85
90
95
100
E/µV
(B)
(C)
I/µA
pH
Figure 3.
(
A
) Effects of the bioactive compounds from the pH ~5.2 on the depolarization potential of
the MCF-7 cells. (
B
,
C
) The influence of the voltage on current of the MCF-7 cells as a function of pH.
The cyclic voltammogram measurements conditions were: Scanning from 690 mV to 970 mV at a
scan rate of 10 mV s
1
. MCF-7 cancer cell viability studies of the bioactive compounds extracted at
pH 5.2 at cell concentration of 1 ⇥106cells/well.
3.1.4. Flow Cytometry Analysis of the Active Metabolites on Cell Behavior
Flow cytometry analysis (BD LSRFortessa X-20, BD Biosciences, Le Pont de Claix,
France) was conducted to investigate the mechanism of inhibition of the breast (MC-7)
cancer cells by the metabolites obtained from pH~5.2 and the results compared with a
commercially available drug (Curcumin), as shown in Figure 4. The quadrants referred
to the cell condition, as displayed in the flow cytometry results, after a 48 h incubation
time frame. The symbols Q1, Q2, Q3, and Q4 on the graph were used to represent debris,
dead cells, live cells, and apoptotic cells, respectively. The conditions of the cells before
and after treatment with the drug, as well as the metabolites, and the results compared
with the control indicated that the curcumin showed dramatic cell death at concentration
of 0.02 mg/mL without going through significant apoptosis. Conversely, the metabolites
showed some level of cell death but with significant apoptotic effects.
Separations 2021,8, 26 10 of 14
Separations 2021, 8, x FOR PEER REVIEW 10 of 14
Figure 4. Fluorescence activated cell sorting (FACS) analysis of the inhibitory effects of breast
(MCF-7) cancer cells using bioactive extracts at pH 5.2 (best IC50 concentration). The results were
compared to the inhibitory effects of a commercially-available cancer drug, cucurmin. (a) Un-
treated cells, (b) cells treated with extract, and (c) cells treated with curcumin, all at cell concentra-
tion of 1 × 10
6
cells/well.
4. Discussion
The current work seeks to propose aluminosilicate minerals, HNTs, to be used to
entrap plant metabolites for biomedical applications. The biomimetic material has been
used for various health applications due to its unique natural design consisting of empty
cylinders with widths of about 100 nanometers. The XRD, FTIR, and TGA results clearly
imply that the HNTs was loaded with the extracted crude DR. The reduction of XRD peak
intensities imply the crystallinity of the loaded HNT is reduced due to the immobilization
of bioactive compounds on the HNT and this observation is in conformity to literature
which indicates that crystallinity of HNT decreases when bioactive compounds complex
with the material [42]. The DR extract is expected to be amorphous, hence, the observed
reduction in the peak intensities of the HNT loaded with the DR extract. The FTIR spectra
transmission fingerprints of the HNT and HNT loaded with DR extract confirmed that
the HNTs was loaded with the HNTS. There was no observed shift in the peaks of the
HNT after loading. This implies that the functional groups present in the DR extract may
not have interacted chemically with the functional groups present in HNTs. If there was
any chemical interaction, then it is likely the interaction was not pronounced enough to
cause any significant/observable shift in the peaks. The reversal of the peak intensities
back to that of the empty tubules indicates that the bioactive compounds entrapped in the
lumen of the HNT are transient and reversible. Thus, the plant metabolites can easily be
released using buffer with different pH values, as was reflected in the FTIR and XRD spec-
(a) (b)
(c)
Figure 4.
Fluorescence activated cell sorting (FACS) analysis of the inhibitory effects of breast (MCF-7) cancer cells using
bioactive extracts at pH 5.2 (best IC50 concentration). The results were compared to the inhibitory effects of a commercially-
available cancer drug, cucurmin. (
a
) Untreated cells, (
b
) cells treated with extract, and (
c
) cells treated with curcumin, all at
cell concentration of 1 ⇥106cells/well.
