Sherif Ashraf Fahmy1,2, Marwa Y Issa3, Basma M Saleh1, Meselhy Ragab Meselhy3, Hassan Mohamed El-Said Azzazy1. 1. Department of Chemistry, School of Sciences & Engineering, The American University in Cairo, AUC Avenue, P.O. Box 74, New Cairo 11835, Egypt. 2. School of Life and Medical Sciences, University of Hertfordshire Hosted by Global Academic Foundation, R5 New Garden City, New Capital, Cairo 11835, Egypt. 3. Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt.
Abstract
Amphiphilic macrocycles, such as p-sulfonatocalix[6]arenes (p-SC6), have demonstrated great potential in designing synthetic nanovesicles based on self-assembly approaches. These supramolecular nanovesicles are capable of improving the solubility, stability, and biological activity of various drugs. In the present study, the biologically active harmala alkaloid-rich fraction (HARF) was extracted from Peganum harmala L. seeds. Ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC/ESI-MS) analysis of HARF revealed 15 alkaloids. The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis revealed three peaks: peganine, harmol, and harmine. The HARF was then encapsulated in p-SC6 nanocapsules employing a thin-film hydration approach. The designed nanocapsules had an average particle size of 264.8 ± 10.6 nm, and a surface charge of -30.3 ± 2.2 mV. They were able to encapsulate 89.3 ± 1.4, 74.4 ± 1.3, and 76.1 ± 1.7% of the three harmala alkaloids; harmine, harmol, and peganine; respectively. The in vitro drug release experiments showed the potential of the designed nanocapsules to release their cargo at a pH of 5.5 (typical of cancerous tissue). The IC50 values of HARF encapsulated in p-SC6 (H/p-SC6 nanocapsules) were 5 and 2.7 μg/mL against ovarian cancer cells (SKOV-3) and breast adenocarcinoma cells (MCF-7), respectively. The prepared nanocapsules were found to be biocompatible when tested on human skin fibroblasts. Additionally, the antioxidant activity of the designed nanocapsules was 5 times that of the free powder fraction; the IC50 of the H/p-SC6 nanocapsules was 30.1 ± 1.3 μg/mL, and that of the HARF was 169.3 ± 7.2 μg/mL. In conclusion, encapsulation of P. harmala alkaloid-rich fraction into self-assembled p-SC6 significantly increases its antioxidant and cytotoxic activities.
Amphiphilic macrocycles, such as p-sulfonatocalix[6]arenes (p-SC6), have demonstrated great potential in designing synthetic nanovesicles based on self-assembly approaches. These supramolecular nanovesicles are capable of improving the solubility, stability, and biological activity of various drugs. In the present study, the biologically active harmala alkaloid-rich fraction (HARF) was extracted from Peganum harmala L. seeds. Ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC/ESI-MS) analysis of HARF revealed 15 alkaloids. The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis revealed three peaks: peganine, harmol, and harmine. The HARF was then encapsulated in p-SC6 nanocapsules employing a thin-film hydration approach. The designed nanocapsules had an average particle size of 264.8 ± 10.6 nm, and a surface charge of -30.3 ± 2.2 mV. They were able to encapsulate 89.3 ± 1.4, 74.4 ± 1.3, and 76.1 ± 1.7% of the three harmala alkaloids; harmine, harmol, and peganine; respectively. The in vitro drug release experiments showed the potential of the designed nanocapsules to release their cargo at a pH of 5.5 (typical of cancerous tissue). The IC50 values of HARF encapsulated in p-SC6 (H/p-SC6 nanocapsules) were 5 and 2.7 μg/mL against ovarian cancer cells (SKOV-3) and breast adenocarcinoma cells (MCF-7), respectively. The prepared nanocapsules were found to be biocompatible when tested on human skin fibroblasts. Additionally, the antioxidant activity of the designed nanocapsules was 5 times that of the free powder fraction; the IC50 of the H/p-SC6 nanocapsules was 30.1 ± 1.3 μg/mL, and that of the HARF was 169.3 ± 7.2 μg/mL. In conclusion, encapsulation of P. harmala alkaloid-rich fraction into self-assembled p-SC6 significantly increases its antioxidant and cytotoxic activities.
