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Methodology to nanoencapsulate hepatoprotectivecomponents from Picrorhiza kurroa asfood supplement

Dewei Jiaa, Indu Barwalb, Shloka Thakurb, Subhash C. Yadavb,n

aClarendon Laboratory, Physics Department, University of Oxford, OX1 3PU Oxford, UKbTERI-Deakin Nanobiotechnology Centre, The Energy and Resources Institute, Darbari Seth Block, IHC Complex,Lodhi Road, New Delhi 110003, India

a r t i c l e i n f o

Article history:

Received 5 August 2014

Received in revised form

18 October 2014

Accepted 23 October 2014

Available online 13 November 2014

Chemical compounds studied in this article:

Poly(D,L-lactide) (PubChem CID: 612)

Pluronic-F-68 (PubChem CID: 24751)

Methanol (PubChem CID: 887)

Acetone (PubChem CID: 180)

Picroside I (PubChem CID: 6440892)

Picroside II (PubChem CID: 9849283)

Sodium dihydrogen phosphate

(PubChem CID: 23672064)

Di-sodium hydrogen phosphate

(PubChem CID: 24203)

Water (PubChem CID: 962)

Phosphotungstic acid (PubChem CID:

16212977)

Keywords:

Heptoprotective

Picrorhiza kurroa

Nanoencapsulation

Polylactic acid

Pluronic-F-68

a b s t r a c t

Picrorhiza kurroa root and rhizome powder of extract is well-recognized neutraceutical for

healthy liver functioning. The major hepatoprotective components (picrosides I and II) of

P. kurroa extract shows poor intestinal absorption and poor bioavailability due to sparingly

aqueous solubility. To improve bioavailability of these biomolecules, a nanoformulation of this

extract has been developed with pluronic-F-68 copolymer based biodegradable PLA nanopar-

ticles by nanoprecipitation method. This method showed high encapsulation efficiency as

60.172.8% (for picroside I) and 67.277.4% (for picroside II). The hydrodynamic (by zeta size) and

actual size (by TEM) revealed homogenously rounded nanoparticles of size 174734 nm and

154712 nm respectively. This formulation showed an initial burst and half release up to

2773 h, followed by a sustained release (up to 210710 h). Nanoformulation of this traditional

herbal extract provides neutraceutical with value addition property for better hepato-protection

by enhancing intestinal absorption and bioavailability.

& 2014 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.fbio.2014.10.0052212-4292/& 2014 Elsevier Ltd. All rights reserved.

Abbreviations: HPLC, high performance liquid chromatography; DLS, dynamic light scattering; RP-HPLC, reverse phase high performance

liquid chromatography; PLA, polylactic acid; FTIR, Fourier transform infrared spectroscopy; TEM, transmission electron microscopynCorresponding author. Tel.: þ91 11 24682100x2610, mobile: þ91 9418096177; fax: þ91 11 24682144.E-mail addresses: subhashmbu@gmail.com, subhash.yadav@teri.res.in (S.C. Yadav).

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 2 8 – 3 5

1. Introduction

Picrorhiza kurroa (Pk), is a well-known herb native of Hima-layan high altitude (�3000 m) region. This is used as dietarysupplement to maintain healthy bile production and liverfunction by offering multiple pharmacological effects(Nadkarni & Nadkarni, 1976). P. kurroa is effectively recom-mended for the restoration of various liver related complica-tions such as jaundice, nausea anorexia, dyspepsia, viralhepatitis, periodic fevers (Handa, Sharma, & Chakravarti, 1986;Jeyakumar et al., 2008; Pade, 1957; Vaidya, Bhatia, Mehta, &Sheth, 1976; White & Humphrey, 1901), diabetes (Joy & Kuttan,1999), antineoplastic (Rajkumar, Guha, & Kumar, 2011) andliver chemo-preventive activities (Banerjee, Maity, Nag,Bandyopadhyay, & Chattopadhyay, 2008; Joy, Rajeshkumar,Kuttan, & Kuttan, 2000; Rajeshkumar & Kuttan, 2001). Kutkinis the active principal component (for liver protection) of P.kurroa extract and comprised of picrosides I and II glycoside(Fig. 1). The hepatoprotective function of kutkin is mainly due tothe inhibition of oxygen anions generation and scavenging freeradicals similar to superoxide dismutase, metal-ion chelators,xanthine oxidase inhibitors and anti-lipid peroxidative effect(Chander, Kapoor, & Dhawan, 1992; Chander, Singh, Visen,Kapoor, & Dhawan, 1998; Russo, Izzo, Cardile, Borrelli, &Vanella, 2001).