4. Discussion
The current work seeks to propose aluminosilicate minerals, HNTs, to be used to
entrap plant metabolites for biomedical applications. The biomimetic material has been
used for various health applications due to its unique natural design consisting of empty
cylinders with widths of about 100 nanometers. The XRD, FTIR, and TGA results clearly
imply that the HNTs was loaded with the extracted crude DR. The reduction of XRD peak
intensities imply the crystallinity of the loaded HNT is reduced due to the immobilization
of bioactive compounds on the HNT and this observation is in conformity to literature
which indicates that crystallinity of HNT decreases when bioactive compounds complex
with the material [
42
]. The DR extract is expected to be amorphous, hence, the observed
reduction in the peak intensities of the HNT loaded with the DR extract. The FTIR spectra
transmission fingerprints of the HNT and HNT loaded with DR extract confirmed that the
HNTs was loaded with the HNTS. There was no observed shift in the peaks of the HNT
after loading. This implies that the functional groups present in the DR extract may not
have interacted chemically with the functional groups present in HNTs. If there was any
chemical interaction, then it is likely the interaction was not pronounced enough to cause
any significant/observable shift in the peaks. The reversal of the peak intensities back to
that of the empty tubules indicates that the bioactive compounds entrapped in the lumen
of the HNT are transient and reversible. Thus, the plant metabolites can easily be released
using buffer with different pH values, as was reflected in the FTIR and XRD spectra. The
TGA results further confirmed that the HNT was indeed loaded with the DR extract. The
decomposition of the DR extract in the HNT loaded DR was observed between 240–370
C,
Separations 2021,8, 26 11 of 14
and the amount of the DR extract loaded into the HNT was estimated to be approximately
5.1 wt.%.
The captured bioactive compounds fail to show significant bioactivity when the
crude extracts from the optimization parameters are tested on model cancer cell lines.
Though literature reveals DR contains flavonoids, phenolics compounds, alkaloids, and
antioxidants, as confirmed by UV studies and other analytical characterizations, our
studies show the cells are not compromised in their cell viability in the presence of the
metabolites [
6
,
26
,
43
]. However, further studies are required to carry out careful analysis
on the extracts to confirm the presence of these metabolites. Nonetheless, the rationale
of the current study is to develop a simple procedure to capture metabolites mixtures for
further characterization.
We used pH-dependent elution of the bioactive compounds from the HNTs to further
validate the activity of the captured metabolites on cell death. HNTs have SiOH and AlOH
groups, which are found on the outer surface and the inner surface, making the outer and
inner surfaces have different charges, respectively. The charge disparity has drawn interest
from the research community, whereby overall negatively charged proteins taken above
their isoelectric points are mostly loaded into the positively charged tube’s lumen [
16
].
Thus, depending on the pH conditions, aluminol (AlOH) and the siloxane (SiOH) groups
can either be protonated or deprotonated, leading to different affinities towards certain
macromolecules and organic compounds. Our hypothesis here was that partially positive
metabolites will be weakly attracted to the SiOH groups, whereas negatively charged
metabolites will prefer the latter. The pH dependent release of the metabolites from the
HNTs are not statistically different after determining the amount in milligram quantities
and expressing the entrapment efficiency as a percentage value. However, when tested
against the breast (MCF-7) cancer cell lines, the acidic pH elution demonstrates significant
anti-proliferative activity against the cancer cell lines compared to the basic pH metabo-
lites. The most profound activity is found in the pH~5.2, which is supported by IC50
calculated values.
Polarization and depolarization are attributes associated with mitochondrial dysfunc-
tion in most cancer cells and can be used to inform the mechanism of cell death [
44
]. In
this work, cyclic voltammetry measurements are used to probe the extent of polarization
and depolarization by relating the voltage to current surge using electrochemical detection
methods. The results reveal that the metabolites exhibit quasi-reversible redox behavior
and concentration dependent reduction in the applied voltage [
45
]. The currents also
show a triangular modulation with a rise in oxidation current at lower pH, follow by
another rise beyond acidic pH and further reduction in the strongly basic pH conditions.
Metabolites from the pH~5.2 extract require a higher voltage application to generate the
minimum amount of current in the cells, indicating that cell membrane polarization in
the presence of the metabolite is achieved. The extracts from pH~7.4 and pH~8.1, even
though they give higher IC50 values, the voltage required to initiate cell depolarization is
at a minimum. Nonetheless, the highlights of the current studies are that the metabolites
can cause cell death through a polarization/depolarization mechanism, as documented by
other researchers in the literature [46].
