Lipid nanocarriers based on natural compounds: an evolving role in further plant extracts delivery

Research Article

Lipid nanocarriers based on natural compounds: An evolvingrole in plant extract delivery

Ioana Lacatusu1, Nicoleta Badea1, Gabriela Niculae1, Natalita Bordei1, 2, Raluca Stan1

and Aurelia Meghea1

1 Faculty of Applied Chemistry and Materials Science, University POLITEHNICA of Bucharest, Bucharest,Romania

2 S.C. Hofigal Export Import SA, Bucharest, Romania

The vegetable oils and extracts known for their beneficial effects should be identified and used in variousforms for the development of new healthy products. This study was designed to provide furtherinvestigation on new nanocarriersmade with hempseed oil or a blend of amaranth and hempseed oils, for aconcomitant encapsulation and release of the carotenoids enriched plant extract. The size of plant extractloaded lipid nanocarriers ranging between 109 and 130nmwas found to be less influenced by the differentratios of hempseed and amaranth oils. For all of the synthesized nanocarriers, zeta potential values werenegative (�33.4��38.1mV). The scanning calorimetry study has shown that lipid nanocarriers havefavorable lattice defects for plant extract encapsulation. Entrapment efficiency results revealed an increaseof carotenoids entrapment from 57.6 to 83.5% as the amaranth oil percent has been increased. High abilityto scavenge the free oxygenated radicals was distinguished for all free and loaded nanocarriers. The level ofantioxidant activity increase was proportional to the extent of vegetable oil and was ranging between 93.4and 98.1%. The nanocarriersmade with amaranth and hempseed oils have shown amore sustained releaseover time than those prepared with hempseed oil only in association with solid lipids.

Practical applications: The applicability of lipophilic plant extracts enriched in bioactive compoundsencounters serious problems in the food and pharmaceutical sector due to poor bioavailability. Using theconcept of exploiting natural resources in combination with soft nanotechnology, valuable bioactivevegetable mixtures could be formulated into solid colloidal nanoparticles. The bioavailability and thetherapeutic benefit of hempseed and amaranth oils in association with lipophilic plant extract enriched incarotenoids are increased by incorporation into the same nanostructured formulation. Theseformulations result in unique precursors of health products, particularly for nutraceutical industry.The lipid nanocarriers based on natural compounds offer potential applications as natural, low cost andinnovative delivery systems to improve quality and extend shelf‐life of food products. The developedvegetable based lipid nanocarriers offer advantages of a minimum carrier cytotoxicity, good storagestability, synergistic effects, antioxidant and sustained release, easy to scale up production.

Keywords: Antioxidant activity / In vitro carotenoids release / Lipid nanocarriers / Plant extract / Vegetable oils

Received: December 11, 2013 / Revised: March 14, 2014 / Accepted: April 8, 2014

DOI: 10.1002/ejlt.201300488

1 Introduction

Natural products such as vegetable oils and selective plantextracts are considered the most successful discovery ofmodern medicine being valuable sources of bioactivecompounds with multiple health benefices [1, 2]. Numerous

Correspondence: Professor A. Meghea, Faculty of Applied Chemistryand Materials Science, University POLITEHNICA of Bucharest, PolizuStreet No 1, 011061 Bucharest, RomaniaE‐mail: [email protected]: þ40 213154193

Abbreviations: DLS, dynamic light scattering; EE, entrapment efficiency;NLC, nanostructured lipid carrier; PDI, polydispersity index

Additional corresponding author: N. Badea,E‐mail: [email protected]

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studies have emphasized and proved that active compoundsoriginated from plants possess a high spectrum of biologicalactivity [3–5]. Today, the hydrophilic plant extracts arelargely used in the treatment of various diseases in the formof syrups, drops, and tablets. Instead, the applicability oflipophilic plant extracts encounters serious problems in thepharmaceutical and food sectors. Despite having an excellentin vitro bioactivity, the lipophilic extracts demonstrate less orno in vivo actions, due to their low solubility or impropermolecular size, resulting in a poor absorption and bioavail-ability upon oral administration [6–8]. One approach toeffectively alleviate these deficiencies is to lower the particlesize of vegetal extracts to a micro‐ and nanometer scale byencapsulation into appropriate biocompatible systems.

