Inhibitory Effect of Quercetin-Loaded Nanostructure Lipid Carrier on Compound 48/80-Induced
Mast Cell Degranulation
Asit Kumar De, Rajkumar Shil, Santanu Ghosh, and Tanmoy Bera*
Division of Pharmaceutical Biotechnology, India
Submission: August 11, 2017; Published: October 25, 2017
*Corresponding author: Tanmoy Bera, Division of Pharmaceutical Biotechnology, India, Tel: +,
How to cite this article: Asit K D, Rajkumar S, Santanu G, Tanmoy B. Inhibitory Effect of Quercetin-Loaded Nanostructure Lipid Carrier on Compound
48/80-Induced Mast Cell Degranulation. Glob J Nano. 2017; 3(2): 555608. DOI:10.19080/GJN.2017.03.555608
Quercetin is naturally occurring flavanoids, it is abundant in nature and the human beings are taken up it through their daily meals. The different pharmacological activity of quercetin has been evaluated since a long time. The scientific exploration of quercetin revealed that it is effective as anti-oxidant, anti-inflammatory agent; it can reduce the elevated levels of cholesterol, triglycerides, low-density-lipoproteins etc. However, solubility and bioavailability has been an issue for the quercetin. In this experimental work we not only evaluate the antihistaminic activity of quercetin but also developed a nano structure lipid carrier system for quercetin and explore its effectiveness as an antihistaminic agent.
It was found that the quercetin loaded nano structured lipid carrier system was successfully fabricated with adequate drug loading and entrapment efficiency. The intracellular uptake of formulated delivery system by isolated mice peritoneal mast cells has also been studied. The antihistaminic activity of quercetin and quercetin loaded lipid carrier system was compared against the standard antihistaminic agents like prednisolone, cromolyn sodium and it was found that the quercetin loaded lipid carrier system have showed superior activity than the free quercetin and the standard antihistaminic agents.
Allergic diseases and asthma have been prevalent since a long ago and still, now the incidence of these types of disease condition increased in such a manner that it has appeared as a major health concern. As per world health organization, more than 1.8lakhs of mortality is caused by asthma annually . Ages are no bar for the occurrence of this type of disease; it may happen in any age group. The diseases like asthma and allergy are initiated by the process of inflammation. CD4+T helper cell responses are important for the initiation and propagation of the disease condition . Interleukin-4, 5, 9 and 13 are the cytokines which are secreted by TH2 cells and they are the most important mediators of the process of inflammation. Mast-cell degranulation, increased IgE level is the characteristic phenomenon of asthma and allergy related diseases .
The treatment of this kind of disease condition falls into different categories like mast cell stabilizers, corticosteroids, antihistaminic agents. They may act by blocking the mediators responsible for inflammation or by preventing the process which initiates the process of inflammation. The present chemotherapy for this purpose is efficient however long term side effects still be a concern. Skin fragility, immunosuppressant is associated with the long term use of oral corticosteroids. It was also observed that the new generation antihistaminic agents are showed sedative effects [4,5]. Hence, there is always a need for a new antihistaminic agent which could effectively treat the disease condition as well as tolerable in patients in long term.
Searching for a new antihistaminic agent as well as a new drug delivery system for this type of diseases we have developed quercetin loaded nano structured lipid carrier system. Quercetin is a naturally occurring phenolic compound and it is flavonoid
in class. Quercetin is abundant in nature and it is generally
found in the food plants. The flavonoids commonly present
as glycosides. Like, the aglycone quercetin linked to quercetin
as 3-o-glycoside . The beneficial pharmacological effects
of quercetin always draw the attention of many scientists for
several decades. According to Gupta et al, the quercetin has
potent antioxidant activity . Quercetin could scavenge the free
radicals, chelatemetal ions and it shows synergistic activity with
other antioxidants [8-12].
Quercetin could be effective in the treatment of inflammation
and oxidative stress as well . It was found in a study that
regular intake of quercetin could decrease the elevated levels
of cholesterol, triglyceride, and low-density-lipoproteins .
Tzankova et al in his recent study have found that quercetin
possessed hepatoprotective activity . It was reported that
more than 90% of quercetin was metabolized after an hour when
it was administered intraperitoneally. When Justino et al. 
was treated Sprague–Dawley rats with a single intraperitoneal
injection of quercetin at a dose of 50mg/kg body weight, it
was found that the plasma contains very low amount of free
quercetin . Therefore, the development of a drug delivery
system with quercetin which could effectively increase the
solubility and bioavailability of it, have always been a challenge.
