IET Nanobiotechnology

Research Article

ISSN 1751-8741
Development of nanoformulation of picroliv Received on 21st April 2015

isolated from Picrorrhiza kurroa

Revised on 24th August 2015
Accepted on 6th September 2015
doi: 10.1049/iet-nbt.2015.0032
www.ietdl.org

Anika Guliani 1, 2*, Avnesh Kumari 1*, Dharmesh Kumar 3, Sudesh Kumar Yadav 1, 2
1
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur-176061, HP, India
2
Acedemy of Scientific and Innovative Research, New Delhi, India
3
Food Nutraceuticals and Quality Control Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur-176061, HP, India
*
Both the authors have contributed equally to this work.
E-mail: skyt@rediffmail.com

Abstract: Picroliv, a mixture of picroside I and kutkoside isolated from rhizome of Picrorrhiza kurroa has been reported for
many pharmaceutical properties such as hepatoprotective, anticholestatic, antioxidant and immune-modulating activity.
However, picroliv possessed lesser efficacy due to its poor aqueous solubility and lesser bioavailability. To find
solution, picroliv was loaded into biodegradable poly lactic acid nanoparticles (PLA NPs) using solvent evaporation
method. The picroliv-loaded PLA NPs were characterised by UVvis spectroscopy, atomic force microscopy,
transmission electron microscopy, Fourier transform infrared and Zeta sizer. The size of picroliv-loaded PLA NPs was
182 20 nm. Zeta potential of picroliv-loaded PLA NPs was 23.5 mV, indicated their good stability. In vitro picroliv
release from picroliv-loaded PLA NPs showed an initial burst release followed by slow and sustained release. The
efficacy of picroliv-loaded PLA NPs was assessed against KB cell lines. Blank PLA NPs showed no cytotoxicity on KB
cells. The picroliv-loaded PLA NPs showed more cytotoxic activity on KB cells as compared to the pure drug. Hence,
the developed picroliv nanoformulation would find potential application in pharma-sector.

1 Introduction The solvent evaporation method was used for encapsulating

There are several plant-derived natural products possessing many
pharmaceutical and therapeutic properties, obtained from the
underground parts of the plant. Picroliv (Fig. 1a) or kutkin of
Picrorhiza kurroa, is one such compound possessing
hepatoprotective, immunomudulatory and antiviral activities [1]. It
is also an antioxidant [2], antiallergic [3], antidiabetic [4],
anticarcinogenic [5] and superoxide scavenging [6] in nature.
Picroliv, a mixture of picroside I and kutkoside is isolated from
the rhizome of P. kurroa [7]. In spite of its wide pharmacological
properties, use of picroliv in pharmaceutical eld is limited due to
its poor aqueous solubility [8]. There are similar other
phytochemicals like curcumin and ellagic acid, whose use as a
drug is limited because of their poor aqueous solubility and
bioavailability. The sensitivity to light and temperature is also
