Encapsulation of Ficus deltoidea Extract in Nanostructured Lipid Carrier for Anti-melanogenic Activity
Siti Maria Abdul Ghani1&Nur Zatul Iradah Roslan1&Rohaiza Muda1&Azila Abdul-Aziz2,3
#Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Nanostructured lipid carrier (NLC) was studied as a system to transportFicus deltoideaextracts through the skin. The thin film ultra-sonication dispersion (TFUD) technique and double-emulsion solvent diffusion (DESD) techniques were performed to compare encapsulation efficiency (EE) of the two methods. The DESD displayed superior performance for encapsulation of F. deltoideaextracts which was 67.92 ± 10.55%. Next, different types of lipids were assessed to determine their effects on the characteristics ofF. deltoidea–loaded NLC prepared via the DESD method. Fatty acids were superior to triglycerides for the formulation of NLC. The final NLC formulation selected was stearic acid (SA) as solid lipid and oleic acid (OA) as liquid lipid, at a total ratio of 1:1, and poly(vinyl alcohol) (PVA) as a stabiliser. The delivery system exerted exceptional transdermal transport properties.F. deltoidea–loaded NLC successfully and more effectively reduced melanin, when compared with non-entrapped F. deltoideaextract.
Keywords Ficus deltoidea. Nanostructured lipid carrier (NLC) . Fatty acids . Anti-melanogenic activity
1 Introduction
Ficus deltoideais widely found in Southeast Asia, including Malaysia and Indonesia. Almost every part ofF. deltoideahas been utilised in traditional medicine practices [1]. It is be- lieved that chewing the fruits can relieve headache, toothache, and cold, while the root and leaves can heal wounds and sores, apart from relieving rheumatism [2]. The decoction of F. deltoideais consumed to strengthen the uterus after giving birth and to improve menstrual flow [3]. Recently, F. deltoidea extracts have been proven to possess anti- malaria activity [4]. F. deltoidea extracts can also elevate
testosterone level in diabetic rats [5] and hence has the poten- tial for application as ergogenic aids by athletes.
Oh et al. [6] reported that F. deltoidea water extract exhibited anti-melanogenic activity by directly inhibiting tyrosinase activity and suppressing gene-encoding tyrosi- nase, which is a key transcription factor. Melanin is a nat- ural pigment that defines the colour of the skin, the hair, and the eye of living things, apart from ultimately provid- ing protection against UV radiation, skin burn, and cancer [7]. Nevertheless, abnormal biosynthesis of melanin can cause irregular hyperpigmentation, such as freckles, lentiginous, melasma, or age spots [8]. Melanogenesis in- hibitors in F. deltoidea can be used in treating cases of hyperpigmentary disorders. Since F. deltoidea is a good source of antioxidants [9], the extract has a high potential to be utilised in skincare products.
To be effective,F. deltoideaextracts need to penetrate skin barrier to reach melanocytes. The outermost skin layer (stra- tum corneum (SC)) functions as a barrier to protect skin from undesired substances in the environment, aside from preventing the body from losing water, minerals, and dis- solved protein [10]. The SC is made up of 78% neutral lipid, 18% of sphingolipid (ceramides) with a small amount of polar lipid, and about 11% of non-polar material. Nanostructured lipid carrier (NLC), which is composed of physiological and biodegradable lipid ingredients, is similar in SC composition
* Azila Abdul-Aziz [email protected]
1 Department of Bioprocess and Polymer Engineering, School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor Darul Takzim, Malaysia
2 Department of Chemical and Environmental Engineering, Malaysia –Japan International Institute of Technology, Universiti Teknologi Malaysia, 54100 Kuala Lumpur, Malaysia
3 Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 Skudai, Johor Darul Takzim, Malaysia
https://doi.org/10.1007/s12668-020-00786-2
Published online: 29 October 2020
[10]. The fatty acids in the lipid components of NLC may also serve as skin penetration enhancers [11].
NLC is a second-generation solid lipid nanoparticle (SLN) composed of solid lipid and liquid lipid in an aqueous disper- sion medium. NLC has been introduced to address issues re- lated to low loading capacity of active expulsion during SLN storage [12]. Both SLN and NLC are low in toxicity, are biocompatible, and have high bioavailability [12]. The bio- compatible lipid components in NLC exhibit the ability to protect chemically labile drugs, besides allowing controlled release of active ingredients [10].
The lipid structure of NLC is loosely ordered, hence resulting in more space for active ingredients, in comparison with the perfectly ordered structure of SLN. The solubility of hydrophilic substances is comparably low in NLC [13] due to the hydrophobic behaviour of NLC. In this study, NLC was applied to encapsulateF. deltoideawater extract. Thin film ultra-sonication dispersion (TFUD) is a simple method for NLC preparation. However, since the extract was hydrophilic, double-emulsion solvent diffusion (DESD) was also consid- ered in this research work.
The influence of lipids on the characteristic of NLC was also examined in this study. The evaluated solid lipids were stearic acid (SA) and tristearin (TRI), while oleic acid (OA), virgin coconut oil (VCO), and soybean oil (SO) were assessed as liquid lipids. SA is a saturated fatty acid with 18-carbon chain, while TRI is an ester derived from three SAs and a glycerol. OA is monounsaturated long-chain fatty acid, while VCO and SO are complex mixtures of fatty acids. The effect of poly(vinyl alcohol) (PVA) on the improvement of NLC dispersion stability was investigated as cross-linked PVA and was hypothesised to offer stability to the delivery system [14]. Thus, the present study is aimed to analyse the effect of F. deltoideaencapsulation using NLCs and its effect on melanogenic activity on the skin.