The mixture of Picrorhiza glycosides in alcoholic extract aremore potent hepatoprotective than single pure functionalcompound (Hussain, Shadma, Maksood, & Ansari, 2013). Dueto poor aqueous solubility and intestinal absorption (Li et al.,2012; Park, Lee, Oh, Lee, & Lee, 2009; Upadhyay, Dash,Anandjiwala, & Nivsarkar, 2013; Wang, Wang, Bligh, White,& White, 2004), this herbal neutraceutical was prescribed tobe given more frequently in higher quantity (3–5 g/day) (Bone,1995; Hussain et al., 2013; Upadhyay et al., 2013). Higherquantity shows activation of immune regulatory systems aswell as increase of effective cost (Ansari et al., 1988). This isone of the serious concerns that limits the frequent applica-tion of this herb in liver related diseases. Nanoencapsulationof complete herbal extract would enable us to reduce thedosage with better therapeutic potential by creating moresolubility and higher intestinal absorption. By nanoencapsu-lation, the active principal components are protected fromexternal conditions and are released later in a controlled way.The maximum efficacy could be achieved by nanoencapsula-tion for kutkin and dose dependent side-effects could bereduced simultaneously (Kumar et al., 2014). In this manu-script, we report the nanoencapsulation of alcoholic extractfrom dried and powdered rhizome of P. kurroa into pluronic-F-68-PLA nanoparticles.

2. Materials and methods

2.1. Materials

Poly-D,L-lactide (PLA) (MW¼75,000–120,000) and Pluronic-F-68(polyoxyethylene–polyoxypropylene block copolymer) were pur-chased from Sigma–Aldrich and used as received. HPLC grademethanol was procured from Merck Limited (India). All thesolutions were prepared using water filtered through a Milli-Qwater system (Millipore, Bedford, MA). Dried and powdered rootand rhizome of P. kurroa was provided by Institute of HimalayanBioresources Technology (IHBT) Palampur HP India.

2.2. Extraction of P. kurroa with methanol

The fine powder (10 g) of dried root and rhizome was soakedin 100 ml aqueous methanol (80% v/v) overnight in stirringcondition at room temperature. The clear supernatant wascollected by high speed centrifugation and filtered. Thissolution was stored at 4 1C for later use and was referred asP. kurroa extract in this manuscript. UV–vis spectroscopy scananalysis of 100 times diluted extract was performed to detectthe absorption wavelength maxima in 1 ml quartz cuvette.

2.3. Nanoencapsulation of P. kurroa extract

P. kurroa extract encapsulated PLA nanoparticles were preparedby using nanoprecipitation method. The methanol extract wassubjected for rotary evaporation using controlled temperatureand pressure. After the removal of methanol for 5–6 h dependingupon the quantity, the appropriate dilution of aqueous extractwere treated with appropriate amount of PF-68 to make it final0.5% w/v. In brief, 5 ml PLA (10mg/ml in acetone) was addeddrop wise into 20ml aqueous P. kurroa extract (after methanolremoval) containing PF-68 (0.5% w/v) with a constant stirring at590 rpm and dropping rate of one droplet (2 ml) per second. Thenewly formed nanoparticle suspension was allowed to completethe reaction for 30min before acetone was removed by rotaeva-poration. PLA nanoparticles (blank) were synthesized by sameprotocol. The resulting nanoparticles were collected by centrifu-gation at 16,000g for 10min and gently washed twice with milliQwater. The nanoparticles were stored at 4 1C until further use.