In these circumstances, the nano structured lipid carrier system
(NLC) for quercetin was developed to evaluate its antihistaminic
property which would open up a new beneficial pharmacological
effect of the naturally occurring flavonol.
Gattefose India Pvt. Ltd. supplied Comparitol® 888 ATO.
Other substances like soy lecithin, compound 48/80, triolein,
o- phthalaldehyde, sorbitol, Pluronic F68, and quercetin were
purchased from Sigma- Aldrich. Fetal bovine serum, RPMI1640
and medium 199 were supplied from HiMedia, India. All the
solvents used in the experiment were of analytical grade.
BALB/c mice (20-25gm) of either sex or apparently
same ages were used for the studies. Animals were housed in
polypropylene cage. All of the experiments, where animals
were used, were done according to the guidelines of Jadavpur
University Animal Ethics Committee. Animals were fed with
standard diet and water and they were exposed to normal day
and night cycle.
Preparation of Cedrol-loaded NLCs: A combination of melt
emulsification along with homogenization and ultrasonication
method was employed to fabricate the quercetin loaded NLC
(qNLC) . Briefly, according to the drug and lipid ratio, the
weighted amount of comparitol® 888 ATO (1.5mg) [(glycerol
mono behenate 15%, glyceryl tribehenate 35% and glyceryl di
behenate 50%)], triolein (0.31mg) and quercetin were melted
together at about 85 ˚C. That melted mixture was stirred
occasionally for 15mins to develop transparent and uniform
oil phase. It was observed that there was no degradation of
quercetin happened at that particular temperature condition.
The aqueous phase was prepared by dissolving Pluronic F68
and soya lecithin (0.31mg) at the same temperature condition
and for the same period of time in a 60 ml double distilled water.
Now, the aqueous phase was added drop-wise into the oil phase
during homogenization at 20000rpm for 10mins. The formed
emulsion was then sonicated for 10mins in an ultra sonicator at
85 ˚C. The formed NLC was cooled at 4 ˚C for overnight followed
by membrane filtration through the 0.45μm membrane filter.
After frozen at -80 ˚C NLC suspension was lyophilized to obtain
a dry finished product.
Blank NLC was prepared by the same technique except
adding the quercetin (Table 1).
Particle size, zeta potential, and poly dispersity index:
The average particle size, zeta potential, and polydispersity
index (PDI) of formulated NLCs were determined by the dynamic
light scattering (DLS) technique by the Zetasizer Nano ZS90
(Malvern Instrument ltd., UK) using the software Data transfer
assistance (DTS). For these studies, 1 mg of the formulations
were dispersed in 2ml of Milli Q water and then sonicated for
half an hour before the analysis.
Quercetin loading and entrapment efficiency study: To
carry out the drug loading and entrapment efficiency study, 5mg
of qNLC was dispersed in 5 ml of 0.5% tween 80 in PBS buffer
(pH 7.4). The dispersion was sonicated for 15mins. After that, it
was centrifuged at 20000rpm for 15min. The supernatant was
collected and the quantity of quercetin was determined by the
liquid chromatographic method. For that purpose, C18 column
(4.6 × 250 mm, 5μ) was used. Acetonitrile and phosphate buffer
(pH 4.0 maintained by o- phosphoric acid) in a ratio of 50:50 was
used as a mobile phase and the flow rate was 1ml/min. Quercetin
was detected at a detecting wavelength of 292nm. Drug loading
and the entrapment efficiency was calculated by the following
Where Wtotal was the weight of drug added in the system,
Wfree was the analyzed weight of drug in the supernatant and
Wlipids weight of lipids added to the system.
Field emission scanning electron microscopic study: The
surface morphology of the formulated NLC was evaluated by field
emission scanning electron microscopy. The lyophilized product
was used for taking the SEM images. Briefly, lyophilized NLC was
spread on the carbon tape then subjected to gold coating. After
completion of the coating, particles were observed under FESEM
(Model-JSM-6700F; JEOL, Tokyo, Japan).
In vitro cumulative drug release study:In vitro cumulative
drug release study of formulated qNLC was carried out for 21
days. Briefly, 5mg of qNLC was suspended in 5 ml PBS buffer
pH 7.4 and kept it in the dialysis bag (MW cutoff 12000 kDa).