2 Materials and Methods 2.1 Materials
Stearic acid (n-octadecanoic acid) 95%, glycerol TRI (1, 2, 3–
propantriol), OA ((9Z)-octadec-9-enoic acid), soybean oil (SO), tween 80 (polysorbate 80), span 80 (sorbitan oleate), poly(vinyl alcohol) (Mw ~ 85,000), ethyl acetate, dichloro- methane, monopotassium phosphate, dipotassium phosphate, potassium bromate (KBrO3), Folin–Ciocalteu reagent (naphthoquinone-4-sulfonate), melanin synthetic, 1-propanol, ethanol, and uranium acetate were purchased from Sigma- Aldrich (Germany). Deionised water was obtained on-site from a Barnsted water purification system; VCO and F. deltoidea water extract were supplied by Institute of Bioproduct Development (IBD), Universiti Teknologi
Malaysia, Skudai, Johor; Sephadex G-50 mini-column from Epoch Life Science (USA); nylon syringe filter (0.45 μm) from Whatman (UK); and reconstructed human pigmented epidermis (RHPE) from EpiSkin™(France). All chemicals were cosmeceutical, pharmaceutical, or analytical reagent grades and were used as received unless otherwise noted.
2.2 Preparation of NLC Using Double-Emulsion Solvent Diffusion
Preparation of NLC using the DESD (w/o/w) method was carried out by following the methods described by Cohen- Sela et al. (2009) [15] and Meng et al. [16] with some modi- fications. The primary emulsion of w/o was first prepared by dissolving 0.43% (w/v) of solid lipid (SA/TRI) and 0.43%
(v/v) liquid lipid (OA/VCO/SO) at ratio 1:1 in ethyl acetate (6 mL). After solid and liquid lipids were mixed, 0.05% (v/v) Span 80 was slowly added into the solution and 0.05% (v/v) PVA was added subsequently.F. deltoideaextract was dis- solved in 1% (w/v) deionised water and was added slowly into the lipid phase. The mixture was sonicated with a probe sonicator at 25% amplitude or at 20 W for 90 s. The primary emulsion was mixed with phosphate buffer saline (PBS) con- taining 8% (v/v) Tween 80 and sonicated with a probe sonicator at 40% amplitude for 3 min. The resultant double emulsion was stirred for 1.5 h to remove the remaining solvent under a fume hood. Finally, the formed NLC was stored at room temperature for further analysis.
2.3 Preparation of NLC Using the Thin Film Ultra- sonication Dispersion Method
By using the TFUD method, NLC was prepared based on the methods described by Kamble et al. [17] and Liu et al. [18]
with modifications. Solid lipid (SA/TRI), liquid lipid (OA/
VCO/SO), andF. deltoideaextract were dissolved in dichlo- romethane in a round-bottom flask and placed in a rotary evaporator to remove the solvent, leaving behind thin film of lipids. PBS with 2% (v/v) Tween 80 and 2% (v/v) Span 80 at 60 °C was applied to hydrate the thin film. Next, the disper- sion was sonicated using a probe sonicator at 40% amplitude for 3 min. The dispersion was filtered with 0.45-μm syringe filter. The filtrate dispersion was cooled down to room tem- perature to solidify and form the NLC.
2.4 Particle Size and Zeta Potential Measurements
Both particle size and zeta potential were measured using dynamic light scattering (Zeta Sizer Nano ZSP, Malvern, Germany). The detector was positioned at 173°, and the de- tection process was performed based on the non-invasive backscatter technology. The samples were diluted 1:9 indeionised water prior to analysis. Following this, the measure- ments were recorded in triplicates.
2.5 Encapsulation Efficiency
By applying the methods described by Zhang et al. [19], en- capsulation efficiency (EE) measurements were performed using a spin mini-column packed with Sephadex G-50 (Epoch Life Science, USA). Initially, the spin mini-column was centrifuged using a mini centrifuge at 492gfor 2 min to remove excess water. After that, 0.1 mL of NLC dispersion was loaded into the mini-column. The mini-column was cen- trifuged again at 492g for 2 min to elute the NLC.
Subsequently, the eluted samples were mixed with a mixture of ethanol and 1-propanol (6:4) to disrupt the lipid matrix of NLC and to release the entrapped F. deltoidea extract.
Validation for the spin mini-column methodology was per- formed by loading onlyF. deltoideaextract solution into the spin mini-column to prove that freeF. deltoideaextract was retained in the packed Sephadex G-50 during the procedure.
In this investigation, vitexin was applied as the marker com- pound forF. deltoideaextract. Vitexin was detected using high-performance liquid chromatography (HPLC).
Following that, EE was calculated according to Eq. (1) [20].
Encapsulation efficiency %ð Þ
¼amount of FD entrapped in NLC
total amount of FD extract 100 ð1Þ
2.6 Determination of Vitexin
Vitexin was quantitatively determined using HPLC (Agilent LC1200, USA), which was equipped with a quaternary pump, online degasser, auto-sampler, column heater, and UV detector. The quantification of vitexin on the collected samples was based on the technique d e v e l o p e d b y C h o o e t a l . [2 1] w i t h s e v e r a l modifications.