2.4. RP-HPLC profiling and encapsulation efficiency

HPLC (Shimadzu with photodiode array detector SPD-M20A)profiling was used to validate the encapsulation of P. kurroacomponents. Supernatant (after separation of complete nano-particles) of nanoencapsulated extract and PLA nanoparticles, as

Fig. 1 – Chemical structure of (a) picroside I and (b) picroside II.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 2 8 – 3 5 29

well as equimolar concentration of P. kurroa extract (initialconcentration) were filtered with 0.22 mm membrane. HPLCprofile of such filtered solution (20 ml injection) was experimen-tally determined with 60% (v/v) aqueous methanol (mobilephase) at 1ml/min isocratic flow rate. Analytical reverse-phaseC18 column (150mm�4.6mm, 5 mm size) at room temperature(pressure o350 psi) was used for HPLC profiling. Optimal detec-tion wavelength of 285 nm was decided with 3D data from aphotodiode array (PDA) detectors range. Linearity of the HPLCsystem, expressed as a function between extract amount andpeak area at 285 nm was tested and calibrated with a series ofsample from the original extract concentration (128 times dilu-tion). Encapsulation efficiency was determined as a function ofthe peak area/height corresponds to picrosides I and II at 285 nmas below:

Encapsulation efficiency %ð Þ ¼ Peak area of supernatantPeak area of P: kurroa extract

� 100

2.5. Zeta analysis of the nanoparticle system'selectrodynamics property

Zetasizer (Nano ZS, Malvern) with 5 mW He–Ne lasers wasused to determine the average hydrodynamic diameter and

zeta potential of the nanoparticle. The measurements wereperformed at 25 1C with an equilibration time for 120 s.Particle size was calculated with correlation functions fromscattered light collected by photomultiplier tubes. Averagediameter was yielded together with poly-dispersity index toindicate the homogeneity of particle system.

2.6. FTIR spectrophotometric characterization ofencapsulated nanoparticles

P. kurroa extract supernatant after nanoencapsulation, PLANPs and extract loaded PLA nanoparticles were lyophilizedovernight for Fourier transform infrared (FTIR) spectra ana-lysis. The FTIR spectra of lyophilized powders were recordedin the scanning range of 400–4000 cm�1 at 1 cm�1 resolutionwith Nicolet 6700 FTIR spectroscopy (Thermo Scientific, USA)using a Smart OMNI sampler Accessory.

2.7. Morphological characterization by transmissionelectron microscopy

Morphological analysis of extract encapsulated PLA nanopar-ticles and PLA nanoparticles were evaluated by TEM (TecnaiT20, FEI Company, 120 kV). Nanoparticle suspension of 10 ml

Fig. 2 – HPLC characterization of extract components: (a) response surface from the HPLC PDA 3D data and (b) elution profiledetected at 285 nm.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 2 8 – 3 530

on carbon coated copper grid was dried in desiccator andnegatively stained by 0.1% phosphotungstic acid. The imagewas taken at 3500� magnification on TEM mode.

2.8. In vitro release assay of P. kurroa extractencapsulated on PLA nanoparticles

P. kurroa extract encapsulated PLA nanoparticles (50 mg) wereincubated in 20ml 0.1 M PBS at pH 7.4 in mild stirring condi-tions at 37 1C. 1 ml of the sample was taken at previouslyselected time and centrifuged (19,000g for 10min) to removePLA nanoparticles. The supernatant was lyophilized, dissolvedin 1ml of 60% methanol, and centrifuged at 19,000g for 10 min.The release amount of picroside was relatively calculated basedon the peaks height/area of picrosides I and II at selected time.The picrosides I and II content in the supernatant wererelatively measured by taking peak height/area of validatedHPLC using same protocol as previously described. Release ratiowas subsequently decided as the ratio of released amount ofpicrosides I and II to total amount of picrosides I and II

encapsulated in nanoparticles, formulated as

Release ratio %ð Þ ¼ Released amount of picrosides I=II at time tTotal amount of encapsulated picrosides I=II in NP

� 100

The encapsulation and measurement were performed intriplicates and statistically represented as mean7SD.