Although delivery systems such as liposomes, micelles,nanoemulsions, microemulsions, lipid nanoparticles, andbiopolymer nanoparticles have found numerous applica-tions [9, 10], their use as vehicle for the delivery of naturalbioactive extracts is relatively new in the Pharmaceutical andNutraceutical industry. In the last years, only few studieshave been completed to extend the vast area of micro‐ andnanotechnology to encapsulate various herbal medicines, e.g.curcumin extract [11], soybean extract enriched in daidzeinand genistein [12] grape marc extract [13] and eugenol‐richclove extract [14].

Among delivery systems, the use of nanostructured lipidcarriers (NLC) offers unique advantages [15, 16] andpromising means to enhance the solubility and the bioavail-ability of poorly soluble substances such as herbal extracts.Generally, these lipid nanocarriers (NLC) are composed of asolid lipid core consisting of a mixture of solid lipids blendedwith a synthetic liquid lipid in order to form a nanosizedunstructured matrix [17, 18]. Nowadays, a fundamentaldifferent group of dispersions is also under investigation,namely lipid nanodispersions based on vegetable oils. Lipidmatrix with a certain content of spatially incompatiblevegetable oils come out to be the most promising encapsula-tion and delivery system for complex mixtures of lipophilicplant extracts. For instance, in the previous studies of theauthors, lipid nanocarriers loaded with various nutraceuticalsand prepared with grape seed oil have shown greatantioxidant and antibacterial activities, as well as a prolongeddrug delivery, as compared to NLC prepared with individualoil [19–21].

Using the concept of exploiting natural resources,insoluble vegetable mixtures can be formulated as colloidallipid nanoparticles, of solid nature. Moreover, these nano-structured lipid systems present many advantages ascompared to other colloidal systems, e.g. overcome a numberof carrier‐related drawbacks, such as the limitations of theside effects due to the matrix material of the carriers, assure aprotection against toxicity, and permit better controlled drugrelease due to the increased mass transfer resistance [22].

On this background, the aim of the present study was toinvestigate the association of a natural carotenoids mixture,

e.g. lyophilic extract derived from Marigold plant (Tagetespatula), with lipid nanocarriers based on vegetable oils. Apartfrom the appropriate accommodation space assured bythe complex lipid core, therapeutic benefit inherent to theassociation of the two vegetable oils and one herbal extractincorporated into an unique formulation, would result inhighly efficient precursors of health products, particularly forNutraceutical and Pharmaceutical industries.

The carotenoid extract offers potential applications as anatural, low cost and innovative pigment to improve qualityand extend shelf‐life of food products without the use ofsynthetic additives. The carotenoids mixture has been usedto treat inflammatory conditions of internal organs andgastrointestinal ulcers [23]. Carotenoids were also found toreduce the risks of age‐related macular degeneration(AMD) [24], manifest antimicrobial activity [25], antioxidanteffectiveness [26], and help fight and prevent cancer [27].

In addition to carotenoid extract benefits, the selectedvegetable oils used in the preparation of lipid core bringadditional properties. Amaranth oil has antioxidant andhepatoprotective activities [28, 29], can lower cholesterol inanimal models owing to its rich content of PUFA andSqualene, being an effective remedy for coronary heartdisease and hypertension [30]. Hempseed provides a well‐balanced and rich source of dietary omega‐6 and ‐3 EFA andappears to be a valuable source of food [31, 32]. Hempseedhas unique nutritional properties and has commonly beenclaimed to be one of the most nutritionally completefoods [33].

The combination of lipophilic herbal extract with lipidnanocarriers comprising in a final content of vegetable oil upto 20% is an innovative approach in the design of an efficientlipid formulation. This study intends to underline the abilityand the synergistic effect of vegetable oils and extracts toconfer multifunctional properties to the entire developedbioactive lipid formulation. In this respect, the design and theoptimization of new delivery nanocarriers based on one oil(e.g. Hempseed oil enriched in v‐6 fatty acids) or on a blendof two vegetable oils (e.g. Amaranth and Hempseed oils),for simultaneous encapsulation and release of plant extractenriched in carotenoids are thus presented.