Chromatographic separation was performed using a C-18 column (250 mm × 4 mm × 4μm) with the column tempera- ture fixed at 30 °C. The following gradient elution profile was employed: 0–12 min (15–100% B), followed by 12–15 min of 100% B. Mobile phase B was methanol , while mobile phase A was 1% formic acid. The flow rate was 1.0 mL/
min. The injection volume was set at 10μL. The detection wavelength was 330 nm.
To validate the separation method, the spiking method, also known as the standard addition method, was performed.
A known volume and concentration of vitexin was combined with a fixed volume ofF. deltoidea extract and diluted to equal volume. The mass (μg) of vitexin inF. deltoideaextract was calculated by multiplying the quantitate concentration
(μg/mL) of HPLC analysis with the total dilution volume (5 mL). Figure 1 shows the chromatogram of F. deltoidea extract. The peak of vitexin was amplified after the sample was spiked using vitexin standard.
2.7 Fourier-Transform Infrared Analysis
In determining the interaction (if any) between F. deltoidea water extract and lipid during the preparation of NLC, Fourier-transform infrared (FTIR) spectroscopic analyses were performed. The FTIR spectra ofF. deltoidea extract, empty NLC, and F. deltoidea–loaded NLC were recorded on KBr pellet in a scanning range of 400–4000 cm−1. These spectra were recorded using a Fourier-transform spectropho- tometer (Perkin Elmer).
2.8 Morphology Observation
The morphology of NLC loaded withF. deltoideaextract was determined with the aid of a transmission electron microscope (TEM) (JOEL JEM 210 LaB6, Japan), operating at a voltage of 100 kV. A tiny droplet of NLC dispersion was dropped on top of a copper grid and was dried for 3 min at room temper- ature. The sample was stained with uranium acetate for 1 min prior to viewing under the TEM.
2.9 In Vitro Release Studies
An in vitro release study of loaded NLC carrier was carried out for NLC–SA–OA–PVA withF. deltoidea extract and non-encapsulated F. deltoideaextract. An amount of 5 mL of each NLC and F. deltoidea extract was pipetted into a dialysis tube (MWCO 12,000 kDa). The tube was immersed in 50 mL of PBS (0.1 mol/L, pH 6.8) solution in a 100-mL graduated test tube and kept at 37 °C in an incubator shaker.
The samples were rotated at 100 rpm and sampled every 2 h until 24 h. Aliquots of 2 mL were sampled thrice (n= 3), and an equal amount of fresh buffer was replenished. The phenolic content was determined using Folin–Ciocalteu assay. The in vitro study was repeated thrice (n= 3). Calculation of the release rate was carried out using the following equation:
Q¼ðMn=MÞ100% ð2Þ
whereQis the release rate,Mnis the phenolic content at a certain time, andMis the phenolic content initially entrapped in the NLC.
2.10 In Vitro Skin Permeation Studies
Skin permeation studies were carried out using side-by-side Franz diffusion cell [22]. Rat skins from 8-week-old female Sprague Dawley species were used. The abdominal parts of
the rats were initially shaved and cut into squares. The subcu- taneous fat layers of the rats were removed, leaving only the epidermis and dermis layers. The excised skins were stored at
−10 °C and pre-equilibrated in PBS solution at 4 °C overnight prior to the experiment. The approval to carry out this study was obtained from the animal ethics committee of Universiti Kebangsaan Malaysia (UTM/2013/AZILA/15 MAY/513- MAY-2013-DEC-2014-AR-CATZ).
For equilibration, the diffusion cells were continuously stirred at 300 rpm at 37 °C for 30 min. Next, the samples were
added to the donor compartments. Subsequently, 1-mL ali- quot of the sample was collected at predetermined time inter- vals from the receptor chamber. In order to maintain a constant volume inside the chamber throughout the experiment, fresh buffer of the same amount of the collected aliquot was added to the receptor chamber.
The collected samples were mixed with a mixture of etha- nol and 1-propanol (6:4) at the ratio of 1:3 and vortexed for 5 min. After that, the samples were analysed using total phe- nolic compound (TPC) assay to quantify the amount of Fig. 1 aChromatogram ofF. deltoideaextract. Vitexin retention time (Rt) = 8.206. The peak of vitexin in the extract is highlighted.bChromatogram of F. deltoideaextract after spiking with vitexin standard . The peak was higher, indicating that the concentration of vitexin was amplified
F. deltoideain the samples expressed as gallic acid equivalent (GAE). The TPC assay was conducted based on the method by Almey et al. [23] in dark condition. A total of 0.1 mL of the sample was added to 0.75 mL of 10-fold dilution of Folin–Ciocalteu reagent and was left for 5 min.
Next, 0.75 mL of 6% (w/v) sodium carbonate was added, and the absorbance rate was read at 725 nm with a UV-VIS spectrophotometer (Scinco S-1350, USA) after 90 min of incubation.
2.11 Anti-melanogenic Study
The anti-melanogenic study was based on the method described by Yoon et al. [24]. RHPE type VI from EpiSkin™ (0.5-cm2 diameter) was used for this part of the study. The RHPEs were transferred from the initial 12-well plate into a 6-well plate containing the provided maintenance medium and were incubated in a CO2 in- cubator at 37 °C and 5% CO2 overnight. The duration of this investigation was 6 days. The samples were ap- plied to the RHPEs only for the first 4 days, while the maintenance medium was changed daily.