Cancer
is the second most common cause of death in developed and
developing countries, with a steady increase in the number of new
cases each year. The number of patients diagnosed with cancer is predicted
to increase by 2-fold within the coming 30 years.[1] Currently, synthetic chemotherapeutic agents are used in
cancer therapy either alone or in combination with surgical resection
and/or radiation therapy.[2−7] However, direct use of classical chemotherapeutic agents and procedures
has many drawbacks and generates severe adverse effects and resistance.[8−10] However, encapsulation of the anticancer drugs into nanocarriers
could provide effective drug delivery and minimize their side effects.[11−13] Since more than 50% of the currently available anticancer drugs
are from natural products or developed derivatives, natural products
should be recognized as a potential source of potent and less toxic
anticancer agents. Many studies have been reported on this path on
employing natural products to treat several diseases.[14,15]Peganum harmala L. (Zygophyllaceae)
is a small herbaceous plant known to grow as an invasive weed on poor
soils in the Middle East and North Africa.[16] The mature harmala seeds are rich in alkaloids (about 6% of total
dry weight), including some β-carboline and quinazoline alkaloids,
jointly known as harmala alkaloids.[17] Major
harmala alkaloids include harmine, harmane, harmol, harmaline, harmalol,
and peganine. These alkaloids possess a broad spectrum of biological
activities, including antibacterial, antioxidant, antiplatelet, immunomodulatory,
and cytotoxic activities.[16,18] However, their clinical
applications in cancer therapy are hindered by their hydrophobic nature,[16] and some studies reported toxic effects of harmala
alkaloids on the central nervous system at high doses.[19] Accordingly, carrying out encapsulation of harmala
alkaloids could effectively reduce their harmful effects and/or enhance
their anticancer activities.Various nanocarriers such as polymeric
nanoparticles, dendrimers,
and liposomes were previously reported for the targeted delivery of
different natural and synthetic drugs.[15,20] Recently,
macromolecules, such as p-sulfonato-calix[n]arenes [n = 4 and 6, (p-SC4 and p-SC6)] have demonstrated considerable
impact on the delivery of anticancer drugs.[10]p-SCns are water-soluble, safe (for doses up to
105 μg/kg), biodegradable, and biocompatible. They
are very promising as a potential host molecule for various chemotherapeutics
via host–guest inclusion complexation.[21−24] Additionally, amphiphilic macrocycles
can be used to develop nanocapsules relying on self-assembly methods.
These assemblies are bonded together via weak and reversible bonds,
making them very responsive. Therefore, their amphiphilic nature qualifies
them to encapsulate both water-soluble and oil-soluble chemotherapeutic
agents within their central shells where host–guest complexation
plays a role part in binding the hydrophobic and hydrophilic moieties.[21,25] These assemblies were also reported to exhibit targeted and controlled
release of drugs to the sites of action and hence minimize the undesirable
effects on healthy cells. The first amphiphilic macrocyclic nanocapsules
based on self-assembly advances were designed by Markowitz et al.
(1989) by injecting tetrahydrofuran solution containing p-SC6 into water resulting in the production of nanocapsules with
size ranging from 500 to 1000 nm.[26] Further
studies have been conducted on the design of p-SCn
as potential drug carriers. For example, nanovesicles created by the
self-assembly of tetrahexyloxy-p-sulfonatocalix[4]arene
were utilized to encapsulate paclitaxel (PTX) anticancer drug. The
prepared nanocapsules exhibited much enhanced anticancer activities
on human cervical cancer cells than the unencapsulated PTX, at concentrations
of 1, 10, and 100 μg/mL.[27]In the present work, the harmala alkaloid-rich fraction extracted
from P. harmala L. seeds was encapsulated
in p-SC6 nanocapsules (H/p-SC6)
employing a thin-film hydration approach. Nanocapsules were physically
identified in terms of average particle sizes, surface charge, polydispersity
index (PDI), encapsulation efficiency %, and morphology (employing
transmission electron microscope (TEM)). Also, the chemical structural
features of the nanocapsules were investigated employing Fourier transform
infrared spectroscopy (FT-IR). Additionally, the antioxidant (using
DPPH) and cytotoxic activities against ovarian cancer cells (SKOV-3),
breast adenocarcinoma (MCF-7), and human skin fibroblasts (using sulforhodamine
B (SRB) assay) were evaluated for both encapsulated and unencapsulated
harmala alkaloid-rich fraction.
Results
and Discussion
P. harmala Alkaloids
The three major alkaloids of P.
harmala (peganine, harmol, and harmine) were detected
using HPLC–DAD
and confirmed by UPLC-ESI-MS analysis.