reported to be responsible for reducing efcacy of such molecules [9].
As reported in the literature that the use of nanoparticles (NPs) as a
physical approach helps to improve the pharmacodynamic and
pharmacokinetic properties of various drugs. The therapeutic index
of various water soluble/insoluble bioactive molecules can be
increased by improving their bioavailability, retention time and
solubility by encapsulating them in the biodegradable polymers.
The high drug loading, sustained drug release, increased
persistence inside the blood, and site-specic targeting can be
achieved by encapsulation of the drugs into NPs [10, 11].
Towards this approach, we have documented the improved
aqueous solubility and bioavailability of a hydrophobic antioxidant
quercetin molecule through encapsulation on poly lactic acid
(PLA) NPs [12]. Biodegradable PLA NPs have been extensively
used for improving the physicochemical properties of many useful
small molecules such as savoxepine, progesterone and
amphotericin B [13]. PLA NPs offer advantages such as
biocompatibility, biodegradability, sustained release, strong
mechanical strength and safety and is thus widely used for
nanoencapsulation [14]. In this study, we have tested the
feasibility of encapsulating plant isolated picroliv in PLA NPs.
picroliv in PLA NPs. The picroliv-loaded PLA NPs were
characterised for size and morphology. The surface charge of
picroliv-loaded PLA NPs was measured by Zeta sizer.
Encapsulation efciency and in vitro release of picroliv-loaded
PLA NPs was also quantied by high-performance liquid
chromatography (HPLC). The efcacy analysis of pure picroliv,
blank PLA NPs and picroliv-loaded PLA NPs was carried out
against KB cell lines.
2 Materials
Pircoliv was isolated from the rhizome of P. kurroa at CSIR-IHBT,
Palampur. Dichloromethane (DCM) was purchased from Merck,
India. Poly-D,L-lactide (PLA) with molecular weight of 75,000
120,000, poly(vinyl alcohol) (PVA), HPLC grade acetonitrile,
water, triuoroacetic acid were purchased from Sigma-Aldrich. All
the solutions were prepared using water ltered through a Milli-Q
water system (Millipore, Bedford, MA). Human epidermoid
carcinoma cell line (KB) was obtained from National Centre for
Cell Science (NCCS), Pune. The cells were grown in Dulbeccos
Modied Eagle Medium (DMEM) (Invitrogen Biosciences, India),
supplemented with 10% heat-inactivated fetal bovine serum
(Invitrogen Biosciences, India) and 1% antibiotic antimycotic
solution (Ref. No. 15240-062, Invitrogen Biosciences, India). The
cells were maintained at 37 C in 5% CO2 incubator [15].
3 Experimental
3.1 Synthesis of picroliv-loaded PLA NPs
The solvent evaporation method was used for synthesis of blank
PLA NPs and picroliv-loaded PLA NPs. Different concentrations
of polymer (50150 mg), surfactant (15%) and the bioactive
molecule (210 mg) were used for the optimisation. Best results