Flow cytometer-based analysis shows that the metabolites exhibit dose-dependent
apoptosis of MCF-7 cells. It is noted that exposure of 2 mg/mL concentrations of the
metabolites leads to a greater than two-fold increase in apoptosis in comparison to the
untreated cells. Curcumin is a well-known polyphenol obtained from Curcuma longa,
and it is widely used for its anti-oxidative and anti-cancerous application. Curcumin
effects on the breast cancer cells are also investigated and compared to the results from the
metabolites. It is observed that curcumin improves cell death significantly, without going
through the apoptotic phase, indicating the synergistic effect could be developed when
both metabolites and curcumin are used to treat cancer.
Separations 2021,8, 26 12 of 14
5. Conclusions
In this work, it is demonstrated that optimization of parameters for Dioclea Re-
flexa (DR) extracts immobilization on HNT and subsequent releasing the cargo based
on pH could find important lead metabolites for discovering druggable entities without
going through complex analytical techniques. Such simplified methods will need addi-
tional modified analytical tools to expediate the drug discovery pipeline. The work also
intend to provide plant metabolites database with fundamental information on herbal
medicine isolation and characterization to serve the scientific community in future studies
of herbal medicine.
Author Contributions:
Conceptualization, E.K.T. and S.B.; methodology, B.O.A. and E.N.; software,
P.K.A., R.A., and S.B.; validation, E.K.T., and P.K.A.; formal analysis, E.K.T.; investigation, E.K.T.;
resources, P.K.A.; funding acquisition, E.K.T. and P.K.A. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by Wellcome Trust, grant number 107755/Z/15/Z.
Acknowledgments:
We would like to thank Lily Paemka (Cancer Biology Laboratory) at the West
African Center for Cell Biology of Infectious Pathogens (WACCBIP) for support in providing the
facilities and technical assistants for the work. We also thank Solomon Katu and Shadrack O. Aboagye
for assisting the team conducting the experiments in the Department of Biomedical engineering.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Saqib, Z.; Mahmood, A.; Malik, R.N.; Syed, J.H.; Ahmad, T. Indigenous knowledge of medicinal plants in Kotli Sattian, Rawalpindi
district, Pakistan. J. Ethnopharmacol. 2014,151, 820–828. [CrossRef]
2.
Pal, D.; Mandal, M.; Senthilkumar, G.; Padhiari, A. Antibacterial activity of Cuscuta reflexa stem and Corchorus olitorius seed.
Fitoterapia 2006,77, 589–591. [CrossRef]
3.
Bhandari, P.; Sendri, N.; Devidas, S.B. Dammarane triterpenoid glycosides in Bacopa monnieri: A review on chemical diversity
and bioactivity. Phytochemistry 2020,172, 112276. [CrossRef]
4.
Ge, Y.-W.; Wang, S.-M.; Zhang, L.-Y. Label-free small molecule probe and target discovery of traditional Chinese medicine.
Zhongguo Zhong Yao Za Zhi 2019,44, 4152–4157. [PubMed]
5.
Benattia, F.K.; Arrar, Z.; Dergal, F.; Khabbal, Y. Pharmaco-Analytical Study and Phytochemical Profile of Hydroethanolic Extract
of Algerian Prickly Pear (Opuntia ficus-indica.L). Curr. Pharm. Biotechnol. 2019,20, 696–706. [CrossRef]
6.
Arthur, P.K.; Yeboah, A.B.; Issah, I.; Balapangu, S.; Kwofie, S.K.; Asimeng, B.O.; Foster, E.J.; Tiburu, E.K. Electrochemical Response
of Saccharomyces cerevisiae Corresponds to Cell Viability upon Exposure to Dioclea reflexa Seed Extracts and Antifungal Drugs.
Biosensor 2019,9, 45. [CrossRef][PubMed]
7.
Bittner, M.; Schenk, R.; Springer, A.; Melzig, M.F. Economical, Plain, and Rapid Authentication of Actaea racemosa L. (syn.
Cimicifuga racemosa, Black Cohosh) Herbal Raw Material by Resilient RP-PDA-HPLC and Chemometric Analysis. Phytochem.
Anal. 2016,27, 318–325. [CrossRef]
8.