Detection
of Harmala Alkaloids by HPLC–DAD
The alkaloids of P. harmala seeds
were analyzed on HPLC–DAD. The chromatogram showed three major
alkaloids, which could be detected by HPLC–DAD at 320 nm and
corresponded to peganine, harmol, and harmine (Figure ) as previously described.[16] These peaks were compared to standard harmine and the isolated
alkaloidspeganine, harmol, and harmine. The isolated ones were annotated
by their mass and UV spectra (Figures S1 and S2). Moreover, these alkaloids, in addition to the other 15 alkaloids,
were confirmed by liquid chromatography with tandem mass spectrometry
(LC-MS/MS).
Figure 1
HPLC chromatogram of P. harmala seed
total alkaloid extract using a Waters LC 2695 coupled to a Waters
996 diode array detector (DAD) at 320 nm. XTerra MS-C18 column (100
× 4.6 mm2 id, 5 μm). Mobile phase: water containing
triethylamine (pH 8.6) (A) and acetonitrile (B). Gradient elution
from 0% B (100% A) to 20% B in 4 min, 30% B at 9 min, 50% B at 14
min, and 60% B at 16 min. Then, 2 min washing with 100% B and reequilibration
for 4 min. A flow rate of 1 mL/min, column temperature of 25 °C,
and injection volume of 20 μL were applied. The peaks were compared
to standard harmine and the isolated alkaloids (peganine, harmol,
and harmine).
HPLC chromatogram of P. harmala seed
total alkaloid extract using a Waters LC 2695 coupled to a Waters
996 diode array detector (DAD) at 320 nm. XTerra MS-C18 column (100
× 4.6 mm2 id, 5 μm). Mobile phase: water containing
triethylamine (pH 8.6) (A) and acetonitrile (B). Gradient elution
from 0% B (100% A) to 20% B in 4 min, 30% B at 9 min, 50% B at 14
min, and 60% B at 16 min. Then, 2 min washing with 100% B and reequilibration
for 4 min. A flow rate of 1 mL/min, column temperature of 25 °C,
and injection volume of 20 μL were applied. The peaks were compared
to standard harmine and the isolated alkaloids (peganine, harmol,
and harmine).
Analysis
of Harmala Alkaloid-Rich Fraction
by Ultraperformance Liquid Chromatography–Electrospray Ionization–Tandem
Mass Spectrometry (UPLC/ESI-MS)
The phytochemical identification
of the harmala alkaloid-rich fraction (HARF) was performed using UPLC-ESI-MS
analysis. Fifteen alkaloids were timidly detected using their m/z, fragmentation pattern, their elution
order from the HPLC column, and by comparing their mass spectral data
with those reported. The annotated alkaloids are either β-carboline
or quinazoline-type alkaloids. Alkaloids of both types are major compounds
in this plant.[28] The major β-carboline
alkaloids annotated were harmine (m/z [M + H]+ 213.1546) and harmaline (m/z [M + H]+ 215.5396), with MS2 product
patterns at 198 and 170 (for harmine) and 200 and 174 (for harmaline).
However, quinazoline alkaloids were represented mainly by vasicine
(peganine) and vasicinone (m/z [M
+ H]+ 189.2218 and 203.1254; MS2 ions at 171,
154, 144, and 117 “for vasicine”, and 203 and 185 “for
vasicinone”). Other perceived major harmala alkaloids were
pegamine, hydroxylated harmine, and tetrahydroharmine. In addition,
some minor alkaloids such as vasicinone hexoside, harmalol, deoxyvasicinone,
hydroxylated harmaline, harmol, and harmalanine are presented in Table and Figure . The structures of the major
detected alkaloids are depicted in Figure , and the MS2 fragmentation pattern
of selected alkaloids is represented in Figure S3.
Table 1
Tentatively Identified Peaks in the
LC-MS Spectrum of the Total Alkaloids Fraction of P.
harmala Seed Extract
UPLC-ESI-MS/MS chromatogram (in positive-ion mode) of the total
alkaloids fraction of P. harmala separated
on an ACQUITY UPLC-BEH C18 (1.7 μm, 2.1 × 50 mm2) column.
Figure 3
Major alkaloids detected in HARF by ultraperformance
liquid chromatography–electrospray
ionization–tandem mass spectrometry (UPLC/ESI-MS).
UPLC-ESI-MS/MS chromatogram (in positive-ion mode) of the total
alkaloids fraction of P. harmala separated
on an ACQUITY UPLC-BEH C18 (1.7 μm, 2.1 × 50 mm2) column.Major alkaloids detected in HARF by ultraperformance
liquid chromatography–electrospray
ionization–tandem mass spectrometry (UPLC/ESI-MS).