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were obtained with the following combinations and used in this
study for synthesis. PLA (50 mg) and picroliv (5 mg) was added
to 2 ml DCM and sonicated (Sonics Vibra cell) at 40% amplitude
for 30 s in pulsed mode at room temperature. The oil in water
(o/w) emulsion was formed by adding 4 ml of 3% (w/v) PVA
solution and again sonicated. The emulsion was further diluted to
80 ml with 0.1% of PVA. Under partial vacuum conditions, the
organic phase (DCM) was evaporated using a rotatory evaporator
for 20 min. The NPs were then separated by centrifugation
(13,500 rpm, 20 min, 10 C) and washed for ve times with
distilled water. The same process was applied for the synthesis of
blank PLA NPs. The puried NPs were lyophilised and were kept
at 4 C for future use.

3.2 Characterisation by UVvis spectroscopy

UVvis spectrophotometer (Nanodrop ND-1000) with path length of

1 mm and 2048 element linear silicon CCD array detector was used
to obtain UVvis spectra. The spectroscopic scan of blank PLA NPs,
picroliv-loaded PLA NPs, supernatant of picroliv-loaded PLA NPs
and pure picroliv (0.625 g/ml) was performed at wavelength 220
700 nm to check the picroliv encapsulation in PLA NPs

3.3 Atomic force microscopy

To study the surface morphology by atomic force microscopy (AFM,

Nanoscope-III, Veeco Instruments, Singapore), the samples were
prepared by placing the aqueous solution of picroliv-loaded PLA
NPs and blank PLA NPs on a freshly cleaved clean glass surface
followed by drying at room temperature. The images were
captured in tapping mode using a silicon probe cantilever at a
scanning rate of 1 Hz. To conrm the reproducibility of results, a
minimum of ten images of each sample in ve replicates were taken.
3.4 Transmission electron microscopy (TEM)
The TEM was used to determine the shape and size of the
synthesised NPs. The aqueous suspension of picroliv-loaded PLA
NPs and blank PLA NPs was placed on copper grid and then
negatively stained with 2% (w/v) ammonium molybdate solution.
The images were obtained with a Tecnai, Twin 200 KV (FEI,
Netherlands) at a bias voltage of 200 kV.
3.5 Zeta potential analysis
To measure the surface charge and stability of NPs in the aqueous
suspension, zeta potential measurements of picroliv-loaded PLA
and blank PLA NPs were carried out using Zeta sizer (Nano ZS;
Malvern Instruments Ltd.). Approximately 1 ml of aqueous
suspensions of blank PLA NPs and picroliv-loaded PLA was put
into disposable zeta potential cells for the estimation of surface
charge of NPs.
3.6 Fourier transform infrared spectroscopy (FTIR)
FTIR spectroscopy (Nicolet 5700, Thermo, USA) was used to
conrm the chemical functional groups present on the surface of
picroliv and PLA polymer showing their interactions with each
other. The samples were analysed using KBr pellet. The FTIR
spectra of the blank PLA NPs, picroliv-loaded PLA NPs and pure
picroliv were recorded in the scanning range of 4004000 cm1 at
1 cm1 resolution.
3.7 Determination of encapsulation efficiency (EE) using
HPLC
A HPLC system ( Waters) equipped with UV-detector was employed
for determining the EE of picroliv-loaded PLA NPs. The mobile
phase consists of acetonitrile and water (25: 75) using C-18
column (150 mm 4.6 mm, 5 m size) as a stationary phase,
Fig. 1 Chemical structure of
a Picroliv
b UVvis spectrum of blank PLA NPs, picroliv-loaded PLA NPs, supernatant of
picroliv-loaded PLA NPs and pure picroliv
maintained at 25 C with a ow rate of 1 ml/min at a detection
wavelength of 272 nm. 20 l of supernatant of picroliv-loaded
PLA NPs as well as blank PLA NPs was ltered through 0.22 m
lter and directly injected into the HPLC column. The per cent of
picroliv EE (%) analysis was expressed as the percentage of drug
in the synthesised NPs with respect to initial amount (mg) used for
formulation of NPs. The standard curve was drawn by preparing
different amount of picroliv (0.50.015 mg/ml) versus peak area of
the eluted peak and was found to be linear with a correlation
coefcient of 0.999. The area of eluted peak of supernatants of
picroliv-loaded PLA NPs and blank PLA NPs were integrated and
used for picroliv quantication in picroliv-loaded PLA NPs. The
EE and the actual molecule loading were calculated using the
formula


amount of entrapped picroliv
%EE = 100 (1)
Total amount of picroliv in the formulation


amount of picroliv entrapped
Picroliv loading(%) = 100
mass of nanoparticles recovered
(2)
3.8 In vitro picroliv release assay
The in vitro release of picroliv from picroliv-loaded PLA NPs was
carried out at 37 C by incubating 10 mg of NPs in 10 ml of
phosphate buffer saline (PBS) buffer (0.1 M, pH 7.4). The NPs
suspension was continuously stirred in a thermostat (50 rpm) at
37 C. At regular time intervals (0, 0.5, 2, 4, 6, 8, 12 and 24 h),
1 ml of the sample was removed from the buffer solution. The
sample was centrifuged at 8000 rpm for 15 min. The collected
supernatant was lyophilised and dissolved in l ml of acetonitrile:
water mixture (25:75) to calculate the picroliv release using the
HPLC method.