2.12 In Vitro Melanin Content Determination
Melanin assay was based on the method described by Yoon et al. [24]. The RHPEs were placed in PBS buffer and then were centrifuged. Following that, the pellet was solubilised in 1 mL of 1 N NaOH/10% (v/v) DMSO for 2 h at 80 °C. After that, the solution was centrifuged at 492g for 10 min at room temperature before the supernatant was transferred to fresh tubes.
The absorbance of the supernatants was measured at 470 nm. Melanin synthetic was used as the standard.
2.13 Statistical Analysis
All analyses including determination of particle size, zeta potential, polydispersity index (PDI), encapsulation efficiency, permeability study, and anti-melanogenic study were done in triplicate in accordance with the International Conference of Harmonisation (ICH) recom- mendation for validation of analytical procedures, where at least three batches of experiments were conducted.
Statistical significance of the results was assessed using Student’s t test. In the analysis, p< 0.05 indicated sig- nificance of the value and p> 0.05 indicated no signif- icant difference between the compared results. Statistical analyses were performed using Microsoft Office Excel, 2013.
3 Results and Discussion
3.1 Comparison of the Double-Emulsion Solvent Diffusion and Thin Film Ultra-sonication Dispersion Techniques
NLC was prepared using the DESD and TFUD techniques to compare entrapment efficiency of the NLC obtained using the two methods. The DESD technique is composed by the fol- lowing steps: primary emulsification ofF. deltoideaextracts in lipid solutions, re-emulsification of primary emulsion in the second aqueous phase, and solidification by removing ethyl acetate, a partially water-soluble organic solvent [16]. The double-emulsion system was expected to minimise expulsion of hydrophilic extract to the aqueous medium. The TFUD technique involved formation of a thin lipid film in a reduced pressure condition, hydration with an aqueous solution, and ultra-sonication to down size the formed particles [17,18].
Emulsifiers were applied to stabilise the NLC suspension.
For both methods, TRI was used as the solid lipid and OA as the liquid lipid. The characteristics of the obtained NLC are summarised in Table1.
The favourable particle size for NLC dispersion for the purpose of cosmetic formulation ranges between 100 and 300 nm. Particles smaller than 100 nm need to be subjected to additional cytotoxicity screening [25], while particles ex- ceeding 300 nm cannot effectively penetrate into deeper skin as it only reaches the SC [26]. The particle size for NLC prepared using DESD was 155.9 ± 7.11 nm, which is consid- ered more appropriate for cosmetic formulation. The PDI for both techniques exhibited insignificant variance (p> 0.05), as the values of PDI for DESD and TFUD were 0.25 ± 0.03 and 0.28 ± 0.03, respectively. The presence of Tween 80 in both preparation techniques facilitated the monodispersity of NLC dispersion [27].
Zeta potential for DESD was lower than that of TFUD. The values for both DESD and TFUD were−9.4 ± 1.60 mV and− 22.4 ± 0.55 mV, respectively. Electrostatically stable disper- sions were only obtained if zeta potential exceeded ± 30 mV [28]. Despite the low zeta potential, incorporation of Tween 80 while preparing NLC stabilised the dispersion due to the presence of polyoxyethylene groups [29]. The physical barrier of the adsorbed molecules hindered the particles from coalesc- ing [30,31]. The difference in zeta potential of both methods might be attributed to the dissimilarity in the NLC structure obtained using two different methods. For NLC prepared using DESD, it contained Span 80 in the inner aqueous phase and Tween 80 stabilised the NLC suspension, whereas both Tween 80 and Span 80 in NLC prepared via TFUD stabilised the NLC suspension.
The EE of NLC prepared using DESD (67.92 ± 10.55%) was higher than the NLC prepared using TFUD (26.85 ± 3.50%). The presence of Span 80 in the inner aqueous phase
stabilisedF. deltoideawater extract and minimised its escape from the inner phase, hence improving its encapsulation effi- ciency and stability (Cohen-Sela et al. 2009). For the rest of the study, NLC was prepared using the DESD technique.
3.2 Influence of Lipids on Particle Size and Zeta Potential
In the remaining part of the research, NLCs were prepared using different solid and liquid lipids to assess the effects of the lipids on the characteristics of NLCs. The NLCs were composed of either SA or TRI as the solid lipid and either OA, VCO, or SO as the liquid lipid. Both particle size and zeta potential of the NLCs were analysed over a period of 90 days (see Fig.2).
The particle sizes for all formulations that utilised SA and TRI ranged between 150 and 200 nm from day 1 until day 30.
On day 60, the particle size for NLC formed with TRI as solid lipid and VCO or SO as liquid lipid exhibited an increase of approximately 300 nm and 500 nm, respectively. On day 90, the particle size for NLC composed of TRI as solid lipid and VCO or SO as liquid lipid further increased to about 700 nm and nearly reached 1μm, respectively. On the contrary, for- mulation of NLC using VCO or SO as liquid lipid and SA as solid lipid remained stable until day 90 (see Fig. 2a). The particle size for NLC formulation using OA as liquid lipid, and either SA or TRI as solid lipid, remained stable until day 90.