2 Materials and methods

2.1 Materials

The non‐ionic and ionic surfactants and co‐surfactants,Synperonic PE/F68 (block copolymer of polyethylene andpolypropylene glycol) and L‐a‐Phosphatidylcholine wereobtained from Sigma–Aldrich Chemie GmbH (Germany),Polyoxyethylenesorbitan monolaurate (Tween 20) wasobtained Merck (Germany). The solid lipids, GlycerolMonostearate (GM) and Cetyl Palmitate (CP) were pur-chased from Cognis GmbH (Germany) and Acros Organics

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(USA), respectively. The chemiluminescence reagents,Tris[Hydroxymethyl] aminomethane, 5‐amino‐2,3‐dihy-dro‐1,4‐phthalazinedione (Luminol) were purchased fromSigma–Aldrich Chemie GmbH and hydrogen peroxide wasobtained from Merck (Germany). The oily CarotenoidsExtract (CE) originated from Marigold plant (Tagetes patula)was provided by Hofigal S.A. Company (Romania). Thecarotenoids content (expressed in b‐carotene) determinedby HPLC (HPLC Dionex, UVD – 340U, 200–600nm,Licrosorb PR8 column, l¼ 460nm) and UV–VIS Spectro-scopy (UV–VIS Spectrophotometer Cintra 6, l¼ 460nm)was of 110mg/100 g Hempseed oily extract.

The Amaranth oil (AO) obtained by cold pressed methodfrom Amaranthus seeds spp. have been analyzed by liquid andGC in order to determine the Squalene and the fatty acidscontent. The Squalene content has been determined byHPLC (Lachrom Merck with DAD detector), by usingKromasil C18 column and a mixture of methanol: iso-propanol: acetic acid as the mobile phase, at lmax¼ 214nm.The fatty acids from AO have been analyzed (after trans-esterification with KOH/MeOH 2M) by GC (AgilentTechnologies with split‐splitless injector and FID detector),using HP 88 (88% cyanopropyl –methyl polysiloxane) as thestationary phase and nitrogen carrier gas (flow rate of 1.5mL/min). The bio‐active composition of Amaranth oil (AO) is:6.43% squalene, 24.54% linoleic acid, 17.29% oleic acid,9.32% palmitic acid, 1.58% stearic acid and 0.39% linolenicacid. The Hempseed oil (HO) (Cannabis sativa L.) analysiswas performed using a Gas Chromatograph (ThermoElectron Corporation Focus), HP‐5MS column, carrier gasHe (flow rate of 1mL/min), injection sample volume of0.1mL. Detection was performed using the Mass Spectrom-eter (Thermo Electron Corporation‐DSQII). The main fattyacids determined in the Hempseed oil were: 40.92% linoleicacid, 23.28% linolenic acid, 20.91% oleic acid, 7.41%palmitic acid, 4.28% stearic acid.

2.2 Synthesis

The lipid nanocarriers loaded with different amounts ofcarotenoids extract (noted as CE‐NLC 1� 7) were synthe-

sized by using a combination of high shear and high pressurehomogenization techniques, as previously reported byauthors [20, 21]. Briefly, an aqueous phase (consisting ofdouble‐distilled water and 2.5% surfactant mixture in aweight ratio of 1:0.2:0.2 Tween 20:Phosphatidylcholine:Block copolymer) and various lipid phases (mixture of solidlipids, includingGM, CP, and vegetable oils –HO or blend ofHO and AO) were separately prepared (Table 1). All NLCformulations have been prepared starting from an initialconcentration of 10% lipid phase. Various amounts ofcarotenoids oily extract were added in the lipid phase toform a clear uniform oil phase (Table 1). Both aqueous andlipid phases were heated under stirring at 80°C for 5min.The aqueous phase was gradually added to the oil phase withintense stirring. The resulted emulsions have been kept for15min (at 80°C) in order to ensure efficient entrapment ofthe carotenoids extract inside the lipid nanocarriers.Subsequently, they were subjected to a high shearhomogenization stage (High‐Shear Homogenizer PRO250type; 0�28.000 rpm; power of 300W, Germany), byapplying 12 000 rpm for 1min and then were subjected toa high pressure homogenization (APV 2000 Lab Homoge-nizer, Germany) at 600 bar for 196 s. The resultedformulations were cooled at RT to obtain the free‐ andcarotenoid extract loaded lipid nanocarriers. The aqueousnanocarriers dispersions were lyophilized using an Alpha 1‐2LD Freeze Drying System Germany. The NLC dispersionswere frozen at �25°C overnight before lyophilization for72 h, at �55°C.