3. Results and discussion

3.1. P. kurroa extract encapsulation on PLA nanoparticles

P. kurroa is widely accepted as dietary supplement to main-tain healthy functioning of liver and mainly recommended touse in the form of dried powder/syrup (Kant, Walia,Agnihotri, Pathania, & Singh, 2013). The active hepatoprotec-tive component was best extracted in alcoholic extract at50 1C (Kalaivani, Rajasekaran, & Mathew, 2010). Alcoholicextract of P. kurroa contains many potent antioxidant biomo-lecule effective in treatment of various liver related diseases(Kalaivani et al., 2010; Hussain et al., 2013). The most activeand functional biomolecules of extract (picrosides I and II) are

20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

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52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98

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Fig. 3 – FTIR profiles of lyophilized powder of (a) PLA nanoparticles, (b) P. kurroa extract and (c) PLA nanoparticles with P. kurroaextract encapsulation.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 2 8 – 3 5 31

sparingly soluble in water (CSIR/RRL, 1989–1990). Due to this,these components are poorly absorbed in the intestine andshowed poor bioavailability (Li et al., 2012; Park et al., 2009;Upadhyay et al., 2013; Wang et al., 2004). To improve thehepatoprotective potential by enhancing solubility/intestinalabsorption and for reduction of dosage quantity/frequency, thisextract was nanoencapsulated on intestinal biocompatible andUS FDA approved PLA (as food supplement) nanoparticles. PLAnanoparticles coating with US FDA approved surfactant PF 68protects these nanoparticles from reticuloendothelial system(RES) leading to enhanced permeability and retention (EPR),bioavailability as well as intestinal absorption (De Jaeghereet al., 2000). Copolymer pluronic-F-68, as a surfactant, suffi-ciently prevents the particle aggregation to stabilize the particlefor long term storage (Bhattacharyya, Paul, & Khuda-Bukhsh,

2010). This nanoparticle is well known carrier for bettersustained release of encapsulated biomolecule inside tissueand thus expected to be a desirable system for picrosidedelivery due to high loading efficiency (El Fagui, Dubot,Loftsson, & Amiel, 2013; Shive & Anderson, 1997). To minimizethe loss of functional activities of picrosides and to stabilizepicroside I conversion to aglycones due to hydrolysis (Wanget al., 2004), the nanoprecipitation method was used becausethis method do not require high temperature and sonicationfor nanoencapsulation.

The isocratic (60% aqueous methanol) mobile phase waseventually established to get best HPLC resolution (at 285 nm)profile of picrosides I (14.5 min) and II (8.5 min) (Fig. 2). Ourresults also confirmed by previously reported ratio (9.71:5.85)of picrosides I and II in Indian condition using relative HPLC

Fig. 4 – Zeta particle size distribution of (a) PLA NPs and (b) P. kurroa extract encapsulated PLA nanoparticle. Zeta potential of (c)PLA NPs and (d) P. kurroa extract encapsulated PLA nanoparticle.

Fig. 5 – TEM images of (a) PLA NPs (141710 nm) and (b) P. kurroa extract encapsulated PLA nanoparticle (154712 nm).

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 2 8 – 3 532

peak areas (Patil, Sachin, Shinde, & Wakte, 2013). The relativeencapsulation efficiency was calculated by subtracting theHPLC peak average area/height of picrosides I and II fromnanoparticle synthesis mixture (initial) and supernatant afterseparation of extract loaded PLA nanoparticles (Fig. 2). Theencapsulation and measurement were performed in tripli-cates and revealed the encapsulation efficiency for picrosidesI and II are 60.172.8% and 67.277.4% respectively (Fig. 2).

The encapsulation of picroside extract was also confirmedqualitatively by FTIR analysis of pure lyophilized P. kurroa, PLAnanoparticles without encapsulation and with extract encapsu-lated nanoparticles (Fig. 3). The major characteristic peaks ofP. kurroa extract [–O–H (3307.2 cm�1), –C–H (2925.0 cm�1), and –

C¼O (1701.5 cm�1)] were present in lyophilized extract andencapsulated PLA nanoparticles while absent in blank PLAnanoparticle. PLA characteristic peaks of –C¼O stretching(1747.3 cm�1) and –C–O stretching (1182.9 cm�1, 1127.6 cm�1,1081.7 cm�1) were present in extract encapsulated PLA nano-particles as well. The presence of P. kurroa characteristic peaks

on extract encapsulated PLA nanoparticle in FTIR is a directsupport of successful incorporation of P. kurroa multiple hepa-toprotective ingredients into the nanoparticle system.