Due to the highly hydrophobic nature of TRI, the stability of NLC dispersion was affected probably due to the difference
Legend: day 1 day 14 day 30 day 60 day 90 0
50 100 150 200
OA VCO SO
Particle Size (nm)
liquid lipids
solid lipid: SA a)
-20 -15 -10 -5 0
OA VCO SO
zeta potential (mV)
liquid lipids
solid lipid: SA b)
b)
0 200 400 600 800 1000
OA VCO SO
Particle size (nm)
liquid lipids
solid lipid: TRI
*
*
*
* c)
-15 -10 -5 0
OA VCO SO
zeta potential (mV)
liquid lipids
solid lipid: TRI
d) Fig. 2 The stability of
nanostructured lipid carriers (NLC) prepared with SA or TRI as solid lipid, oleic acid (OA), virgin coconut oil (VCO), and soybean oil (SO) as liquid lipids with respect to particle size and zeta potential. Graphs a and b show the particle size and zeta potential variation of NLC pro- duced using SA as solid lipid and OA, VCO, or SO as liquid lipids over the period of 90 days.
Graphs c and d summarise the particle size and zeta potential of NLC developed using TRI as solid lipid and OA, VCO, or SO as liquid lipids over the period of 90 days. Each value represents the mean ± SD (n= 3). *p< 0.05 in graph c indicates significant dif- ference in comparison with graph a
Table 1 Characteristics of NLC prepared using double-emulsion solvent diffusion (DESD) and thin film ultra-sonication dispersion (TFUD) Preparation technique Particle size (nm) pvalue PDI pvalue Zeta potential (mV) pvalue Encapsulation efficiency (%) pvalue
DESD 155.9 ± 7.1* 0.003 0.25 ± 0.03 0.001 −9.4 ± 1.6* 0.048 67.92 ± 10.6* 0.025
TFUD 93.7 ± 0.3* 0.012 0.28 ± 0.03 0.019 −22.4 ± 0.6* 0.006 26.85 ± 3.5* 0.041
DESD, double-emulsion solvent diffusion method; TFUD, thin film ultra-sonication dispersion method
Both methods utilised tristearin as solid lipid and oleic acid as liquid lipid. Each value represents the mean ± SD (n= 3)
*p< 0.05 as compared with TFUD, indicating statistically significant values
in polarity between TRI and hydrophilicF. deltoideaextract, thus leading to particle aggregation. In comparison, SA, which exhibited good solubility in both polar and non-polar solvents [32], exhibited high possibility of blending well with aqueous F. deltoideaextract to generate more stable NLC dispersion.
OA appeared to be compatible with both solid lipids. This occurrence is attributable to the structure of OA, which is a single unsaturated fatty acid with a single double-bond possessing amphiphilic feature. This is in contract to VCO and SO, which are composed of various types of fatty acids with different lengths of hydrocarbon chains. Perhaps, the binding of a single fatty acid of the liquid and solid lipids resulted in better lipid matrix formation and stable dispersion during storage, when compared with the use of liquid lipid with complex mixture of fatty acids while preparing NLC.
The stability of all NLC dispersions was mostly influenced by steric effect and less likely due to electrostatic effect (see Fig.2band Fig.2d).
3.3 Influence of Poly(Vinyl Alcohol) on Particle Size and Zeta Potential
In the next phase of the study, PVA was incorporated in the inner aqueous phase while preparing NLC to study the effect of PVA on promoting stability of NLC dispersion. In a previ- ous study, PVA had been successfully used as a stabiliser in the preparation of NLC [33].
Figure2a and cillustrate the particle size stability of NLC dispersion with SA or TRI as solid lipids and OA, VCO, or SO as liquid lipids with incorporation of PVA in all formulations.
The particle size for all formulations using SA and TRI ranged from 150 to 200 nm from day 1 until day 30. On day 60, the particle size for NLC formulation using TRI as solid lipid and VCO or SO as liquid lipid increased to approximately 300 nm and 600 nm, respectively (p> 0.05). On day 90, the particle size for NLC formulation using TRI as solid lipid and VCO or SO as liquid lipid did not increase further, as was observed when PVA was excluded from the formulation (see Fig.2c).
NLC dispersion using VCO or SO as liquid lipid and SA as solid lipid remained stable until day 90. The particle size for NLC formulation using OA as liquid lipid, and either SA or TRI as solid lipid, remained stable until day 90 (p< 0.05).
Figure3b and dshow the zeta potentials for NLCs formu- lated with SA and TRI as solid lipid and OA, VCO, or SO as liquid lipid, respectively. All the NLC formulations demon- strated low zeta potentials from day 1 to day 90, similar to NLC dispersions obtained without the addition of PVA.
The stability of the size of NLC formulated with incorpo- ration of PVA (see Fig.3) demonstrated an almost similar trend as the NLC formulated without PVA (see Fig.2). The presence of PVA was supposed to enhance the stability of the dispersion due to its cross-linking behaviour [14]. However, in this study, incorporation of PVA left no impact on the
stability of NLC formed using SA as the solid lipid, as the NLC dispersions were already stable. As for NLC formed using TRI as the solid lipid, PVA failed to arrest increment in the size of NLC formed using VCO or SO as liquid lipid from day 30 to day 60, but PVA managed to prevent further increment in size from day 60 to day 90.
The characteristics of NLCs prepared using SA as the solid lipid and OA, VCO, or SO as the liquid lipid with and without PVA are reported in Table2.
