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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

Anika Guliani 1, 2*, Avnesh Kumari 1*, Dharmesh Kumar 3, Sudesh Kumar Yadav 1, 2
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur-176061, HP, India
Acedemy of Scientific and Innovative Research, New Delhi, India
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.

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

picroliv in PLA NPs. The picroliv-loaded PLA NPs were
There are several plant-derived natural products possessing many characterised for size and morphology. The surface charge of
pharmaceutical and therapeutic properties, obtained from the picroliv-loaded PLA NPs was measured by Zeta sizer.
underground parts of the plant. Picroliv (Fig. 1a) or kutkin of Encapsulation efciency and in vitro release of picroliv-loaded
Picrorhiza kurroa, is one such compound possessing PLA NPs was also quantied by high-performance liquid
hepatoprotective, immunomudulatory and antiviral activities [1]. It chromatography (HPLC). The efcacy analysis of pure picroliv,
is also an antioxidant [2], antiallergic [3], antidiabetic [4], blank PLA NPs and picroliv-loaded PLA NPs was carried out
anticarcinogenic [5] and superoxide scavenging [6] in nature. against KB cell lines.
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 2 Materials
its poor aqueous solubility [8]. There are similar other
phytochemicals like curcumin and ellagic acid, whose use as a Pircoliv was isolated from the rhizome of P. kurroa at CSIR-IHBT,
drug is limited because of their poor aqueous solubility and Palampur. Dichloromethane (DCM) was purchased from Merck,
bioavailability. The sensitivity to light and temperature is also India. Poly-D,L-lactide (PLA) with molecular weight of 75,000
reported to be responsible for reducing efcacy of such molecules [9]. 120,000, poly(vinyl alcohol) (PVA), HPLC grade acetonitrile,
As reported in the literature that the use of nanoparticles (NPs) as a water, triuoroacetic acid were purchased from Sigma-Aldrich. All
physical approach helps to improve the pharmacodynamic and the solutions were prepared using water ltered through a Milli-Q
pharmacokinetic properties of various drugs. The therapeutic index water system (Millipore, Bedford, MA). Human epidermoid
of various water soluble/insoluble bioactive molecules can be carcinoma cell line (KB) was obtained from National Centre for
increased by improving their bioavailability, retention time and Cell Science (NCCS), Pune. The cells were grown in Dulbeccos
solubility by encapsulating them in the biodegradable polymers. Modied Eagle Medium (DMEM) (Invitrogen Biosciences, India),
The high drug loading, sustained drug release, increased supplemented with 10% heat-inactivated fetal bovine serum
persistence inside the blood, and site-specic targeting can be (Invitrogen Biosciences, India) and 1% antibiotic antimycotic
achieved by encapsulation of the drugs into NPs [10, 11]. solution (Ref. No. 15240-062, Invitrogen Biosciences, India). The
Towards this approach, we have documented the improved cells were maintained at 37 C in 5% CO2 incubator [15].
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 3 Experimental
used for improving the physicochemical properties of many useful
small molecules such as savoxepine, progesterone and 3.1 Synthesis of picroliv-loaded PLA NPs
amphotericin B [13]. PLA NPs offer advantages such as
biocompatibility, biodegradability, sustained release, strong The solvent evaporation method was used for synthesis of blank
mechanical strength and safety and is thus widely used for PLA NPs and picroliv-loaded PLA NPs. Different concentrations
nanoencapsulation [14]. In this study, we have tested the of polymer (50150 mg), surfactant (15%) and the bioactive
feasibility of encapsulating plant isolated picroliv in PLA NPs. 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 Fig. 1 Chemical structure of
NPs and blank PLA NPs on a freshly cleaved clean glass surface a Picroliv
followed by drying at room temperature. The images were b UVvis spectrum of blank PLA NPs, picroliv-loaded PLA NPs, supernatant of
captured in tapping mode using a silicon probe cantilever at a picroliv-loaded PLA NPs and pure picroliv
scanning rate of 1 Hz. To conrm the reproducibility of results, a
minimum of ten images of each sample in ve replicates were taken.
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
3.4 Transmission electron microscopy (TEM) 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
The TEM was used to determine the shape and size of the
picroliv EE (%) analysis was expressed as the percentage of drug
synthesised NPs. The aqueous suspension of picroliv-loaded PLA
in the synthesised NPs with respect to initial amount (mg) used for
NPs and blank PLA NPs was placed on copper grid and then
formulation of NPs. The standard curve was drawn by preparing
negatively stained with 2% (w/v) ammonium molybdate solution.
different amount of picroliv (0.50.015 mg/ml) versus peak area of
The images were obtained with a Tecnai, Twin 200 KV (FEI,
the eluted peak and was found to be linear with a correlation
Netherlands) at a bias voltage of 200 kV.
coefcient of 0.999. The area of eluted peak of supernatants of
picroliv-loaded PLA NPs and blank PLA NPs were integrated and
3.5 Zeta potential analysis used for picroliv quantication in picroliv-loaded PLA NPs. The
EE and the actual molecule loading were calculated using the
To measure the surface charge and stability of NPs in the aqueous formula
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 amount of entrapped picroliv
suspensions of blank PLA NPs and picroliv-loaded PLA was put %EE = 100 (1)
Total amount of picroliv in the formulation
into disposable zeta potential cells for the estimation of surface
charge of NPs.
amount of picroliv entrapped
3.6 Fourier transform infrared spectroscopy (FTIR) Picroliv loading(%) = 100
mass of nanoparticles recovered
FTIR spectroscopy (Nicolet 5700, Thermo, USA) was used to (2)
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 3.8 In vitro picroliv release assay
spectra of the blank PLA NPs, picroliv-loaded PLA NPs and pure
picroliv were recorded in the scanning range of 4004000 cm1 at The in vitro release of picroliv from picroliv-loaded PLA NPs was
1 cm1 resolution. 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
3.7 Determination of encapsulation efficiency (EE) using suspension was continuously stirred in a thermostat (50 rpm) at
HPLC 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
A HPLC system ( Waters) equipped with UV-detector was employed sample was centrifuged at 8000 rpm for 15 min. The collected
for determining the EE of picroliv-loaded PLA NPs. The mobile supernatant was lyophilised and dissolved in l ml of acetonitrile:
phase consists of acetonitrile and water (25: 75) using C-18 water mixture (25:75) to calculate the picroliv release using the
column (150 mm 4.6 mm, 5 m size) as a stationary phase, HPLC method.