That dialysis bag was further kept into a 100 ml 0.5% v/v tween
80 with PBS buffer (pH 7.4). That system was kept in a shaker
incubator. 1 ml sample aliquots were collected and 1ml of fresh
release medium was incorporated for a predetermined time
period (45 mins, 90mins, 3h, 6h, 12h, 24h and up to 504h). The
collected samples were analyzed by the similar chromatographic
condition as previously described.
Mast cell isolation and culture: Peritoneal mast cells were
isolated from BALB/c mice . Briefly, after sacrificing the mice
by cervical dislocation, 5mL of heparinized (10 IU/mL) calciumand
magnesium-free Hank’s solution (HBSS) was injected into the mice peritoneal cavity. After 2mins, the peritoneal fluid was
collected and centrifuged at 1,500rpm for 10minutes at 4 °C. The
supernatant was discarded and the cells were resuspended in
fresh HBSS containing 0.1% protease-free bovine serum albumin
and then it was purified by continuous isotonic Percoll gradient
(72%) for isolation of mast cell.
Mast cells thus purified were resuspended in fresh HBSS
media containing calcium and magnesium. Toluidine blue and
trypan blue exclusion staining were performed to evaluate the
purity and the viability of mast cells, respectively.
After the tests for purification and viability, mast cells were
transferred to RPMI-1640 medium containing 15% fetal bovine
serum and 5μg/mL streptomycin and 50U penicillin. After
every 3 days sub culturing was carried out, and the culture was
maintained for 2 months.
Trypan blue exclusion assay of mast cell viability: Mast
cell viability was assessed by trypan blue staining. For this,
0.2ml of mast cells were transferred aseptically into a 1.5ml
eppendr of tube along with an equal amount of 0.4% (w/v)
trypan blue solution prepared in 0.8% (w/v) sodium chloride
and 0.06% (w/v) dibasic potassium phosphate and incubated it
for 3min at room temperature. Cells were counted by using the
hemocytometer under a light microscope. The count of viable
and nonviable cells was recorded separately.
Mast cell sensitization by compound 48/80: HEPESTyrode’s
buffer containing mast cell suspension was incubated
with 4μg/ml of compound 48/80 for the period of 10mins at 37
°C. Mast cells smears were prepared, fixed and stained with 0.1%
(w/v) toluidine blue and kept it for 10mins. The morphology of
the mast cells was observed under a light microscope .
Cellular uptake study: To carry out the cellular uptake
study, coumarin-6 loaded NLC was prepared it was used instead
of qNLC. For this study, sterile cover slips were placed in each
well of the 24 well plate was 4×105 cells/well were seeded
with coumarin-6 loaded NLC (0.5mg/mL) and incubated in a
5% CO2 incubator at 37 °C for 20min. After the specific period
of time, the cover slips were collected, washed for three times
and then observed under confocal microscope (TCS SP8; Leica
Microsystems, Wetzlar, Germany) using Leicasuitelas X software.
The magnification of the objective was 63×.
Effects of anti-asthmatic drugs, quercetin, and qNLC on
histamine release assay from mast cells: For the stabilization
purpose, the isolated and purified mice peritoneal mast cells
(2×105 cells/mL) were incubated at 37 °C in a 5% CO2 incubator
for 10mins. Standard anti-asthmatic drugs, quercetin, and qNLC
at variable concentrations were added to the pre-incubated
media containing the mast cells and further incubated at 37
°C. After the incubation period of 24h, the cells were washed
and further incubated with 4μg/ml of compound 48/80 for the
period of 20minutes. After that, the test tubes were kept in ice water. The released histamines were separated from the mast
cells by the process of centrifugation at 2,000rpm for 5minutes at
4 °C. 0.05% (v/v) Triton X-100 was added to liberate the residual
histamine. Then, 0.02% opt-methanol solution was added and
histamine content in the supernatant and in the pellets of cells
was determined spectrofluorimetrically (excitation 360nm,
emission 450nm). For the estimation of the spontaneous release
of histamine (spontaneous), exactly the same procedure without
adding drugs and samples was followed. The following equation
was used to calculate the release percentage of histamine
Statistical analysis: Experimental data were expressed
as a mean ± standard deviation. One way analysis of variance
was used to calculate the statistical significance. P <0.05 was
considered as significant and P >0.05 was considered as not
The average particle size, zeta potential, PDI values were
calculated for blank-NLC and qNLC nanoparticles. According to
the Table 2 the both types of formulated nanoparticles were in
nano size range and their average particle sizes were less than
100nm. The percentage yields of the NLCs were more than
adequate. The PDI values were found to be 0.213±0.05 and
0.257±0.03 for the Blank-NLC and qNLC, respectively. The NLCs
have shown negative zeta potential values like -17.3±0.03 for
Blank-NLC and -34.2±0.019 for qNLC.