3.4 Fourier-Transform Infrared Analysis
The FTIR patterns ofF. deltoideaextract, NLC, and NLC–
F. deltoideaare shown in Fig.4.F. deltoideaextract exhibited bands at 3411 cm−1(O–H), 2929 cm−1(C–H), 1621 cm−1(C=C), 1419 cm−1(C–H), 1272 cm−1(C–O), and 1075 cm−1(C–N). Blank NLC showed lower C–H peak at 2900 cm−1, when compared with NLC-FD. The increase in C–H stretching peak indicated the presence ofF. deltoideaextract in NLC-FD. Similar trend was observed in the FTIR spectra of phenyl ethyl resorcinol–
loaded NLC [34]. X-ray diffraction (XRD) and energy disper- sive X-ray (EDX) non-destructive analyses are usually ap- plied to gain explanatory information on the inner structure of the nanoparticles. Physical characterisation of NLC using XRD and EDX is suggested for future research to obtain the information on crystalline structure and to verify the purity of elements in the formulation.
3.5 Morphology
With the aid of TEM, the morphology of NLC–SA–OA–PVA loaded with F. deltoidea water extract was observed. The NLC was spherical in shape with size ranging from 100 to 200 nm (see Fig.5). The sizes obtained by using TEM were similar to the sizes obtained from the DLS method.
3.6 In Vitro Release Studies
The estimated cumulative rate was calculated and plotted as a function of time for non-encapsulated F. deltoidea loaded NLC–SA–OA–PVA (Fig.6). Data were analysed from differ- ent fitting models of controlled released mechanisms includ- ing zero-order equation, first-order equation, and diffusion- controlled model using Higuchi and Korsmeyer–Peppas (Table3).
In this study, after a period of 10 hours, the release of more than 60% of the extract was achieved and the release levelled off after approximately 14 hours (Fig. 5A). In Table3, the fitted equations and coefficients of correlation of each control release model are shown. The best fit model was determined by means of the highest correlation coefficient R2. The best fit for the non-encapsulatedF. deltoidea extract was the zero order equation (R2 = 0.9544). The first order equation,
Korsmeyer-Peppas equation and Higuchi equation fitted the data of non-encapsulatedF. deltoideawith lower coefficients of correlation.
The best fit for NLC-SA-OA-PVA loaded with F. deltoidea extract was also zero order equation (R2= 0.9627) (Table3).
The release behaviour was nearly linear and the release rate reached 50% after 10 hours (Figure5).
3.7 Permeation Studies
Prior to the experimental work on skin, cytotoxicity and skin irritation tests were conducted on F. deltoidea extracts
(company confidential data not disclosed). The concentration ofF. deltoideaextracts used in this study was in the safe range based on the tests. NLC–SA–OA–PVA was selected for pen- etration study using rat skin from Sprague Dawley species.
Rat skin was used because the thickness and the composition of rat skin mimic those of the human skin [35]. The transport properties of NLC–SA–OA–PVA loaded withF. deltoidea water extract and non-encapsulatedF. deltoideawater extract are listed in Table4.
F. deltoidea–loaded NLC exhibited better transport prop- erties, when compared with non-encapsulated F. deltoidea water extract. This finding is supported by Verma et al. [26], Legend: day 1 day 14 day 30 day 60 day 90
0 50 100 150 200
OA VCO SO
Particle size (nm)
liquid lipid
solid lipid: SA a)
-20 -15 -10 -5 0
OA VCO SO
Zeta potential (mV)
liquid lipids
solid lipid: SA b)
0 100 200 300 400 500 600
OA VCO SO
Particle size (nm)
liquid lipids
solid lipid: TRI c)
*
**
*
-20 -15 -10 -5 0
OA VCO SO
Zeta potential (mV)
liquid lipid
solid lipid: TRI d)
Fig. 3 Stability study of nanostructured lipid carriers (NLC) prepared with SA or TRI as solid lipid, and oleic acid (OA), virgin coconut oil (VCO), or soybean oil (SO) as liquid lipids with incorporation of poly(vinyl alcohol) (PVA) in all formula- tions with respect to particle size and zeta potential. Graphs a and b show the particle size and zeta potential variation of NLC when prepared using SA as solid lipid and OA, VCO, or SO as liquid lipids. Graphs c and d summarise the particle size and zeta potential variation of NLC that utilised TRI as solid lipid and OA, VCO, or SO as liquid lipids. Each value represents the mean ± SD (n= 3).*p< 0.05 in graph c indicate a significant difference as com- pared with graph a
Table 2 Characteristics of NLCs prepared using stearic acid as the solid lipid and various lipids as the liquid lipid with and without PVA
Lipids PVA Particle size (nm)
pvalue PDI pvalue Encapsulation efficiency (%)
p value
Oleic acid X 151.3 ± 6.15 0.029 0.19 ± 0.03 0.018 49.75 ± 8.36 0.042
Virgin coconut oil
X 136.4 ± 38.54 0.041 0.21 ± 0.05 < 0.001 51.87 ± 6.70 0.007
Soybean oil X 152.7 ± 3.54 0.002 0.21 ± 0.05 0.022 54.85 ± 3.22 0.035
Oleic acid ✓ 168.4 ± 0.10 < 0.001 0.19 ± 0.03 0.017 56.02 ± 4.50 0.044 Virgin
coconut oil
✓ 149.2 ± 1.27 0.005 0.20 ± 0.02 0.021 51.55 ± 9.40 0.031
Soybean oil ✓ 164.3 ± 2.61 < 0.001 0.22 ± 0.01 < 0.001 60.68 ± 3.12 0.028
who reported that vesicles with particle size≤300 nm were small enough to penetrate and deliver active compounds to some extent into the skin.