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3.9 Cytotoxicity evaluation of picroliv and
picroliv-loaded PLA NPs by sulphorodamine B (SRB)
assay
The viable KB cells were incubated at a density of 20,000 cells/well
in a 96-well microtitre plate with 100 l of complete medium. Pure
picroliv, blank PLA NPs and picroliv-loaded PLA NPs at three
different concentrations (10, 25 and 50 g/ml) were added in 100
l of complete media. Vinblastine (1 M) was used as positive
control, whereas cells alone supplemented with complete medium
were used as negative control. Plates were incubated at 37 C for
24, 48 and 72 h in a CO2 incubator. After incubation period, 50 l
of 50% trichloroacetic acid was added to each well and the
microtitre plate was kept at 4 C for 1 h. The plate was icked,
washed ve times with water and then air-dried. Subsequently,
100 l SRB solution was added and incubated for 30 min at room
temperature. After incubation, the plate was washed ve times
with 1% acetic acid, air dried and was added with 10 mM tris base
(Sigma Aldrich, India). The absorbance was measured at 540 nm
using microplate reader (BioTeK Synergy H1 Hybrid Reader) [16].
3.10 Statistical analysis
All experiments were carried out in triplicate. Statistical analysis was
done using Microsoft excel. All results are expressed as mean
standard deviation (SD).
Fig. 2 AFM images of
a Blank PLA NPs
b Picroliv-loaded PLA NPs
c TEM images of blank PLA NPs
d Picroliv-loaded PLA NPs, HR-TEM (inset)
AFM images of air-dried NPs were captured on Nanscope-III in the tapping mode, scale bar 4.00 m. For TEM analysis, a drop of NPs was placed on copper grid and negatively
stained with 3% ammonium molybdate. Images were recorded on Tecnai T20 Twin TEM at 200 kV
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4 Results and discussion
4.1 Synthesis of picroliv-loaded PLA NPs
Picroliv-loaded PLA NPs were synthesised by a solvent evaporation
method [17]. The method is based on the emulsication of organic
solution of a polymer in an aqueous phase followed by the
evaporation of organic solvent (DCM) leading to the precipitation
of the polymer as NPs. As picroliv is hydrophobic in nature, this
method was chosen to encapsulate picroliv into PLA [18].
Entrapment of many water insoluble drugs like Diazepam,
Hydrocortisone into PLA polymer using solvent evaporation
method has been reported [19]. PLA NPs have earlier been used as
carriers for hydrophobic molecules [20, 21]. In NPs synthesis,
surfactants play important role in stabilisation of the emulsions
formed. At higher concentration (>2.5%), there occurs an increase
in viscosity which provides an increased surfactant activity of PVA
leading to the formation of stable emulsion containing smaller-sized
NPs [22]. The solvent evaporation method results in the formation
of monodisperse emulsion. Monodisperse emulsion has a more
dened behaviour and release pattern of entrapped active agent than
polydisperse emulsion [23]. The UVvis spectra of picroliv-loaded
PLA NPs represents a characteristic peaks like turbidity indicating
the formation of NPs (Fig. 1b). The spectrum of picroliv is quite
different after inclusion in PLA NPs. Free picroliv powder showed
an absorption band at 272 nm, whose intensity decreased
signicantly in the spectrum of picroliv-loaded NPs (Fig. 1b).
3
4.2 Characterisation of developed picroliv-loaded
PLA NPs
The picroliv-loaded PLA NPs were characterised in terms of mean
size, morphology and size distribution. AFM measurement is also
an effective method to provide the surface morphology and more
accurate size as well as size distribution. AFM images of blank
PLA NPs and picroliv-loaded PLA NPs showed the NPs of uniform
and almost spherical in shape. AFM analysis also revealed that the
size of blank PLA NPs (Fig. 2a) was larger than that of
picroliv-loaded PLA NPs (Fig. 2b). The results indicated that the
picroliv-loaded PLA NPs were smaller in size than blank PLA NPs.
The TEM was used for nal characterisation of size and shape of
picroliv-loaded PLA NPs. The average size of blank PLA and
picroliv-loaded PLA NPs consecutively synthesised by the same
method was found to be 220 18 and 182 20 nm, respectively.
TEM image of blank PLA (Fig. 2c) and picroliv-loaded PLA NPs
(Fig. 2d) showed that the NPs were spherical in shape. However,