The scanning electron microscopic study was carried out to
evaluate the size and shape of the formulated NLC. It could be
found from the Figure 2 that the formulated qNLC was in nano
size range and particles were almost spherical in shape.
The confocal study was carried out to evaluate the
formulated NLC uptake by the isolated and purified mast cell.
From the Figure 4, it was observed that within the period of
20mins coumarin-6 loaded NLC was taken up by the mast cells.
The green fluorescence appeared in the Figure 4B & 4C were the
Table 3 represented the IC50 values of Prednisolone,
cromolyn sodium, quercetin and qNLC which indicated that
at the particular concentration of the drug or formulation the
histamine release from the mast cells was 50% inhibited. It was
observed from the Table 3 that the IC50 of free quercetin was
19.84±2.76μM which was less than the standard antihistaminic
agents and the qNLC formulation of quercetin decreased the
IC50value to 7.06±2.14μM (Figure 5).
Fabrication of quercetin loaded NLC was carried out by a
combination of melt emulsification along with homogenization
and ultrasonication method. It was observed from the character
studies that the formed drug loaded particles were in nano size
range. From Table 2 and Figure 1A it could be observed that the
average particle size of qNLC was 90.06±0.57nm. The FESEM
analysis was also confirmed the formation of nano size ranged particles of NLC. From Table 2 it was also observed that the
both the blank and qNLC possessed negative surface charge.
Zeta potential is an important parameter which could depict the
stability of the nanoparticles inside the formulation.
It was found from the Table 2 and Figure 1B that the zeta
potential of qNLC was -29.2±0.019 mV which indicated that the
particles in the formulation would not agglomerate in suspension
if it will remain suspended for an extended time period .
When the quercetin loading into the NLC was evaluated it was
found that the formulation of qNLC exerted superior drug
loading of 19.3±0.72% with 98.7±0.92% entrapment efficiency.
In vitro percentage cumulative drug release study was
carried out for an extended period of time. It was an important
parameter as it will indicate the amount of drug release for the
predetermined period of time as well as the pattern of release. It
was observed from Figure 3 that the qNLC formulation released
the 88.7±1.5% quercetin after 504h.
As the mast cell was our target so uptake of NLC by the mast
cells was an essential parameter to evaluate and for that purpose
confocal microscopy study was carried out. It was clearly
observed from the Figure 4 that the coumarin-6 loaded NLC
was well taken up by the isolated and purified mast cells within
20mins. Here the coumarin-6 was mimicking the quercetin
which meant that quercetin will be taken up also when it will
deliver through the NLC system.
Histamine released from the mast cells and inhibition of that
phenomenon will be a property of an antihistaminic agent. For
that purpose, 50% inhibitory concentrations of the free quercetin
and qNLC formulation were determined and compared the data
against the data generated from the standard antihistaminic
drug like prednisolone and cromolyn sodium. From Table 3 and
Figure 5 it was observed that the IC50 values of prednisolone,
cromolyn sodium, and quercetin were 25.86±3.21μM,
23.15±3.04μM, and 19.84±2.76μM, respectively and it was
also found that there were no significant differences among
them which could indirectly indicate the similar antihistaminic
property of quercetin like the standard drugs. Moreover, when
the quercetin was loaded inside the lipid core of the NLC it was
found that the qNLC exerted superior inhibition of histamine
release from the mast cells with an IC50 value of 7.06±2.14μM.
There was an extremely significant difference obtained among
the IC50 value of qNLC and the other drugs.
From this experimental work, it could be concluded that the
qNLC was successfully fabricated. The formulated NLCs were
in nano size range. The uptake study revealed the adequate
homing of the drug of interest inside the mast cells and the qNLC
was efficiently inhibited the histamine release from the mast
cells and the quercetin loaded NLC delivered significantly higher
efficacy than the standard antihistaminic agents.