Identification
of the Designed Harmala Alkaloid-Rich
Fraction/p-SC6 (H/p-SC6) Supramolecular
Nanocapsules
Particle Size, Polydispersity
Index (PDI),
ζ-Potential, and Encapsulation Efficiency (EE)
The
average size and PDI of the designed H/p-SC6 system
were investigated by dynamic light scattering and were found to be
264.77 ± 10.56 nm and 0.26 ± 0.02, respectively (Table ). These values lie
within the range of the particles with nano sizes of 200–600
nm previously reported as ideal chemotherapeutics carriers with boosted
passive accumulation, due to enhanced permeability and retention (EPR),
into the permeable vasculature of tumor cells.[38−40] The ζ-potential
of the designed nanocapsules was obtained via laser Doppler velocimetry
and exhibited a high negative surface charge of −30.33 ±
2.15 mV. This is attributed to the presence of the anionic p-SC6 amphiphile, which is highly negatively charged.[21] The increased surface negativity on the nanocapsules
is expected to impart high stability as it inhibits the aggregation
of the particles, and hence keeps them suspended for a longer period.
Encapsulation efficiencies on the three alkaloids are presented in Table .
Table 2
Average Size, PDI, ζ-Potential,
and Encapsulation Efficiency of H/p-SC6 Nanocapsules
encapsulation efficiency (%) ± SD
formula
average size (nm)
PDI
ζ-potential (mV) ± SD
harmine
harmol
peganine
H/p-SC6
264.77 ± 10.56
0.26 ± 0.02
–30.33 ± 2.15
89.34 ± 1.41
74.39 ± 1.30
76.10 ± 1.66
The high encapsulation efficiency of HARF is attributed to the
unique chemical structure of the amphiphilic p-SC6
that possesses a para-substituent of a phenolic ring on the upper
edge, a phenolic hydroxyl group on the lower edge, and a hydrophobic
π electron-rich central cavity (central annulus). Thus, their
amphiphilic properties qualify them to encapsulate the water-insoluble
harmala alkaloids, within their central cavities.[21,41] The ability to encapsulate high concentrations of drugs inside nanovesicles
supports sustained drug release.[42]
Morphological Features
The TEM
images presented in Figure show HARF loaded in amphiphilic p-SC6 nanocapsules,
which have spherical shapes with smooth surfaces.
Figure 4
TEM image for H/p-SC6 nanocapsules at a scale
of 200 nm showing sizes of (A) 275.43 nm and (B) 250.41 nm.
TEM image for H/p-SC6 nanocapsules at a scale
of 200 nm showing sizes of (A) 275.43 nm and (B) 250.41 nm.The self-assembly of amphiphilic p-SC6 is capable
of producing spherical nanovesicles in aqueous solutions. The unique
amphiphilic nature of p-SC6 permits the solubilization
of the harmala alkaloid-rich fraction in the inner hydrophobic core
while revealing its hydrophilic sulfonate groups found at the upper
rims to the exterior medium.[21]
FT-IR Analysis
The FT-IR spectra
of unencapsulated HARF, p-SC6, and the designed nanocapsules
were compared to demonstrate the encapsulation of HARF into the cavity
of the p-SC6 (Figure ). Three major sharp bands, for pure HARF, are observed
at 3444.02 cm–1 (−OH stretching vibrations),
1639.55 cm–1 (C=O asymmetry stretching vibration),
and 1014.59 cm–1 (C–O stretching). These
spectral bands agree with those reported previously.[43] Upon encapsulation of HARF in p-SC6 nanocapsules,
it is observed that the C–O stretching band at 1014.59 cm–1 had disappeared compared to HARF. Thus, this could
indicate the encapsulation of HARF inside the cavity of p-SC6.[27]
Figure 5
Fourier transform infrared (FTIR) spectra
of (A) pure HARF, (B) p-SC6, and (C) H/p-SC6 nanocapsules.
Fourier transform infrared (FTIR) spectra
of (A) pure HARF, (B) p-SC6, and (C) H/p-SC6 nanocapsules.
In Vitro Release Study
The release of harmala major
alkaloids from p-SC6-based
nanocapsules was studied at pH 7.4 of healthy cells and pH 5.5 of
cancer cells (Figure A–C).[44] The released three alkaloids
(peganine, harmol, and harmine) were determined using the above-mentioned
HPLC–DAD method. As expected from ζ-potential measurements,
H/p-SC6 nanocapsules exhibited outstanding stability
at pH 7.4 with about 28.3, 39.9, and 35.2% of the loaded harmala alkaloids
released after 48 h at 37 °C. At pH 5.5, 89.2, 88.9, and 90.8%
of the loaded harmala alkaloids were released after 48 h at 37 °C.