narrow range of size distributions was observed for the synthesised
NPs and exhibited a relatively monodisperse distribution. Similarly,
smaller-sized quercetin-loaded PLA NPs compared to the blank
PLA NPs have been observed in our previous study [12].
The size of NPs is an important factor to decide the cellular
uptake, intracellular fate and release of encapsulated molecule
[24]. Moreover, polymeric particles can be administered in various
Fig. 3 FTIR spectroscopy of
a Blank PLA NPs
b Picroliv-loaded PLA NPs
c Pure picroliv
FTIR spectra of blank and picroliv-loaded PLA NPs was recorded on Thermo Nicolet 6700 FTIR spectroscopy (Thermo, USA)
4 & The Institution of Engineering and Technology 2015
dosage forms, including intravenous and oral routes to increase the
bioavailability and to reduce the associated adverse effects [25, 26]
Narrowly distributed stable polymeric NPs with an average
diameter less than 200 nm have been reported ideal as they can
easily pass through the blood capillary [27, 28]. Hence, smaller
than 200 nm sized picroliv-loaded PLA NPs developed in this
study could be ideal for effective use.
4.3 Zeta potential indicated the development of stable
picroliv-loaded PLA NPs
Zeta potential of NPs is an important criterion for determining their
stability [23]. The zeta potential of blank PLA NPs was 3.60 mV
(Fig. S1a) and picroliv-loaded PLA NPs was 23.5 mV (Fig.
S1b). The zeta potential value indicated that encapsulation of
picroliv in PLA NPs has increased their stability. The extent of
phagocytosis also increases with increase in zeta potentials [29].
Negatively charged particles get adsorbed at cationic sites on cell
membrane through electrostatic interactions, leading to localised
charge neutralisation and further membrane bending to enhance
endocytosis [30]. As the surface charge of endothelial cells is
negative and the picroliv-loaded PLA NPs also have negative
surface charge, therefore the half-life of negatively charged NPs
would be more in the circulating blood.
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Table 1 FTIR peaks of picroliv-loaded PLA NPs and pure picroliv
Sl. no. Frequency band, cm1 Bond Functional group
1 3416 OH stretch alcohol
2 1626 C=C stretch alkenes
3 1756 C=O stretch esters
4.4 FTIR analysis documented the safe encapsulation of
picroliv
FTIR analysis is one of the important and efcient methods used to
analyse the intermolecular interaction of the encapsulated chemicals
into the polymer [29]. The FTIR spectra of blank PLA NPs,
picroliv-loaded PLA NPs and pure picroliv are shown in Fig. 3
and Table 1. The characteristic peaks of picroliv for a hydroxyl
group (3416 cm1), a C=C double bond (1626 cm1) and an ester
functional group (1756 cm1) were present in pure and
encapsulated picroliv. These peaks were absent in FTIR spectra of
blank PLA NPs. Thus, the presence of the characteristic peak
of picroliv on picroliv-loaded NPs conrms the encapsulation of
picroliv on PLA NPs. Also the similar peaks in pure picroliv and
picroliv-loaded PLA NPs documented no alteration in the
chemical structure of picroliv on its encapsulation in PLA.
4.5 Encapsulation efficiency of picroliv-loaded PLA NPs
To achieve the maximum EE and to minimise the loss of molecule
loading in NPs, various parameters like polymer concentration and
amount of surfactant used need to be optimised [21].
Encapsulation efciency of picroliv-loaded PLA NPs was
quantied using the validated HPLC method [31]. The calibration
curve was generated with different concentrations of pure picroliv
(Fig. S2). The EE of picroliv-loaded PLA NPs was found to be
12.5%. This was determined by using the pre-calibration equation
y = 1E + 07x + 95790 where y is the arbitrary area of the
picroliv-eluted peak and x is the concentration of picroliv in mg.
The difference of the picroliv amount in initial and nal (after NPs
separation) solutions was the basis for determination of
encapsulation efciency. The loading of hydrophobic picroliv
molecule was found to be 3.1% on to the PLA NPs. The
interaction of lactides and ester groups of polymer with
hydrophobic molecule ultimately determines the solubility of
molecule encapsulated in NPs [32]. The EE of NPs depends on
the synthesis method, type of NPs and interaction of bioactive