2.3 Characterization of vegetable nanostructuredcarriers

2.3.1 Dynamic light scattering

Particle size measurements were done by dynamic lightscattering (DLS) using a Zetasizer Nano ZS (MalvernInstruments Ltd., United Kingdom), equipped with asolid‐state laser (670 nm). The mean particle size (Zave)and the polydispersity index (PDI) of the aqueous NLCdispersions were measured at 90° scattering angle and a

Table 1. Composition and EE of NLCs loaded with carotenoids extract

Composition Surfactants mixture (%) GM%a) CP% HO% AO% CE%b) EE%

CE‐NLC 1 2.5 3.5 3.5 3 – 0.11 41.3CE‐NLC 2 2.5 4 4 2 – 0.11 57.6CE‐NLC 3 2.5 4.5 4.5 1 – 0.11 69.1CE‐NLC 4 2.5 3.5 3.5 2.5 0.5 0.092 62CE‐NLC 5 2.5 3.5 3.5 2 1 0.073 78.4CE‐NLC 6 2.5 3.5 3.5 1.5 1.5 0.055 83.5CE‐NLC 7 2.5 3.5 3.5 1 2 0.037 79.7

a)Values are given in w/w (%) of components/total weight of aqueous dispersion.b)Values are given in w/w (%) of CE/total weight of oils.

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temperature of 25°C. Before measurements, all NLCdispersions were diluted with deionized water to an adequatescattering intensity. The particle size data were evaluatedusing intensity distribution. The average diameters (based onStokes–Einstein equation) and the PDI were given as averageof three individual measurements.

2.3.2 Transmission electron microscopy

The morphology of lipid nanocarriers loaded with carote-noids extract was examined using a transmission electronmicroscope (Philips 208 S, Netherlands). The lipid nano-carriers were diluted with water (1:100, v/v) beforeexamination. Samples were prepared by simply depositingthree drops of the nanocarrier aqueous suspension on thesurface of a copper grid and kept for 24 h before TEMexamination. Analysis was performed without staining.

2.3.3 Stability of lipid nanocarriers loaded withcarotenoids extract

Determination of the zeta potential (ZP) used to assess thecharge stability of all lipid nanocarriers was achieved bymeasuring the electrophoretic mobility of the nanoparticles inan electric field, using the Helmholtz‐Smoluchowsky equa-tion (Zetasizer Nano ZS, Malvern Instruments Ltd., U.K.).Before zeta potential measurements, all the NLC dispersionswere adjusted with a 0.9% NaCl solution, in order to correctthe conductivity to 50mS/cm. All measurements wereperformed at 25°C, in triplicate and the mean value wasreported.

2.3.4 Entrapment efficiency of carotenoids extractinto lipid nanocarriers

The procedure used for the determination of carotenoidsentrapment efficiencies (EE%) into lipid nanocarriersprepared with two kinds of vegetable oils is based on thestandard spectrophotometric determinations. The EE% wasdetermined by measuring the concentration of free carote-noids in the dispersion medium. The lyophilized lipidnanocarriers were dispersed into ethanol by gentle shakingin order to dissolve the free carotenoids. The resultingdispersions were centrifugated for 15min at 15 000 rpm. Thecarotenoids content in the supernatant was measured by UV–Vis–NIR spectrophotometer type V670 (Jasco, Japan) atlmax¼ 452nm. The entrapment efficiency (EE) was calcu-lated using the equation:

%EE ¼ W a �W s

W a� 100

where, Wa is the weight of carotenoids added into lipidnanocarriers and Ws is the analyzed weight of carotenoids insupernatant.

2.3.5 Differential scanning calorimetry

Thermal analysis of the lyophilized free‐ and carotenoidextract loaded NLCs was performed in order to investigatethe changes in the crystalline state of the lipid core. Thethermograms of loaded and free‐NLC were recorded using adifferential scanning calorimeter Jupiter, STA 449C(Netzsch, Germany). The samples (10mg) were weighedinto standard alumina pans using an empty pan as reference.The temperature was increased 5°C/min between 20 and100°C.