Wang, H.; Zhao, X.; Wang, S.; Tao, S.; Haibo, W.; Wang, Y. Fabrication of enzyme-immobilized halloysite nanotubes for affinity
enrichment of lipase inhibitors from complex mixtures. J. Chromatogr. A 2015,1392, 20–27. [CrossRef][PubMed]
9.
Liu, H.; Wang, D.; Ji, L.; Li, J.; Liu, S.; Liu, X.; Jiang, S. A novel TiO2 nanotube array/Ti wire incorporated solid-phase
microextraction fiber with high strength, efficiency and selectivity. J. Chromatogr. A 2010,1217, 1898–1903. [CrossRef]
10.
Gianni, E.; Avgoustakis, K.; Papoulis, D. Kaolinite group minerals: Applications in cancer diagnosis and treatment. Eur. J. Pharm.
Biopharm. 2020,154, 359–376. [CrossRef]
11.
Saif, M.J.; Asif, H.M.; Naveed, M. PROPERTIES AND MODIFICATION METHODS OF HALLOYSITE NANOTUBES: A
STATE-OF-THE-ART REVIEW. J. Chil. Chem. Soc. 2018,63, 4109–4125. [CrossRef]
12.
Park, K.; Lee, J.; Chang, J.H.; Hwang, K.H.; Lee, Y. Characterization of Surface-Modified Halloysite Nanotubes by Thermal
Treatment Under Reducing Atmosphere. J. Nanosci. Nanotechnol. 2020,20, 4221–4226. [CrossRef]
13.
Tas, C.E.; Ozbulut, E.B.S.; Ceven, O.F.; Tas, B.A.; Unal, S.; Unal, H. Purification and Sorting of Halloysite Nanotubes into
Homogeneous, Agglomeration-Free Fractions by Polydopamine Functionalization. ACS Omega
2020
,5, 17962–17972. [CrossRef]
[PubMed]
14.
Jang, S.H.; Jang, S.R.; Lee, G.M.; Ryu, J.H.; Park, S.I.; Park, N.H. Halloysite Nanocapsules Containing Thyme Essential Oil:
Preparation, Characterization, and Application in Packaging Materials. J. Food Sci. 2017,82, 2113–2120. [CrossRef]
15.
Lisuzzo, L.; Wicklein, B.; Lo Dico, G.; Lazzara, G.; Del Real, G.; Aranda, P.; Ruiz-Hitzky, E. Functional biohybrid materials based
on halloysite, sepiolite and cellulose nanofibers for health applications. Dalton Trans. 2020,49, 3830–3840. [CrossRef]
Separations 2021,8, 26 13 of 14
16.
Tully, J.; Yendluri, R.; Lvov, Y. Halloysite Clay Nanotubes for Enzyme Immobilization. Biomacromolecules
2016
,17, 615–621.
[CrossRef]
17.
Barman, M.; Mahmood, S.; Augustine, R.; Hasan, A.; Thomas, S.; Ghosal, K. Natural halloysite nanotubes /chitosan based
bio-nanocomposite for delivering norfloxacin, an anti-microbial agent in sustained release manner. Int. J. Biol. Macromol.
2020
,
162, 1849–1861. [CrossRef][PubMed]
18.
Bonifacio, M.A.; Gentile, P.; Ferreira, A.M.; Cometa, S.; De Giglio, E. Insight into halloysite nanotubes-loaded gellan gum
hydrogels for soft tissue engineering applications. Carbohydr. Polym. 2017,163, 280–291. [CrossRef]
19.
Bottino, M.C.; Yassen, G.H.; Platt, J.A.; Labban, N.; Windsor, L.J.; Spolnik, K.J.; Bressiani, A.H.A. A novel three-dimensional
scaffold for regenerative endodontics: materials and biological characterizations. J. Tissue Eng. Regen. Med.
2013
,9, E116–E123.
[CrossRef]
20.
De Oliveira, T.; Guégan, R.; Thiebault, T.; Milbeau, C.L.; Muller, F.; Teixeira, V.; Giovanela, M.; Boussafir, M. Adsorption of
diclofenac onto organoclays: Effects of surfactant and environmental (pH and temperature) conditions. J. Hazard. Mater.