SC is composed of keratin filaments and water, enclosed within a cell envelope made of dense cross- linked protein layers, wherein the cell envelope func- tioned as an interface between hydrophilic corneocytes and lipophilic intercellular non-polar lipids [36]. Since F. deltoidea water extract displayed hydrophilic
properties, it would face difficulties in penetrating the intercellular part of the SC due to the difference in polarity, thus increasing the lag phase of the skin diffu- sion. On the contrary, F. deltoidea that was loaded within NLC required shorter time to diffuse through the skin based on its size and the incorporation of OA in the NLC formulations. OA also can act as skin pen- etration enhancer through the phase separation mecha- nism, depending on the polarity of the molecule [37].
OA is a major unsaturated fatty acid that appears in SC lipid bilayer. This explains why OA can modulate the properties of lipid bilayer of SC, hence providing better skin penetration [38].
The final NLC formulation was optimised and validated using a classic optimisation method (data not shown). The final concentration of the non-ionic surfactant to stabilise the NLC in suspension was 13.4%. Non-ionic surfactants displayed low skin sensitisation potential, thus are preferable for dermal application. The surfactant also seemed to affect skin permeability. Non-ionic surfactants at concentrations ranging from 0.1 (w/w) to 40% (w/w) have been reported to function as penetration enhancers (Iti Som et al. 2012 [39]).
Hence, Tween 80, which can serve as penetration enhancer, was also involved in the penetration of NLC–SA–OA–PVA through the skin.
For topical application, the depth of penetration de- pends on particle size. Adib et al. [40] reported that SLN smaller than 100 nm gave the most promising
Absorbance
20
15
10
5 ) 4 ( e l p m a s
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400
Wavenumber
Fig. 4 FTIR spectrum ofa. NLC-F. deltoidea (uppermost spectrum),b. Blank NLC (middle spectrum),c. F. deltoidea extract (lowermost spectrum)
Fig. 5 Representative transmission electron microscopy (TEM) image of NLC with stearic acid as solid lipid and oleic acid as liquid lipid and loaded with F. deltoidea water extract. The morphology observed was round in shape under 100-kV resolution. Magnification: × 50,000
outcomes to penetrate into deep skin layers. This finding is supported by a penetration study of SLN conducted by Jenning et al. [41], where SLN of approximately 200 nm was found mostly in the upper skin layers. They discov- ered that the active ingredients reached a high level in the viable epidermis without parallel increase in the deeper layers of the skin. Based on these findings, it was expect- ed that a negligible amount of F. deltoideaextract might reach systemic circulation, if any. This discussion
supports the assumption that NLC–SA–OA–PVA will reach melanocyte cells located in viable epidermis.
3.8 Anti-melanogenic Properties of
Ficus deltoidea The efficacy ofF. deltoidea–loaded NLC was studied using type 6 RHPE. Type 6 RHPE (Skin Ethics, France) is multi-layered, stratified, and pigmented reconstructed epidermis.F. deltoideaLegend: F. deltoidea NLC-SA-OA-PVA
linear (F. deltoidea) linear (NLC-SA-OA-PVA) R² = 0.9627
R² = 0.9544
0 20 40 60 80 100 120
0 4 8 12 16 20 24
Cumulative release %
Time (h)
a
R² = 0.849
R² = 0.8497 0.00
0.50 1.00 1.50 2.00 2.50
0 10 20 30
Log cumulative % drug remaining
Time (h)
R² = 0.9106 R² = 0.9099
-40 -20 0 20 40 60 80 100 120
0.00 2.00 4.00 6.00
desaelergurd%evitalumuC
Square root of time (√h)
R² = 0.9496 R² = 0.929
0.00 0.50 1.00 1.50 2.00 2.50
0.00 0.50 1.00 1.50
Log cumulative % drug released
Log time
b
c d
Fig. 6 The release profile of non- encapsulatedF. deltoideaand F. deltoideafrom NLC–SA–OA– PVA containingF. deltoidea. The release profile is fitted with the following release models:a zero order,bfirst order,c Higuchi, anddKorsmeyer–
Peppas. Significance level,p= 0.05
Table 3 Fitted equations of different release models.Q= release rate,k= release constant, t= time (h)
Samples Equation R2 K
F. deltoidea Zero order,Q=k·t 0.9544 0.5164 p= 0.043
First order,Q=a· (1−exp. (−k·t)) 0.8497 2.3646 p= 0.045
Higuchi,Q=k· (t) 0.5 0.9099 1.6354 p= 0.024
Korsmeyer–Peppas,Q=k· tn 0.9290 0.2453 p= 0.002,n= 1.51
NLC–SA–OA–PVA Zero order,Q=k·t 0.9627 1.3255 p= 0.003
First order,Q=a· (1−exp. (−k·t)) 0.8490 2.2595 p= 0.047
Higuchi,Q=k· (t) 0.5 0.9106 25.098 p= 0.001
Korsmeyer–Peppas,Q=k· tn 0.9496 0.1055 p= 0.037,n= 1.55
extract would need to pass through all epidermis layers before reaching the melanocyte cells located in the stratum basal.
The type of samples and the amount applied on the RHPEs are tabulated in Table5. Figure7shows the melanin concen- trations in RHPE after 6 days of treatment. The cells were treated with the samples during the first 4 days, while the medium was changed daily. Deionised water was used as negative control and kojic acid was applied as positive control.