2.3.6 In vitro determination of antioxidant activity

The in vitro antioxidant activity of carotenoid extract‐NLCand free‐NLC has been determined by chemiluminescencemethod using a Chemiluminometer Turner Design TD 20/20, USA. Luminol has been used as light amplifyingsubstance, which emits light when it is oxidized and it isconverted into an excited amino‐phthalate ion in the presenceof ROS such as hydrogen peroxide (H2O2). H2O2 in Tris–HCl buffer solution (pH 8.6) has been used as a generatorsystem for free radicals. Starting from lyophilized samples,ethanol solutions of free‐ and CE‐NLCs with concentrationsranging between 0.015 and 0.37mg/L carotenoids andbetween 13.6 and 40.8mg/L vegetable oils have beensubjected to the free oxygen radicals generated into thechemiluminescence system. Before the antioxidant activitydetermination all the samples were subjected to ultra-sonication (for 3min) to assure complete dissolution. Thepercentage of free radical scavenging of lipid nanocarriers wascompared with the same concentration of vegetable oils andwas calculated by using the relation:

%AA ¼ I0 � I sI0

� 100

where I0 and Is are the chemiluminescence maximum forstandard, and for the samples at t¼ 5 s, respectively.

2.3.7 In vitro release experiments

In vitro release study of carotenoids from NLCs was doneusing vertical Franz glass diffusion cells (25mm in diameter;Hanson Reasearch Corporation, USA). Franz diffusion cellconsists of a donor and a receptor chamber between which acellulose nitrate membrane filter (0.1mm; Whatman,Germany) was placed. The release medium from the receptorchamber, with a volume of 6mL, consisted in ethanol buffersolution (phosphate‐buffered saline, pH 5.5/ethanol, 70:30,v/v). 500mL of NLC dispersion were placed into the donorchamber. The area of diffusion was 0.636 cm2 and thereceptor medium was continuously stirred at 400 rpm and37°C (17). After 1, 2, 3, 4, 5, 6, 7, 8, and 24h, 500mL ofsamples were withdrawn from the receptor chamber and

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discharged, and another 500mL were collected and dilutedwith ethanol for determinations. The volume of releasemedium was maintained constant throughout the study.Samples were analyzed for carotenoids concentration byusing UV spectrophotometry at lmax 452 nm.

The release kinetics were depicted using the followingmathematical models equations [34]: zero order: % R¼ k0t;first order: log(100–%R)¼ k1t/2.303; Higuchi: %R ¼ k2

ffiffi

t2p

;Korsmeyer–Peppas: %R ¼ k2

ffiffi

tnp

; Hixson–Crowell:ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

100�%R3p ¼ k4t, where, %R is percent of carotenoidsrelease at time t; k0, k1, k2, k3, and k4 are the rate constantsfor zero order, first order, Higuchi, Korsmeyer–Peppas, andHixson–Crowell, respectively; n is the release exponent.

3 Results and discussion

3.1 Particle size change and physical stability of lipidnanocarriers according to carotenoid extract loadingand type of vegetable oil

In many previous investigations, the amount of lipids used toprepare NLC systems was between 5 and 30% w/w informulations [17, 35]. In this study, an amount of 10% lipidsmixture (w/w) was used to prepare lipid nanocarriers based onvegetable oils. Whereas one of the aims of this research was toobserve the influence of Hempseed oil and Amaranth oil asliquid lipids to obtain NLC with appropriate size, the weightratio of vegetable oils was varied from 1 to 3% HOw/w fromtotal weight of dispersion (Table 1) and between 0.5 and 2%AOw/w.

Preliminary size characteristics of aqueous systems of lipidnanocarriers loaded with plant extract and the correspondingfree‐NLC have been achieved by DLS. The average particlesize and PDI for the CE loaded –NLC formulations preparedwith Hempseed oil (CE‐NLC 1� 3) or with a mixture ofHempseed and Amaranth oils (CE‐NLC 4� 7) are shown inFig. 1.

The mean size of CE loaded – lipid nanocarriers (e.g.between 112.2 and 131.4 nm for NLC prepared withHempseed oil and of 110.1 and 123.3 nm for NLC preparedwith mixture of vegetable oils) was slightly higher than thoseof the corresponding free‐NLC (e.g. between 109.9 and116.4 nm for the first NLC systems and between 108.2 and111.1 nm for the second one). The combination of vegetableoils with the solid lipids mixture (e.g. CP andGM) resulted ina narrow particle size distribution, most of NLC formulationsshowing PDI< 0.22 (Fig. 1).

