- Email: [email protected]
Assessment of pharmacokinetic parameters of lupeol in Ficus religiosa L. extract after oral administration of suspension and solid lipid nanoparticles to Wistar rats
Journal of Drug Delivery Science and Technology 41 (2017) 58e67
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Assessment of pharmacokinetic parameters of lupeol in Ficus religiosa L. extract after oral administration of suspension and solid lipid nanoparticles to Wistar rats Karunanidhi Priyanka a, Ramoji Kosuru a, Raju Prasad Sharma a, Puran Lal Sahu b, Sanjay Singh a, * a b
Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005, India Indian Pharmacopoeia Commission, Ministry of Health and Family Welfare, Government of India, Sector-23, Raj Nagar, Ghaziabad, 201002, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 April 2017 Received in revised form 24 June 2017 Accepted 25 June 2017 Available online 29 June 2017
In spite of wide applications of herbal drugs as phytochemicals or extracts, their application is limited because of their poor bioavailability. The aim of the present work was to enhance the poor bioavailability of lupeol present in Ficus religiosa L. extract through solid lipid nanoparticles (SLN). Ficus religiosa L. extract loaded SLN were prepared and administered orally to male Wistar rats for bioavailability studies. Developed SLN were characterized for particle size, PDI, zeta potential, entrapment efficiency, in vitro drug release and stability studies. Further, interaction studies were carried out. Bioavailability studies were performed for Ficus religiosa L. extract in suspension form and SLN form. AUC of lupeol was increased by 9.2 - folds in rats treated with SLN compared to suspension. Also, Cmax of lupeol was increased by 3.9 - folds in rats treated with SLN compared to suspension. t1/2 of lupeol was found to be 7.3 ± 1.0 h in suspension and 15.3 ± 1.3 h in SLN. SLN enhanced AUC and Cmax and prolonged t1/2 of lupeol in Ficus religiosa L. extract which in turn may lead to dose reduction, prolonged duration of action and also enhanced therapeutic efficacy. © 2017 Elsevier B.V. All rights reserved.
Chemical compound studied in this article: Lupeol (PubChem CID: 259846) Keywords: Ficus religiosa L. Bioavailability Lupeol Solid lipid nanoparticles
1. Introduction Herbal drugs as single phytochemical or as extracts are known for their mixed pharmacological actions with the benefit of no or less harmful side effects. Phytochemicals such as curcumin [1], berberine [2] and paclitaxel [3] are well known for their effective pharmacological actions without or with less side effects and extracts such as Artichoke leaf extract [4], Cimicifuga foetida extract [5], Andrographis paniculata extract [6] and Ginkgo biloba extract [7] are widely used in the treatment of various diseases. The availability of synthetic drugs is more in the market than herbal drugs. The presence of several phytochemicals in a single plant or extract causes difficulty in carrying out qualitative and quantitative analyses. For qualitative and quantitative analyses, there is a need of reference compounds for each phytochemical present which results in higher cost of the experiment. Also, due to unavailability of
* Corresponding author. E-mail addresses: [email protected] (P.L. Sahu), [email protected], [email protected] (S. Singh). http://dx.doi.org/10.1016/j.jddst.2017.06.019 1773-2247/© 2017 Elsevier B.V. All rights reserved.