These results support the ability of self-assembled calixarenes to
release their cargo selectively by a pH-triggered mechanism at a typical
cancerous tissue pH of 5.5.[45]
Figure 6
Time-dependent
release profiles of the three harmala alkaloids
from H/p-SC6 nanocapsules: (A) peganine, (B) harmol,
and (C) harmine at 37 °C, into pH 5.5 (triangle) and pH 7.4 (square)
phosphate buffer media.
Time-dependent
release profiles of the three harmala alkaloids
from H/p-SC6 nanocapsules: (A) peganine, (B) harmol,
and (C) harmine at 37 °C, into pH 5.5 (triangle) and pH 7.4 (square)
phosphate buffer media.These designed nanocapsules
hold much promise for future cancer
therapy because of their capability to release the encapsulated drugs
inside the tumor tissue while shielding them from early decomposition
in the systemic circulation of pH 7.4.
Antioxidant
Activity
The antioxidant
activity of free HARF and H/p-SC6 nanocapsules was
investigated utilizing 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
free radical scavenging assay. Trolox, a powerful antioxidant, was
used as a positive control and showed an IC50 of 56.82
± 0.87 μg/mL (Figure ). It was shown that the IC50 of the H/p-SC6 nanocapsules (30.10 ± 1.30 μg/mL) exhibited
about 5 times that of the free HARF (169.3 ± 7.2 μg/mL),
as presented in Figure .
Figure 7
IC50 values for DPPH radical scavenging assay of Trolox
(positive control), harmala alkaloid-rich fraction (HARF), and H/p-SC6 nanocapsules.
IC50 values for DPPH radical scavenging assay of Trolox
(positive control), harmala alkaloid-rich fraction (HARF), and H/p-SC6 nanocapsules.The nanocapsules’ enhanced antioxidant activity compared
to harmala alkaloid-rich fraction might be attributed to enhancing
the hydrophilic nature of harmala alkaloid-rich fraction upon its
encapsulation within the p-SC6 self-assemblies.[46,47] Additionally, calixarenes are reported to have antioxidant properties
combined with the antioxidant abilities of harmala alkaloid-rich fraction,
giving rise to more pronounced free radical scavenging activities.[48] These findings are very promising because powerful
antioxidants have many biomedical applications and might play a vital
role in protecting normal cells, especially during cancer treatment.
In Vitro Cell Viability
Assay
The cytotoxicity of p-SC6, free HARF,
and H/p-SC6 nanocapsules was assessed by SRB assay
and ovarian cancer cells (SKOV-3), breast adenocarcinoma cells (MCF-7),
and normal human skin fibroblasts.[23,24]p-SC6 was used as host control, and it showed a negligible decline
in cell viability, and HARF was used as the positive control. After
48 h incubation, the harmala extract nanocapsules (H/p-SC6) exerted remarkable in vitro cytotoxicity compared
to the free powder extract (positive control). The cytotoxic activities
(IC50 in μg/mL, computed by Sigma plot) of p-SC6, free HARF, and H/p-SC6 nanocapsules
against cancer noncancer cell lines are presented in Table . The IC50 values
of H/p-SC6 were about 1.2- and 3-fold less than that
of the HARF against SKOV-3 and MCF-7 cells, respectively. On the other
hand, testing H/p-SC6 nanocapsules on HSF noncancer
cells confirmed their biocompatibility compared to free harmala seed
fraction. The previous studies reported the ability of different host
molecules to encapsulate anticancer drugs and selectively deliver
them to cancer cells with minimal toxic effects on normal cells.[23,24,45−47,49−51] These results agree with the in vitro release study demonstrating the pH-triggered release
of the HARF from the self-assembled H/p-SC6 nanocapsules.
Cancerous cells have an acidic pH of 5.7, normal cells possess a pH
of 7.4, whereas late endosomes and lysosomes have pH values of 4.5–5.5.
This pH variation is vital because the pH-sensitive carriers would
selectively release their cargo in the acidic tumor microenvironment.[45,52,53] Additionally, HARF showed increased
cytotoxic activities as its encapsulation within the p-SC6 nanocapsules overcame the extract’s hydrophobic nature
and improved its bioavailability.[23,24,46] This study also highlights the importance of using P. harmala seed alkaloid-rich fraction as a possible
natural anticancer drug. Harmala alkaloid-rich fraction’s anticancer
activity is attributed to the β-carboline alkaloids that inhibit
DNA topoisomerases via DNA intercalation and induction of apoptotic
pathways.[54,55] Additionally, harmine alkaloid has been
shown to possess antiangiogenic activity, reduce the expression of
numerous proangiogenic factors, and diminish the propagation of vascular
endothelial cells.[56] Thus, these findings
shed more light on the significance of using self-assembled p-SC6 as a possible supramolecular carrier for the natural
promising anticancer agent, P. harmala seed alkaloid-rich fraction.