This study has also shown that there were no significantsize differences among lipid nanocarriers prepared withdifferent ratios between Hempseed oil and Amaranth oil. Anoptimal ratio between particle size and PDI was obtained forCE loaded‐NLCs prepared with 3% Hempseed oil (e.g.Zave¼ 112.2�0.305 and PDI¼ 0.193� 0.004) and a mix-ture of 2.5% Hempseed oil and 0.5% Amaranth oil (e.g.Zave¼ 111.5�0.005 and PDI¼ 0.191�0.007). These lastlipid nanocarriers have been analyzed by electronmicroscopy.The TEM photomicrographs revealed the presence of non‐aggregated and almost spherical nature of lipid nanocarriersprepared with Hempseed oil or a blend of vegetable oils(Fig. 2). It is also worth to note that the mean particle sizeobtained from the TEM measurements are well correlatedwith those evaluated by DLS measurements.

The nanoparticles are thermodynamically unstable sys-tems and for their stability a zeta potential value between�30and �60mV is desirable in order to avoid aggregation ofparticles [36]. In the present study, the zeta potential profile ofNLC was negative and indicated the existence of an efficientrepulsion among particles, which prevent the aggregation ofnanocarriers prepared with high amount of vegetable oils.When discussing about the lipid nanocarriers stability, it wasrevealed that the incorporation of carotenoid extract did notsignificantly affect the zeta potential value, only a slowincrease being observed for CE – loaded NLC as comparedwith those obtained for free – NLC (Fig. 3a).

Regarding the influence of the vegetable oil, it isinteresting to note that a decrease of the hemp oil contentleads to higher values of electrokinetic potentials (e.g. from�38.1� 0.737mV toward �34.5� 0.551mV, for the nano-carriers prepared with Hempseed oil and from �37.2� 2.10mV toward �33.4� 1.36mV, for the nanocarriers preparedwith mixed vegetable oils. Thus, the best stability has beenobtained for CE‐NLC 1 (prepared with Hempseed oil) andCE‐NLC 4 (prepared with Hempseed and Amaranth oils).The selected graphs related to the electrokinetic potentialdistribution of the prepared nanocarriers are shown in Fig. 3b.

3.2 Evaluation of lipid crystallinity and entrapmentefficiency as function of type and amount of vegetableoil

In general, the solid state of the lipid core in nanoparticulatesystems is important to achieve controlled release properties.

Figure 1. The average diameters of free‐ and CE loaded lipidnanocarriers synthesized with Hempseed oil (CE‐NLC 1� 3) andblend of Hempseed and Amaranth oils (CE‐NLC 4� 7).

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Consequently, the solid state of the synthesized lipidnanocarriers was confirmed by scanning calorimetry study.The use of a single vegetable oil (e.g. Hempseed oil) or ablend of two vegetable oils (e.g. Hempseed and Amaranthoils) resulted in the obtaining of solid lipid nanostructureswith strongly disordered network, as confirmed by theendothermic peak allure. All the prepared free – andcarotenoids extract – loaded NLC show a broad region ofthe endothermic peak, in the 45–60°C melting range.Complex composition of the lipid core containing solidlipids blended with vegetable oils is underlined by theappearance of three shoulders in the range of 46–48, 53–55,and 57–59°C, respectively.

The perturbation of the lipid network after carotenoidsextract entrapment is obvious by comparing the DSCbehavior of both free‐ and loaded‐NLC (Fig. 4). Thisbehavior points out that lipid nanocarriers synthesized byusing different amounts of vegetable oils are characterized bya lipid network with many imperfections, and consequently,with significant effect on the entrapment efficiency (EE) ofcarotenoids extract. For instance, contrary to expectations,the use of 1%HO for the preparation of lipid nanocarriers has

led to a less ordered crystalline network as compared to thoseprepared with 2 and 3% HO (Fig. 4a). This behavior is wellcorrelated with the EE results, which revealed an increase ofcarotenoids entrapment as the Hempseed oil percent hasbeen decreased from 30 to 10% (e.g. CE‐NLC prepared witha mixture of solid lipids and hemp oil depicted EE rangedbetween 41.3 and 69.1%, Fig. 5). Beside the influence ofvegetable oil percent, the initial amount of CE could beresponsible for these results (e.g. CE‐NLC 1 has been loadedwith high amount of CE, Table 1).