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3.9 Cytotoxicity evaluation of picroliv and 4 Results and discussion
picroliv-loaded PLA NPs by sulphorodamine B (SRB)
assay 4.1 Synthesis of picroliv-loaded PLA NPs

The viable KB cells were incubated at a density of 20,000 cells/well Picroliv-loaded PLA NPs were synthesised by a solvent evaporation
in a 96-well microtitre plate with 100 l of complete medium. Pure method [17]. The method is based on the emulsication of organic
picroliv, blank PLA NPs and picroliv-loaded PLA NPs at three solution of a polymer in an aqueous phase followed by the
different concentrations (10, 25 and 50 g/ml) were added in 100 evaporation of organic solvent (DCM) leading to the precipitation
l of complete media. Vinblastine (1 M) was used as positive of the polymer as NPs. As picroliv is hydrophobic in nature, this
control, whereas cells alone supplemented with complete medium method was chosen to encapsulate picroliv into PLA [18].
were used as negative control. Plates were incubated at 37 C for Entrapment of many water insoluble drugs like Diazepam,
24, 48 and 72 h in a CO2 incubator. After incubation period, 50 l Hydrocortisone into PLA polymer using solvent evaporation
of 50% trichloroacetic acid was added to each well and the method has been reported [19]. PLA NPs have earlier been used as
microtitre plate was kept at 4 C for 1 h. The plate was icked, carriers for hydrophobic molecules [20, 21]. In NPs synthesis,
washed ve times with water and then air-dried. Subsequently, surfactants play important role in stabilisation of the emulsions
100 l SRB solution was added and incubated for 30 min at room formed. At higher concentration (>2.5%), there occurs an increase
temperature. After incubation, the plate was washed ve times in viscosity which provides an increased surfactant activity of PVA
with 1% acetic acid, air dried and was added with 10 mM tris base leading to the formation of stable emulsion containing smaller-sized
(Sigma Aldrich, India). The absorbance was measured at 540 nm NPs [22]. The solvent evaporation method results in the formation
using microplate reader (BioTeK Synergy H1 Hybrid Reader) [16]. 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
3.10 Statistical analysis PLA NPs represents a characteristic peaks like turbidity indicating
the formation of NPs (Fig. 1b). The spectrum of picroliv is quite
All experiments were carried out in triplicate. Statistical analysis was different after inclusion in PLA NPs. Free picroliv powder showed
done using Microsoft excel. All results are expressed as mean an absorption band at 272 nm, whose intensity decreased
standard deviation (SD). signicantly in the spectrum of picroliv-loaded NPs (Fig. 1b).