Table 3
In Vitro Cytotoxic
Activities of p-SC6, Harmala Alkaloid-Rich Fraction,
and H/p-SC6 Nanocapsules against SKOV-3, MCF-7, and
Human Skin Fibroblastsa
in
vitro cytotoxic activity (IC50 in μg/mL)
cells
p-SC6
harmala alkaloid-rich fraction
(positive control)
H/p-SC6
SKOV-3
>300
6.7 ± 1.6
5 ± 1.3
MCF-7
>220
6.7 ± 0.7
2.3 ± 1.1
human skin fibroblasts
>300
4.6 ± 1.5
>300
The treatment period
was 48 h. Data
represent the mean ± standard deviation of triplicate.
The treatment period
was 48 h. Data
represent the mean ± standard deviation of triplicate.
Conclusions
In the present work, the harmala alkaloid-rich fraction was extracted
from P. harmala seeds, and 15 alkaloids
were annotated using ultraperformance liquid chromatography–electrospray
ionization–tandem mass spectrometry (UPLC/ESI-MS). Reversed-phase
high-performance liquid chromatography coupled with diode array detector
(HPLC–DAD) analysis of the alkaloid-rich fraction revealed
three major peaks: peganine, harmol, and harmine. This fraction was
encapsulated in amphiphilic p-SC6 self-assembled
nanovesicles employing a thin-film hydration approach. The designed
nanocapsules possessed an average particle size of 264.77 ± 10.56
nm, a surface charge of −30.33 ± 2.15 mV, and were found
to encapsulate 89.34 ± 1.41, 74.39 ± 1.3, and 76.1 ±
1.66% of the three harmala alkaloids, peganine, harmol, and harmine,
respectively. In vitro drug release experiments showed
the potential of the designed nanocapsules to release their cargo
in mildly acidic media. This is very promising for future cancer therapy
because of their ability to release the encapsulated drugs inside
the acidic cancer tissue while keeping them stable in the systemic
circulation of pH 7.4. The antioxidant activity of the H/p-SC6 nanocapsules was 5 times that of harmala alkaloid-rich fraction.
Additionally, the cytotoxicity study revealed that the IC50 values of the designed nanocapsules were about 1.2- and 3-fold less
than that demonstrated by harmala alkaloid-rich fraction against SKOV-3
and MCF-7 cells, respectively. The prepared nanocapsules were found
to be biocompatible when tested on human skin fibroblasts. Thus, encapsulation
of harmala alkaloid-rich fraction into self-assembled supramolecular
nanocapsules dramatically enhances the natural anticancer and antioxidant
effects of harmala alkaloids.
Materials and Methods
Materials
p-Sulfocalix[6]arene
was purchased from WuXi LabNetwork, China. Diphenyl-1-picrylhydrazyl
radical (DPPH), Harmine standard, HPLC-grade deionized water, chemicals,
and solvents were obtained from Sigma-Aldrich (St. Louis, MO). Streptomycin,
penicillin, fetal bovine serum, trichloroacetic acid (TCA), Dulbecco’s
modified Eagle’s medium (DMEM) SRB, and tris(hydroxymethyl)aminomethane
(TRIS) were obtained from Lonza, Basel, Switzerland.
Plant Material
Dried mature seeds
of P. harmala L. were purchased from
the Egyptian local market. A voucher specimen was deposited (18.1.17)
at the Department of Pharmacognosy Herbarium, Faculty of Pharmacy,
Cairo University (Egypt).
Methods
Extraction and Isolation of Major P. harmala Alkaloids
P. harmala seeds
(2.5 kg) were ground to a fine powder and then extracted by
soaking in 70% ethanol (3 × 5 L) at ambient temperature overnight.