On the other hand, the DSC thermograms of the lipidnanocarriers prepared with blend of Amaranth and Hemp-seed oils revealed a small difference in the endothermic peaksas well as in the lipid crystallinity, in particular for EC‐NLC5,6, and 7. These results, in an initial interpretation, suggest aslight disturbance of lipid network by incorporating herbalmixture, which would result in a poor encapsulation degree ofcarotenoid extract. Fortunately, the encapsulation efficiencywas found to be in this case independent of the lipidcrystallinity. This behavior indicates that the lipid corecontaining a mixture of Hempseed and Amaranth oilspresents appropriate lattice defects, which are more efficient

Figure 2. TEM images of NLC formulations based on vegetable oils: (a) CE‐NLC 1 (3% HO); (b) CE‐NLC 4 (2.5% HO and 0.5% AO).

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for herbal extract entrapment than NLC prepared with onevegetable oil. For most of the mixtures tested, NLC preparedwith 1% HO and solid lipids provided 69.1% encapsulationefficiency, while the highest capacity to entrap the carotenoidsextract (EE¼ 83.5%) was encountered for NLC made frommixture of 1.5% HO and 1.5% AO as the liquid lipid of thelipid core (Fig. 5). Two potential reasons that could lead tosuch differences in encapsulation efficiencies are the decreaseof initial CE loading and the vegetable oil composition. Amoderate percent of omega‐6 fatty acids (�25%) from AO inassociation with solid lipids, assures more suitable spaces forcarotenoids mixture than a high percent of omega‐6 fattyacids encountered in Hempseed oil (�64%).

3.3 Comparative evaluation of in vitro antioxidantactivity and carotenoid release from nanocarriers withone vegetable oil and blends of vegetable oils

For this part of experiments, the vegetable oils, free‐ andNLCloaded with carotenoids extract were subjected to free oxygenradicals in situ generated into chemiluminescence system.

Most of the NLC prepared with HO or blend of HO and AOmanifested an excellent ability to scavenge the free radicals(Fig. 6). The average antioxidant value obtained for NLCmade with Hempseed oil was ranged between 94.9 and97.1%, while for those prepared with vegetable blend rangedbetween 93.4 and 97%. The level of antioxidant increase wasproportional with the extent of vegetable oil. The greatestantioxidant activity was distinguished for EC‐NLC 1 (e.g.98.1�0.14%) and for EC‐NLC 4 (e.g. 97� 0.4%).

Figure 3. Physical stability of free‐ and carotenoid extract – loadedNLC prepared with vegetable oils. (a) Electrokinetic potentialvalues; (b) Selected electrokinetic potential distribution for CE‐NLC1 and 4.

Figure 4. DSC thermograms of free‐ and loaded carotenoidsextract – NLCs prepared with a blend of solid lipids and vegetableoils. (a) NLCs with 3, 2, and 1% Hempseed oil; (b) and c. Mixt NLCprepared with Hempseed and Amaranth oils.

Figure 5. Entrapment efficiency of carotenoids inside the vegetable– lipid nanocarriers.

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An interesting result was observed by comparing theantioxidant activity values of empty and loaded – lipidnanocarriers. There were no significant differences betweenthem, only a slight increase being determined for CE‐loadedNLC. These high antioxidant values can be explained bythe presence of different amounts of vegetable oils withappropriate fatty acids composition as well as by the decreaseloading of carotenoids mixture nanoconfined inside the lipidchains. It is also considered that Hempseed oil having higherv‐6 fatty acids content is more efficient to scavenge thefree oxygen radicals.

Unlike the antioxidant properties, the carotenoids releasefrom the developed lipid nanocarriers is drastically influencedby the presence of Amaranth oil. The results of the in vitrorelease profiles of the carotenoids from three representativenanocarriers (loaded with different amounts of herbal extract)through the Franz diffusion cells revealed a faster release ofcarotenoids from NLC prepared with one vegetable oil thanfrom those prepared with a blend of Amaranth andHempseedoils (Fig. 7). In the first case, a highest content of carotenoidshas been achieved after 1h of release experiment (75.8%).These results could be associated to the non‐encapsulatedcarotenoids and it can be explained by the drug‐enriched shell

model [37], where the drug is not entirely incorporated withinthe lipid matrix being adsorbed onto the particles surface.

In contrast, nanocarriers made with both Amaranth andHempseed oils, showed a gradual slow release over time(Fig. 7). CE‐NLC 6 released about 29% carotenoids at24 h, whereas CE‐NLC 5 has reached a cumulative amountof about 44% carotenoids in ethanol buffer solution asreceptor media (Fig. 7). At 24 h of release, the slowestamount of carotenoids released was obtained for NLC withan equal proportion of Amaranth oil and Hempseed oil (e.g.CE‐NLC 6).