molecules with NPs [28]. Steric hindrance due to bulky structure
of picroliv might be responsible for its low EE [28].
Fig. 4 Release prole of picroliv from picroliv-loaded PLA NPs determined
by HPLC. Release curve was obtained by plotting % of picroliv released
versus time
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Fig. 5 Cytotoxicity of pure picroliv, picroliv-loaded PLA NPs and blank
PLA NPs against KB cells after 24, 48 and 72 h of incubation
4.6 Picroliv-loaded PLA NPs show slow and sustained in
vitro release
In vitro release studies were carried out in PBS to mimic
physiological conditions. Release studies are also important to
know the encapsulation or adsorption of bioactive molecules on
NPs. The percentage of picroliv released from the picroliv-loaded
PLA NPs at different time intervals was analysed. The in vitro
release study was carried out in PBS to mimic physiological
condition of the living organism. The release rate of picroliv was
burst release (023.4%) within rst 2 h, after that sustained release
of the entrapped picroliv was observed (Fig. 4). The maximum
release of picroliv was observed to be 46.9% after 24 h (Fig. 4).
The rapid initial burst release of picroliv from picroliv-loaded PLA
NPs might be because of the surface release of picroliv which is
generally adsorbed on or close to the surface of NPs. Thereafter
slow and sustained release of picroliv could be attributed to the
polymer around the molecule. Thus, in vitro release of picroliv
from NPs is the cumulative effect of diffusion, dissolution and
erosion [3334]. It has been earlier reported that drug loaded by
the incorporation method have relatively small burst effect and
better sustained release characteristics [12]. Release studies reveal
that picroliv-loaded PLA NPs can be used for nanomedicine
formulation with controlled and sustained release. Similar
sustained release of quercetin from the PLA NPs has been
reported [12]. Being amenable to surface modication,
picroliv-loaded PLA NPs can also be used for targeted delivery.
4.7 Cytotoxicity of picroliv and picroliv-loaded PLA NPs
The cytotoxicity of pure picroliv and picroliv-loaded PLA NPs was
tested in vitro against KB cells using three different concentrations
(10, 25 and 50 g/ml) by SRB assay [28] for 24, 48 and 72 h
(Fig. 5). The pure picroliv did not show signicant activity at any
concentration at 24, 48 and 72 h (Fig. 5), while picroliv-loaded
PLA NPs showed remarkable activity at 10, 25 and 50 g/ml
(79.2 0.3, 67.4 2.0 and 65.8 1.8, respectively) at 24 h. The
growth inhibitory activity of picroliv-loaded PLA NPs was higher
than pure picroliv against KB cells. Data has indicated that
bioavailability of picroliv is increased with nanoencapsulation into
PLA NPs. Similar results were obtained for nanoencapsulated
podophyllotoxin and etoposide on PLA NPs [28]. This has
conrmed that therapeutic action of picroliv-loaded PLA NPs was
more signicant than the pure picroliv.
5 Summary
Picroliv was successfully encapsulated on PLA NPs. The average
size of blank PLA NPs and picroliv-loaded PLA NPs was 220
18 and 182 20 nm, respectively. Encapsulation efciency of
picrolivPLA NPs was found to be 12.5%. The release rate of
picroliv was burst release (up to 023% within initial 2 h), after
which sustained release of the entrapped picroliv was observed.
The release of picroliv observed after 24 h of incubation was
found to be 46.9%. Rapid initial release was normally attributed to
the fraction of picroliv which was adsorbed on or close to the
5
surface of the NPs. The cytotoxicity assay against KB cell lines
conrmed that the blank PLA NPs were safe and showed no
activity. The activity of picroliv was enhanced after loading on
PLA NPs. The picroliv-loaded PLA NPs showed better activity on
KB cells than the pure picroliv. The activity was attributed to
picroliv as blank PLA NPs were non-toxic to KB cells. Slow and
controlled release make it suitable candidate for development of
nanoformulation of picroliv.
6 Acknowledgments
Authors acknowledge the Director of CSIR-IHBT for his valuable
guidance and support. Financial assistance in the form of project
grant BSC-112 from the Council of Scientic and Industrial
Research (CSIR), Government of India is truly acknowledged.
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