The ethanol extracts were filtered, combined, and evaporated under
reduced pressure to yield a dark reddish-brown viscous residue (520
g). The concentrated extract was then dissolved in 5% HCl (2 L), filtered,
and partitioned with dichloromethane (4 × 300 mL). The aqueous
acid layer’s pH was adjusted to pH 9 using NH4OH
and then extracted with dichloromethane (4 × 500 mL). The dichloromethane
layer was washed with water and then evaporated under reduced pressure
at 40 °C to yield 60 g of reddish-brown powder (harmala alkaloid-rich
fraction).[16]
HPLC–DAD
Analysis of the Three Major
Harmala Alkaloids
The analysis of P. harmala alkaloids was performed, according to our previously reported method,[16] by RP-HPLC using a Waters LC 2695 coupled to
a Waters 996 diode array detector (DAD). A 100 × 4.6 mm2 id, 5 μm, XTerra MS-C18 column (Agilent Technologies) was
used for analysis. Chromatographic conditions were water supplemented
with triethylamine (pH 8.6) (A) and acetonitrile (B). The gradient
was programmed from 0% B (100% A) to 20% B in 4 min, then 30% B at
9 min, 50% B at 14 min, and finally to 60% B at 16 min. The separation
was followed by a 2 min washing procedure with 100% B and a reequilibration
period of 4 min. Flow rate: 1 mL/min, column temperature: 25 °C,
injection volume: 20 μL, and absorbance detection: 320 nm. Under
these conditions, the elution order was peganine, harmol, and harmaline.
These peaks were compared to standard harmine, and the isolated alkaloids
were peganine, harmol, and harmine when injected individually on HPLC
(Figure ). The isolated
ones were annotated by their mass and UV spectra (Figures S1 and S2). Calibration curves were constructed for
peganine, harmol, and harmine to be used in the measurement of encapsulation
efficiency and in vitro release studies.
UPLC-MS/MS Analysis of Harmala Alkaloids
The harmala
alkaloid-rich fraction was analyzed using UPLC/ESI-MS.
The sample was dissolved in methanol for HPLC at a final concentration
of 100 μg/mL. Then, it was filtered using a membrane disk filter
(0.2 μm) before subjecting to LC-ESI-MS analysis on UPLC/ESI-MS,
ACQUITY UPLC System (Waters Corporation) using ACQUITY UPLC-BEH C18
(1.7 μm, 2.1 × 50 mm2) column and injection
volume of 10 μL. The solvent system consisted of (A) water containing
0.1% formic acid and (B) methanol containing 0.1% formic acid. Elution
was done using gradient mobile phase starting from 90% A: 10% B that
was maintained for 2 min, it reached 70% A at 5 min, then 30% A at
15 min, 10% A at 22 min which was maintained for 3 min and then was
changed to reach 100% B at 26 min and was upheld for 3 min and then
back to the initial composition at 32 min. The flow rate was 0.2 mL/min.
The parameters for analysis were carried out in positive-ion acquisition
mode using a XEVO TQD triple quadrupole mass spectrometer (Waters
Corporation, Milford, MA). The source temperature, cone voltage, capillary
voltage, and desolvation temperature were 150 °C, 30 eV, 3 kV,
and 440 °C, respectively. The cone gas flow rate was 50 L/h,
and the desolvation gas flow rate was 900 L/h. Mass spectra were detected
in the ESI between m/z 100 and 1000.
The peaks and spectra were processed using the Maslynx 4.1 software
and tentatively identified by comparing their retention times (Rt) and mass spectra with reported data.[16]
Preparation of Harmala
Alkaloid-Rich Fraction-Loaded p-Sulfonatocalix[6]arene
Nanocapsules (H/p-SC6)
H/p-SC6 nanocapsules were produced
using the thin-film hydration approach as described elsewhere[27,49,50] with some modifications. Briefly, p-SC6 (57 mg) was mixed with HARF (1 mg) in a sufficient
volume of methanol in a round-bottom flask. The round-bottom flask
was exposed to evaporation under vacuum for 60 min at 40 °C,
utilizing a Laborotay 4000 rotary evaporator (Heidolph Instruments,
Schwabach, Germany) equipped with a vacuum pump (KNF Neuberger GmbH,
Freiburg, Germany), leaving a thin film. Then, hydration was conducted
using phosphate-buffered saline (PBS, pH 7.4) in a rotary evaporator
without using vacuum for 30 min at 40 °C. Finally, sonication
for 1 min of the produced suspension was carried out employing a bath
sonicator (Elmasonic P30 H, Elma Hans Schmidbauer, Singen, Germany).