Responsible for such slow release behavior may be thepresence of squalene from Amaranth oil that createsappropriate gaps in the lipid lattice, which are more suitablehost spaces for carotenoids mixture. These results supportthe presumption formulated in the previous studies (e.g.scanning calorimetry and EE studies) that indicates alocalization of carotenoids mixture inside the lipid core.

These different types of release pattern encountered forboth kinds of lipid nanocarriers made with HO or a blend ofHO andAO are advantageous because, depending on the areaof application, will ensure the faster onset of action as well asthe controlled delivery of carotenoids for longer period.

The cumulative amounts (%) of carotenoids (CE)released during 1 to 8 h, excluding the plateau from 8 to24 h, from NLC were fitted to different kinetic mathematicalmodels. The correlation coefficient (R2), the release rateconstant (k), and the release exponent (n) of the releasedcarotenoids are presented in Table 2. The study of the in vitrorelease kinetics of carotenoids fromNLC on different modelsshowed that the first order model was the best fitting model.The first order kinetic encountered in the released systemssuch as lipid nanocarriers assures a continuously diminishingrelease rate of the drugs (by diffusion controlled process).Therefore, the synthesized NLCs are advantageous owing tothe improved patient compliance (by decrease of drug dose),a decline of the side effects as well as more prolongedtherapeutic effect.

Figure 6. Effect of lipid nanocarriers loaded with herbal extract onantioxidant activity. (a) NLC made with Hempseed oil; (b) NLCprepared with mixed Hempseed and Amaranth oils.

Figure 7. Influence of NLC prepared with Hempseed and Amaranthoils on the carotenoids release: CE – NLC 1 (3% HO; 0% AO); CE –

NLC 5 (2% HO; 1% AO); CE – NLC 6 (1.5% HO; 1.5% AO).

Eur. J. Lipid Sci. Technol. 2014, 116, 1708–1717 Lipid nanocarriers based on natural compounds 1715

� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

4 Conclusions

In recent years, it has become more evident that thedevelopment of new drugs is not sufficient to ensure progressin human health. The use of natural remedies is highlyapproached in human health, in particular for food andpharmaceutical sectors with an ongoing search for theefficient use of health products with broad biologicalrelevance.

The effectiveness of two vegetable oils with differentcontent of omega‐6 and ‐3 EFA for the synthesis of lipidnanocarriers that are able to encapsulate a complexmixture ofvegetable extract was investigated. The size of carotenoidsextract loaded – lipid nanocarriers was found to be lessinfluenced by the different ratios of Hempseed and Amaranthoils. The EE results revealed an increase of the carotenoidsentrapment as the Amaranth oil percent has been increasedup to 1.5%. Based on the results obtained CE‐NLC 6 (madefrom mixture of 1.5% HO and 1.5% AO) was found to bethe most effective nanocarriers in terms of the EE, in vitroantioxidant and control release properties.

The level of the antioxidant activity was ranged between93.4 and 97.1% for all the synthesized nanocarriers, beingproportional with the extent of vegetable oils. The nano-carriers made with both Amaranth and Hempseed oils havemore merit than those prepared with Hempseed oil ascarotenoid delivery systems. The first one has shown a moresustained release over time.

The research achieved by this study has demonstratedthat – by combining the medical and nutritional propertiesof vegetable oils with those of plant extract enriched incarotenoids and with unique features of lipid nanoparticles –valuable bioactive nanocarriers that comprise a final contentof 20% vegetable oil could be developed.

This association will significantly contribute to extrabiological effects, by the existence of the bioactive Amaranthand Hempseed oils and of the natural antioxidant (plantextract enriched in carotenoids), in the same lipid nano-carrier. Moreover, the lipid nanocarriers made with Hemp-seed and Amaranth oils have a great potential for clinicalapplications as a new delivery system for other lipophilic plantextracts enriched in bioactive compounds.

This work was supported by a grant of the Ministry of NationalEducation, CNCS ‐ UEFISCDI, project number PN‐II‐ID‐

PCE‐2012‐4‐0111. Authors also gratefully acknowledge to theUniversity of Agronomic Sciences and Veterinary Medicine,Faculty of Biotechnology, Bucharest, Romania for providing theAmaranth oil.

The authors have declared no conflict of interest.

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