Kojic acid possessed tyrosinase inhibition activity and free radical scavenging activity, and demonstrated prevention of photo damage. The application of kojic acid at a dose of 6.84μg/cm2resulted in significant reduction of melanin con- centration to 0.471μg/mL, signifying 48.8% of melanin re- duction. The dose of non-entrappedF. deltoideaextract ap- plied on RHPE skin was 4.8 × 10−3 μg/cm2. The melanin concentration in RHPE was reduced to 0.549μg/mL, indicat- ing 40.35% of melanin reduction.
This proves that F. deltoidea is a promising anti- melanogenic agent. In this study,F. deltoideawas encapsu- lated to improve its efficiency. Application of NLC–SA–OA– PVA loaded withF. deltoideaextract at the dose of 6.7 × 10−4μg/cm2led to a reduction of melanin concentration to 0.545 μg/mL, indicating 40.70% of melanin reduction.
Although the dose ofF. deltoideaapplied in the NLC carrier
was lower than the dose ofF. deltoideaextract, the melanin content decreased to a similar level, suggesting that entrap- ment had improved the efficiency of the extract in minimising melanin content. Increment in the dose of F. deltoidea in NLC–SA–OA–PVA to 2.7 × 10−3μg/cm2further decreased the concentration of melanin to 0.333 μg/mL, signifying 64.88% of melanin reduction.
The results suggest that by loadingF. deltoideaextracts in NLC, the delivery ofF. deltoideainto viable epidermis layer was enhanced. This allowedF. deltoideaextract to reach me- lanocytes and effectively inhibit melanin, when compared with non-entrappedF. deltoideaextract.
4 Conclusion
This study demonstrates the possibility of loading a high amount of aqueousF. deltoideaextract in NLC by employing the DESD technique through incorporation of simple fatty acids as solid and liquid lipids to improve the characteristics of NLCs. Stearic acid as solid lipid offered more stability in NLC dispersion for 90 days, when compared with that of TRI.
F. deltoideathat was loaded in NLC with SA as solid lipid, OA as liquid lipid, and inclusion of PVA demonstrated good Table 4 Transport properties ofF. deltoidea–loaded NLC through rat skin
Samples Jss (mg/cm2h) tL(h) D(cm2/h) Kp(cm/h)
NLC–SA–OA–PVA 1.35 × 10−1± 0.04 0.13 ± 0.01 7.48 × 10−3± 0.003 3.19 ± 0.87
F. deltoideaextract 0.33 × 10−1± 0.01* 1.l0 ± 0.19* 0.91 × 10−3± 0.007* 0.79 ± 0.15*
NLC–SA–OA–PVA is the NLC formulation composed of stearic acid (SA) as solid lipid and oleic acid (OA) as liquid lipid with the incorporation of polyvinyl alcohol (PVA). TheF. deltoideaextract tested in this phase was the non-encapsulated extract of the same concentration as the encapsulated F. deltoideaextract in NLC formulation
Jss = flux,tL= lag phase,D= diffusivity,Kp= permeability coefficient Each value represents the mean ± SD (n= 3)
*p< 0.05 indicates statistically significant values
Table 5 List of samples for RHPE EpiSkin™anti- melanogenic study
No. Samples Dose/area (μg/cm2) pvalue
1 Deionised water (negative control) -
2 Kojic acid (positive control) 6.84 0.027
3 F. Deltoideaextract 4.8 × 10−3 0.043
4 NLC–SA–OA–PVA loaded withF. Deltoideaextract 2.7 × 10−3 < 0.001 5 NLC–SA–OA–PVA loaded withF. Deltoideaextract 6.7 × 10−4 0.005 The dose/area was calculated based on the concentration of the compound. The dose/area for sample No. 3 was equal to the initial concentration ofF. deltoideaextract incorporated in NLC formulation. In comparison, the dose/
area for samples No.4 and No. 5 was calculated based on 56.02% encapsulation efficiency of NLC–SA–OA– PVA that was loaded withF. deltoidea. NLC–SA–OA–PVA was the NLC formulation composed of stearic acid (SA) as solid lipid and oleic acid (OA) as liquid lipid with the incorporation of poly(vinyl alcohol) (PVA)
characteristics and transport properties. Anti-melanogenic ef- ficacy study demonstrated thatF. deltoideaextract entrapped in NLC managed to reduce melanin more effectively, when compared with non-entrapped F. deltoidea extract.
Nevertheless, in order to prove the efficacy of this preliminary study, further analyses using mice model are needed.
However, for cosmetic products, animal testing is completely banned in the European countries starting March 2013, thus in vitro data is sufficient. NLC loaded withF. deltoideahas a tremendous potential to be incorporated in a skincare product as a whitening agent.
Acknowledgements The authors are grateful to Universiti Teknologi Malaysia, Malaysia, for providing the necessary facilities, assistance, and support for carrying out the project.
Author Contribution Statements Siti Maria Abdul Ghani conceived and planned the experiments. She wrote the manuscript with support from Nur Zatul Iradah Roslan and Rohaiza Muda. Azila Abdul Aziz encour- aged and supervised the research. All authors discussed the results and contributed to the final manuscript.
Funding This work was supported by the Research University Grant (No: QJI 30000 2544.04H51).
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
Research Involving Humans and Animals Statement None.
Ethical Issues The design of penetration study using rat skin were reviewed and approved by the Animal Ethics Committee of Universiti Kebangsaan Malaysia (ethical code: UTM/2013/AZILA/15 MAY/513- MAY-2013-DEC-2014-AR-CATZ).
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