Characterization of the Designed H/p-SC6 Nanocapsules
The average particle size and
polydispersity index (PDI) of the prepared nanocapsules were determined
using dynamic light scattering employing Zetasizer Nano ZS (Malvern
Instruments Herrenberg, Germany).[57] The
instrument is equipped with a 10 mW HeNe laser allowing for the measurements
to be performed at a wavelength of 633 nm and a detection angle of
173° backscatter. The ζ-potential of the nanocapsules was
determined via laser Doppler velocimetry in a clear disposable folded
capillary cell (DTS1070, Malvern Instruments).The morphological
features of the nanocapsules were studied using transmission electron
microscopy (TEM), employing a JEOL-JEM 2100 electron microscope operating
at 160 kV. A 50 μL aliquot of the nanovesicles diluted to 1:2
(v/v) with PBS were stained with 2% aqueous phosphotungstic acid.
This mixture was placed and dried over a carbon-coated copper 200
mesh grid, imagined, and photographed.The FT-IR spectra of
unencapsulated HARF, p-SC6,
and the designed nanocapsules were subjected to Fourier transform
infrared (FTIR) spectroscopy employing FTIR-8400s (Shimadzu, Japan).
Samples were first compressed with KBr into disks, scanned, and spectra
were recorded in the range of 500–4000 cm–1.
Encapsulation Efficiency
The encapsulation
efficiency (EE) of H/p-SC6 nanocapsules was determined
by centrifugation at 14 000 rpm for 90 min at 4 °C, followed
by ultrafiltration to eliminate the harmala alkaloids loaded pellet.
The quantity of the harmala alkaloids unencapsulated in the ultrafiltrate
was detected using the HPLC protocol described earlier. The EE of
the H/p-SC6 nanocapsules was determined using eq (27,58)
In Vitro Release Study
under Different pH Conditions
The release rate of harmala
alkaloids from H/p-SC6 nanocapsules was studied using
the dialysis membrane method at pH 5.5 and 7.4. Briefly, 1 mL of the
sample was loaded to a dialysis bag (cutoff molecular weight, 3500
Da). The dialysis bag was inserted into 50 mL of PBS at pH 7.4 or
5.5, with 1% Tween and 0.5% FBS in a proper jar. The whole system
was fixed in a shaking incubator (Jeio Tech SI-300, Seoul, Korea),
rotating at 100 rpm with temperature adjusted to 37 °C. At specific
time intervals, a 1 mL aliquot of the sample was withdrawn for analysis
by the above-described HPLC method and immediately replaced with another
equal volume of warmed buffer.
Determination
of Total Antioxidant Activity
The antioxidant activity of
HARF and H/p-SC6 nanocapsules
was evaluated using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate)
free radical scavenging assay as previously reported by Boly et al.[59] with minor modification. Briefly, 100 μL
of freshly prepared DPPH reagent (0.1% in methanol) was added to 100
μL of the sample in a 96-well plate (n = 6)
and the reaction was left at room temperature for 30 min in the dark.
Afterward, the color intensity of the DPPH resulting from its reduction
was recorded at 540 nm using a microplate reader FluoStar Omega (Ortenberg,
Germany). Data are represented as mean ± SD according to eq where Acontrol is the absorbance of the
DPPH and Asample is the absorbance of
the sample.Trolox was considered a reference
standard, and DPPH solution was used as the control. Data were analyzed
using Microsoft Excel, and the IC50 value was calculated
using GraphPad Prism 5 by converting the concentrations to their logarithmic
value and selecting nonlinear inhibitor regression equation [log (inhibitor)
versus normalized response – variable slope equation].[60]
Cell Viability Assay
Cell Culture
Ovarian cancer cells
(SKOV-3), breast adenocarcinoma cells (MCF-7), and human skin fibroblasts
healthy cells were obtained from American Type Culture Collection,
(University Boulevard, Manassas, VA 20110) and maintained in DMEM
supplemented with streptomycin (100 mg/mL), penicillin (100 units/mL),
and 10% heat-inactivated fetal bovine serum. Cells were incubated
in humidified 5% (v/v) CO2 at 37 °C.
Sulforhodamine B Colorimetric Assay
Ovarian cancer
cells (SKOV-3), breast adenocarcinoma cells (MCF-7),
and human skin fibroblasts were treated with different concentrations
of p-SC6, free HARF (positive control), and H/p-SC6. The cell viability of either cancerous or noncancerous
cells was tested using the SRB assay, and the IC50 (in
μg/mL) value was computed using our methods described previously.[23,24,61,62]All trials were conducted in triplicates, and data are demonstrated
as mean ± standard deviation.
Authors: Francisco J Ostos; José A Lebrón; Maria L Moyá; Manuel López-López; Antonio Sánchez; Amparo Clavero; Clara B García-Calderón; Iván V Rosado; Pilar López-Cornejo Journal: Chem Asian J Date: 2017-02-28
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