The size distribution of extract encapsulated PLA nano-particles were analyzed by zeta particle size analyzer andtransmission electron microscope (TEM). Zetasizer measure-ment revealed that nanoparticles have a size of 174734 nmtaken in triplicates, slightly bigger than 152728 nm blank PLAnanoparticles (Fig. 4a and b). The sharp narrow peaks showedmonodispersity of synthesized nanoparticles. Zeta potentialof encapsulated and blank PLA nanoparticles were deter-mined as �27.8772.75 mV and �31.572.46 mV respectively(Fig. 4c and d). This confirms the highly stable nanoparticlessafe for long term storage. TEM measurement has shown thesize of the encapsulated nanoparticles as 154712 nm, while141710 nm of blank ones using Image J software (Fig. 5a and b).

3.2. In vitro release property and other parameters

In vitro release analysis showed a pattern with strong initialburst. Half release was shown at 2773 h and completerelease in 210710 h (Fig. 6). Loosely associated or surfaceadsorbed components of extract (picrosides I and II) on PLAnanoparticle were released in vitro initially and responsiblefor the initial burst (McPhee, Tam, & Pelton, 1993; Oh, Nam,Lee, & Park, 1999). The second stage release has been reduceddue to relatively slow speed of PLA polymer degradation. Thesustained and long release would also be beneficial forreducing the total molecule intake and frequency, thus havethe potential to reduce side effects as well (Freiberg & Zhu,2004; Uhrich, Cannizzaro, Langer, & Shakesheff, 1999).

PLA nanoparticles degradation caused by scission of thepolymer backbone leads finally to polymer erosion, the loss ofmaterial from the polymer nanoparticle. However, it is notnecessarily associated with a constantly decreased particlesize. A study of degradation mechanism (Gopferich, 1996;Park, 1994, 1995) showed that change in composition leads tosize changes in a three phase way. There is a slight increase

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Fig. 6 – In vitro release profile of picrosides I and II fromP. kurroa loaded pluronic-F-68-PLA nanoparticles. It showsthe initial burst due to drug bound to the outer surface anduntil half release at 2773 h and complete release at210710h.

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Fig. 7 – Change of zeta particle size and zeta potential during in vitro release. Particle size and zeta potential shows a trend ofincrease upon incubation up to 12 days.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 2 8 – 3 5 33

in size before the complete degradation, which explains themeasured size change during in-vitro release in the first 300 h(Fig. 7). Zeta size also slightly increases in the process of achanged composition (Fig. 7).

4. Conclusion

Alcoholic P. kurroa extract was successfully encapsulated intopluronic-F-68-PLA nanoparticles by nanoprecipitationmethod. Encapsulation efficiency was determined 60.172.8% and 67.277.4% for picrosides I and II respectively.Measurement with TEM demonstrates that the particles havea uniform size distribution and smooth spherical shape ofaverage size range of 154712 nm. The formulation of PLAencapsulated extract components leads to controlled release,with consistent half release of 2773 h and total release of210710 h in vitro release condition. This release dynamicsprofile suits the intestinal absorption and up-take in thehuman body. It is expected to be a promising approach tohave an enhanced intestinal absorption, biocompatibility andbioavailability, and good dietary supplement.

Conflict of interest

We declare no conflict of interest on this project.

Acknowledgment

We acknowledge Dr. Alok Adholeya, Director, TERI-DeakinNanobiotechnology Center for his support and Dr. NeerajKumar from CSIR-IHBT for providing P. kurroa rhizomepowder. DJ is highly grateful to the Life Science InterfaceDoctoral Training Centre for providing funding to work atTERI-Deakin Nanobiotechnology Center, New Delhi, India. IBhelped in vitro release and compilation of this manuscript. SThelped in repeat of HPLC work for encapsulation.

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