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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4.2 Characterisation of developed picroliv-loaded dosage forms, including intravenous and oral routes to increase the
PLA NPs bioavailability and to reduce the associated adverse effects [25, 26]
Narrowly distributed stable polymeric NPs with an average
The picroliv-loaded PLA NPs were characterised in terms of mean diameter less than 200 nm have been reported ideal as they can
size, morphology and size distribution. AFM measurement is also easily pass through the blood capillary [27, 28]. Hence, smaller
an effective method to provide the surface morphology and more than 200 nm sized picroliv-loaded PLA NPs developed in this
accurate size as well as size distribution. AFM images of blank study could be ideal for effective use.
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 4.3 Zeta potential indicated the development of stable
picroliv-loaded PLA NPs (Fig. 2b). The results indicated that the picroliv-loaded PLA NPs
picroliv-loaded PLA NPs were smaller in size than blank PLA NPs.
The TEM was used for nal characterisation of size and shape of Zeta potential of NPs is an important criterion for determining their
picroliv-loaded PLA NPs. The average size of blank PLA and stability [23]. The zeta potential of blank PLA NPs was 3.60 mV
picroliv-loaded PLA NPs consecutively synthesised by the same (Fig. S1a) and picroliv-loaded PLA NPs was 23.5 mV (Fig.
method was found to be 220 18 and 182 20 nm, respectively. S1b). The zeta potential value indicated that encapsulation of
TEM image of blank PLA (Fig. 2c) and picroliv-loaded PLA NPs picroliv in PLA NPs has increased their stability. The extent of
(Fig. 2d) showed that the NPs were spherical in shape. However, phagocytosis also increases with increase in zeta potentials [29].
narrow range of size distributions was observed for the synthesised Negatively charged particles get adsorbed at cationic sites on cell
NPs and exhibited a relatively monodisperse distribution. Similarly, membrane through electrostatic interactions, leading to localised
smaller-sized quercetin-loaded PLA NPs compared to the blank charge neutralisation and further membrane bending to enhance
PLA NPs have been observed in our previous study [12]. endocytosis [30]. As the surface charge of endothelial cells is
The size of NPs is an important factor to decide the cellular negative and the picroliv-loaded PLA NPs also have negative
uptake, intracellular fate and release of encapsulated molecule surface charge, therefore the half-life of negatively charged NPs
[24]. Moreover, polymeric particles can be administered in various would be more in the circulating blood.

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)

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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

Fig. 5 Cytotoxicity of pure picroliv, picroliv-loaded PLA NPs and blank
FTIR analysis is one of the important and efcient methods used to PLA NPs against KB cells after 24, 48 and 72 h of incubation
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 4.6 Picroliv-loaded PLA NPs show slow and sustained in
and Table 1. The characteristic peaks of picroliv for a hydroxyl vitro release
group (3416 cm1), a C=C double bond (1626 cm1) and an ester
functional group (1756 cm1) were present in pure and In vitro release studies were carried out in PBS to mimic
encapsulated picroliv. These peaks were absent in FTIR spectra of physiological conditions. Release studies are also important to
blank PLA NPs. Thus, the presence of the characteristic peak know the encapsulation or adsorption of bioactive molecules on
of picroliv on picroliv-loaded NPs conrms the encapsulation of NPs. The percentage of picroliv released from the picroliv-loaded
picroliv on PLA NPs. Also the similar peaks in pure picroliv and PLA NPs at different time intervals was analysed. The in vitro
picroliv-loaded PLA NPs documented no alteration in the release study was carried out in PBS to mimic physiological
chemical structure of picroliv on its encapsulation in PLA. 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
4.5 Encapsulation efficiency of picroliv-loaded PLA NPs 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
To achieve the maximum EE and to minimise the loss of molecule NPs might be because of the surface release of picroliv which is
loading in NPs, various parameters like polymer concentration and generally adsorbed on or close to the surface of NPs. Thereafter
amount of surfactant used need to be optimised [21]. slow and sustained release of picroliv could be attributed to the
Encapsulation efciency of picroliv-loaded PLA NPs was polymer around the molecule. Thus, in vitro release of picroliv
quantied using the validated HPLC method [31]. The calibration from NPs is the cumulative effect of diffusion, dissolution and
curve was generated with different concentrations of pure picroliv erosion [3334]. It has been earlier reported that drug loaded by
(Fig. S2). The EE of picroliv-loaded PLA NPs was found to be the incorporation method have relatively small burst effect and
12.5%. This was determined by using the pre-calibration equation better sustained release characteristics [12]. Release studies reveal
y = 1E + 07x + 95790 where y is the arbitrary area of the that picroliv-loaded PLA NPs can be used for nanomedicine
picroliv-eluted peak and x is the concentration of picroliv in mg. formulation with controlled and sustained release. Similar
The difference of the picroliv amount in initial and nal (after NPs sustained release of quercetin from the PLA NPs has been
separation) solutions was the basis for determination of reported [12]. Being amenable to surface modication,
encapsulation efciency. The loading of hydrophobic picroliv picroliv-loaded PLA NPs can also be used for targeted delivery.
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 4.7 Cytotoxicity of picroliv and picroliv-loaded PLA NPs
molecule encapsulated in NPs [32]. The EE of NPs depends on
the synthesis method, type of NPs and interaction of bioactive The cytotoxicity of pure picroliv and picroliv-loaded PLA NPs was
tested in vitro against KB cells using three different concentrations
molecules with NPs [28]. Steric hindrance due to bulky structure
(10, 25 and 50 g/ml) by SRB assay [28] for 24, 48 and 72 h
of picroliv might be responsible for its low EE [28].
(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.
Fig. 4 Release prole of picroliv from picroliv-loaded PLA NPs determined The release of picroliv observed after 24 h of incubation was
by HPLC. Release curve was obtained by plotting % of picroliv released found to be 46.9%. Rapid initial release was normally attributed to
versus time the fraction of picroliv which was adsorbed on or close to the

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surface of the NPs. The cytotoxicity assay against KB cell lines 15 Puc, R.M., Dzul, J.C., Fuentes, E.C.: Bonellia albiora: a mayan medicinal plant
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6 & The Institution of Engineering and Technology 2015