pharmacokinetic parameters of most of the phytochemicals in the current scenario limits the use of natural products. Ficus religiosa L. possesses strong anti-oxidant activity and it is included in several ayurvedic formulations for the treatment of diabetes, epilepsy, inflammatory conditions, microbials, gout, stomatitis, leucorrhea, ulcers and against several microbes [8,9]. Phytoconstituents present in the bark, leaves, fruits, latex and decoction of Ficus religiosa L. are responsible for its pharmacological actions. Alcoholic extracts (ethanolic and methanolic extracts) of Ficus religiosa L. bark were reported to contain phytosterolins such as lupeol, b-sitosterol, b-sitosterol-d-glucoside, stigmasterol, lanosterol and campesterol. Octacosanol, methyl oleonate and lupen-3one were reported in petroleum ether extract of the bark. In the benzene extract of bark, furanocoumarins (bergapten and bergaptol) were reported [10]. Even though several marketed ayurvedic formulations of Ficus religiosa L. are available, there is very less information about its qualitative and quantitative estimations and there is no reported pharmacokinetics data. In this context, we aimed to perform qualitative and quantitative estimations to find out the major
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
compound present in Ficus religiosa L. extract and proceeded to estimate the bioavailability of the major compound and developed a drug delivery system which enhances the bioavailability of the major compound. Solid lipid nanoparticles (SLN) are well known for enhancing bioavailability of poorly water soluble drugs by lymphatic uptake [11]. In the present study, pharmacokinetic parameters of Ficus religiosa L. extract were estimated. To avoid the complexity associated with analyzing each phytochemical present in the extract, this study focused on major phytochemical present in the ethanolic extract of Ficus religiosa L. Further, Ficus religiosa L. extract loaded SLN were developed. Comparison of pharmacokinetic profile of Ficus religiosa L. extract suspension with developed Ficus religiosa L. extract loaded SLN has been carried out and found that the major compound was found to have increased bioavailability in SLN form. Briefly, Ficus religiosa L. extract loaded SLN were prepared by using two lipids separately (glyceryl monostearate and compritol ATO 888) by hot homogenization followed by ultrasonication method using binary surfactant mixture (poloxamer 188 and sodium deoxycholate in the ratio of 25:75). Developed Ficus religiosa L. extract loaded SLN were characterized by particle size and PDI, zeta potential, entrapment efficiency, in vitro drug release and kinetics, fourier transform infra-red spectroscopy (FTIR), differential scanning calorimetry (DSC), powder Xray diffractrometry (PXRD) and stability studies. Based on in vitro characterization, best formulation was selected and bioavailability studies were conducted for Ficus religiosa L. extract suspension and SLN. From the results, it was found that the major compound present in the Ficus religiosa L. has very less bioavailability and the poor bioavailability was tremendously increased by SLN. We strongly anticipate that this study may provide pharmacokinetics parameters of lupeol present in Ficus religiosa L. extract and its bioavailability enhancement through SLN. 2. Materials and methods 2.1. Materials Glyceryl monostearate, poloxamer 188 and sodium deoxycholate were obtained as gift samples from Hospira Pvt. Ltd, Chennai, India. Dialysis membrane (cutoff MW 12,000e14,000 Da) was procured from HiMedia, India. Standard, lupeol (with the purity of 95% by HPLC) was procured from Sigma Aldrich, India. Water used in all experiments was purified by Milli-Q-plus system (Millipore, India). All other chemicals and solvents were of analytical grade. 2.2. Plant studied Plant studied in this study was Ficus religiosa L. Local name of Ficus religiosa L. is peepal tree. The plant name has been checked with www.theplantlist.org mentioning the data of accessing that website. The stem barks of Ficus religiosa L. were collected in the month of November from the locality of Banaras Hindu University, Varanasi, India and authenticated by Prof. R. S. Upadhyay, Department of Botany, Banaras Hindu University, Varanasi, India.
59