2017
,
323 Pt A, 558–566. [CrossRef]
21.
Li, W.; Liu, D.; Zhang, H.; Correia, A.; Mäkilä, E.; Salonen, J.; Hirvonen, J.; Santos, H.A. Microfluidic assembly of a nano-in-micro
dual drug delivery platform composed of halloysite nanotubes and a pH-responsive polymer for colon cancer therapy. Acta
Biomater. 2017,48, 238–246. [CrossRef]
22.
Gorrasi, G. Dispersion of halloysite loaded with natural antimicrobials into pectins: Characterization and controlled release
analysis. Carbohydr. Polym. 2015,127, 47–53. [CrossRef]
23.
Lisuzzo, L.; Cavallaro, G.; Pasbakhsh, P.; Milioto, S.; Lazzara, G. Why does vacuum drive to the loading of halloysite nanotubes?
The key role of water confinement. J. Colloid Interface Sci. 2019,547, 361–369. [CrossRef][PubMed]
24.
Pinto-Junior, V.R.; Osterne, V.J.S.; Santiago, M.Q.; Correia, J.L.A.; Pereira-Junior, F.N.; Leal, R.B.; Pereira, M.G.; Chicas, L.S.;
Nagano, C.S.; Rocha, B.A.M.; et al. Structural studies of a vasorelaxant lectin from Dioclea reflexa Hook seeds: Crystal structure,
molecular docking and dynamics. Int. J. Biol. Macromol. 2017,98, 12–23. [CrossRef]
25.
Pinto-Junior, V.R.; Correia, J.L.; Pereira, R.I.; Pereira-Junior, F.N.; Santiago, M.Q.; Osterne, V.J.; Madeira, J.C.; Cajazeiras, J.B.;
Nagano, C.S.; Delatorre, P.; et al. Purification and molecular characterization of a novel mannose-specific lectin from Dioclea
reflexa hook seeds with inflammatory activity. J. Mol. Recognit. 2016,29, 134–141. [CrossRef]
26.
Ajatta, M.A.; Akinola, S.A.; Otolowo, D.T.; Awolu, O.O.; Omoba, O.S.; Osundahunsi, O.F. Effect of Roasting on the Phytochemical
Properties of Three Varieties of Marble Vine (Dioclea reflexa) Using Response Surface Methodology. Prev. Nutr. Food Sci.
2019
,24,
468–477. [CrossRef][PubMed]
27.
Kazenel, M.R.; Debban, C.L.; Ranelli, L.; Hendricks, W.Q.; Chung, Y.A.; Pendergast, T.H.; Charlton, N.D.; Young, C.A.; Rudgers,
J.A. A mutualistic endophyte alters the niche dimensions of its host plant. AoB Plants 2015,7, plv005. [CrossRef][PubMed]
28.
Gupta, M.; Mazumder, U.K.; Pal, D.K.; Bhattacharya, S. Anti-steroidogenic activity of methanolic extract of Cuscuta reflexa roxb.
stem and Corchorus olitorius Linn. seed in mouse ovary. Indian J. Exp. Boil. 2003,41, 641–644.
29.
Mazumder, U.K.; Gupta, M.; Pal, D.; Bhattacharya, S. Chemical and toxicological evaluation of methanol extract of Cuscuta
reflexa Roxb. stem and Corchorus olitorius Linn. seed on hematological parameters and hepatorenal functions in mice. Acta Pol.
Pharm. Drug Res. 2004,60, 317–323.
30.
Gupta, M.; Mazumder, U.K.; Pal, D.; Bhattacharya, S.; Chakrabarty, S. Studies on brain biogenic amines in methanolic extract of
Cuscuta reflexa Roxb. and Corchorus olitorius Linn. seed treated mice. Acta Pol. Pharm. Drug Res. 2003,60, 207–210.
31.
Gupta, M.; Mazumder, U.; Pal, D.; Bhattacharya, S. Onset of puberty and ovarian steroidogenesis following adminstration of
methanolic extract of Cuscuta reflexa Roxb. stem and Corchorus olitorius Linn. seed in mice. J. Ethnopharmacol.
2003
,89, 55–59.
[CrossRef]
32.
Munge, B.S.; Stracensky, T.; Gamez, K.; DiBiase, D.; Rusling, J.F. Multiplex Immunosensor Arrays for Electrochemical Detection
of Cancer Biomarker Proteins. Electroanalysis 2016,28, 2644–2658. [CrossRef][PubMed]
33.
Nejadnik, M.R.; Deepak, F.L.; Garcia, C.D. Adsorption of Glucose Oxidase to 3-D Scaffolds of Carbon Nanotubes: Analytical
Applications. Electroanalysis 2011,23, 1462–1469. [CrossRef]
34.
Szigeti, Z.; Vigassy, T.; Bakker, E.; Pretsch, E. Approaches to Improving the Lower Detection Limit of Polymeric Membrane
Ion-Selective Electrodes. Electroanalysis 2006,18, 1254–1265. [CrossRef]
35.
de Oliveira, L.A.; Soares, R.O.; Buzzi, M.; Mourão, C.F.A.B.; Kawase, T.; Kuckelhaus, S.A.S. Cell and platelet composition assays
by flow cytometry: basis for new platelet-rich fibrin methodologies. J. Biol. Regul. Homeost. Agents
2020
,34, 1379–1390. [PubMed]
36.
Zhang, Y.; Zhang, X.; Zhang, J.; Sun, B.; Zhengfeng, Y.; Jinling, Z.; Liu, S.; Sui, G.; Yin, Z. Microfluidic chip for isolation of
viable circulating tumor cells of hepatocellular carcinoma for their culture and drug sensitivity assay. Cancer Biol. Ther.
2016
,17,
1177–1187. [CrossRef]
37.
Zhu, L.; Chen, Y.; Wei, C.; Yang, X.; Cheng, J.; Yang, Z.; Chen, C.; Ji, Z. Anti-proliferative and pro-apoptotic effects of cinobufagin
on human breast cancer MCF-7 cells and its molecular mechanism. Nat. Prod. Res. 2018,32, 493–497. [CrossRef]
38.
Sargazi, S.; Kooshkaki, O.; Reza, J.Z.; Saravani, R.; Jaliani, H.Z.; Mirinejad, S.; Meshkini, F. Mild antagonistic effect of Valproic
acid in combination with AZD2461 in MCF-7 breast cancer cells. Med J. Islam. Repub. Iran 2019,33, 175–180. [CrossRef]
39.
Mosmann, T. Rapid colorimetric assay for cellular growth and sur- vival: application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983,65, 55–63. [CrossRef]
40. Strober, W. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. 2015,111, A3. B.1–A3. B.3. [CrossRef]
Separations 2021,8, 26 14 of 14
41.
Owoseni, O.; Nyankson, E.; Zhang, Y.; Adams, S.J.; He, J.; McPherson, G.L.; Bose, A.; Gupta, R.B.; John, V.T. Surfactant-loaded
halloysite clay nanotube dispersants for crude oil spill remediation. Ind. Eng. Chem. Res. 2015,54, 9328–9341.
42.
Bunaciu, A.A.; Udri¸stioiu, E.G.; Aboul-Enein, H.Y. X-Ray Diffraction: Instrumentation and Applications. Crit. Rev. Anal. Chem.
2014,45, 289–299. [CrossRef][PubMed]
43.
Oladele Oladimeji, A.; Adebayo Oladosu, I.; Shaiq Ali, M.; Lateef, M. Dioclins A and B, new antioxidant flavonoids from Dioclea
reflexa. Nat. Prod. Res. 2018,32, 2017–2024. [CrossRef][PubMed]
44.
Wang, B.; Zhang, X.; Wang, C.; Chen, L.; Xiao, Y.; Pang, Y. Bipolar and fixable probe targeting mitochondria to trace local
depolarization via two-photon fluorescence lifetime imaging. Analyst 2015,140, 5488–5494. [CrossRef][PubMed]
45.
Kuralay, F.; Dükar, N.; Bayramlı, Y. Poly-L-lysine Coated Surfaces for Ultrasensitive Nucleic Acid Detection. Electroanalytical
2018
,
30, 1556–1565. [CrossRef][PubMed]
46.
Verhoeven, H.A.; van Griensven, L.J. Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate
(DCA) on THP-1 cells: a multiparameter analysis. J. Bioenergy Biomembr. 2012,44, 91–99. [CrossRef]