completely removed at 70 C using rotary evaporator (IKA RV 10) by nitrogen gas purging. Dried ethanolic extract was used for the study. 2.4. Standardization of Ficus religiosa L. extract For qualitative analysis, LCMS was carried out by using quadrapole time-of-flight spectrometer (G6520B, Agilent technologies) equipped with a electrospray ionisation source in both positive and negative modes. Minimum range and maximum range of 50 and 1500, respectively were used. Other parameters such as scan rate of 1, gas temperature of 300 C, gas flow of 10.0 l/minute and nebulizer pressure of 32 psi were used. Thin layer chromatography (TLC) was performed by using Silicagel G as adsorbent and n-butanol: acetic acid: water (4:0.5:5) as mobile phase. Plate was prepared by pouring silica gel slurry on glass plate and activated by heating at 110 C for 30 min. The spots were detected using vanillin reagent in sulphuric acid and Rf values were calculated. Preparative TLC using silica gel 60F254 precoated TLC plates (Merck) 20 20 cm was carried out by partitioning 25 g of ethanolic extract with 50 ml of petroleum ether. This extract was then concentrated and used for isolating major compound. FTIR analysis of major compound was done by using FTIR-8400S, Shimadzu by conventional KBr disc/ pellet method. An FTIR spectrum was measured over the range of 4000e400 cm1 with resolution of 4 cm1 for 50 scans. Structural elucidation of major compound was obtained from 1H NMR and 13C NMR analyses by using Bruker 400 Avance Spectrometer operating at 400 MHz using tetramethylsilane as internal standard. 2.5. RP HPLC analysis For the quantification of major compound present in Ficus religiosa L. extract, RP HPLC was carried out. RP HPLC setup (Waters, USA) comprising of binary pumps and PDA 2998 detector was used. Spherisorb ODS2 C18 column with dimensions of 250 4.6 mm was used. Acetonitrile: methanol: water combination was used as mobile phase in the ratio of 40:40:20. The mobile phase was filtered through 0.45 mm nylon filters (Millipore, USA). 20 mL volume samples were injected at the flow rate of 1 ml/min and analyzed at lmax of 211 nm. 2.6. Preparation of Ficus religiosa L. extract loaded SLN SLN were prepared by the combined method of hot homogenization and ultrasonication. Composition of all SLN formulations is shown in Table 1. Lipid (either glyceryl monosterate or compritol ATO 888) was melted at 5e10 C above the melting point of lipid and Ficus religiosa L. extract was dispersed into the melted lipid phase. Aqueous phase containing 0.5% concentration of surfactant (combination of poloxamer 188 and sodium deoxycholate in the ratio of 25:75) was poured into the lipid phase which was maintained at the same temperature of lipid phase. Then, homogenized at 12,000 rpm for 30 min using high speed homogenizer (T25, UT, IKA). SLN suspension was then allowed to cool at room temperature. Sonication of formed suspension was performed using
2.3. Preparation of ethanolic extract of Ficus religiosa L. Stem barks of Ficus religiosa L. were dried under sun for 15 days and powdered finely. 50 g of this powder was taken into the porous container of soxhlet apparatus. 500 ml of ethanol was used for extraction; 250 ml of ethanol was taken in a distilling pot and remaining ethanol was poured into porous container. Temperature of 40 C was maintained and soxhleted for 48 h. Further, solvent was recovered and dried the extract. Traces of organic solvent were
Table 1 Composition of SLN formulations. Ingredients
SLN 1
SLN 2
SLN 3
SLN 4
SLN 5
SLN 6
Drug (%) Glycerylmonostearate (%) Compritol ATO 888 (%) Surfactant (%) Distilled water (ml)
0.5 1 e 0.5 50
0.5 2 e 0.5 50
0.5 3 e 0.5 50
0.5 e 1 0.5 50
0.5 e 2 0.5 50
0.5 e 3 0.5 50
60
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
ultrasonic processor (200H, Hielscher) at 50% amplitude for 2.5 min. Then the final SLN suspension was lyophilized using lyophilizer (Decibel Digital Technologies, India) for 36 h at - 60 C temperature and pressure below 15 Pa.
Cumulative percentage drug release was then calculated from the amount of drug released. The results of in vitro release studies were fitted into different kinetic equations. 2.10. Fourier transform infrared studies
2.7. Particle size, polydispersity index, zeta potential and surface morphology Particle size, polydispersity index (PDI) and zeta potential of SLN formulations were measured by using particle size analyzer (Delsa™ Nano C, Beckman coulter). Fixed angle of 165 and temperature of 25 C were used for particle size and PDI measurements. Zeta potential of SLN was measured at 25 C. Analyses were performed in triplicate. Surface morphology of best formulation was assessed by using scanning electron microscope (SEM) (JSM 5600, JEOL) and atomic force microscope (AFM) (NT-MDT Solver NEXT, Russia). For SEM analysis, samples were stained with 2% (w/v) phosphotungstic acid and placed on copper grids for observation and for AFM analysis, few drops of SLN dispersion were placed on clean glass slide to form a film and it was used for observation in noncontact mode at a scanning rate of 0.5 Hz. 2.8. Entrapment efficiency and drug loading Entrapment efficiency (EE) was determined by measuring the concentration of unentrapped drug in aqueous medium. From the unentrapped drug concentration, entrapped drug concentration was determined. Briefly, 1 ml of SLN dispersion was taken in eppendroff tubes and centrifuged in a high speed cooling centrifuge (C-24, Remi) at 18,000 rpm for 45 min at 4 C. After centrifugation, supernatant was separated. The amount of unentrapped drug in supernatant was determined by using RP HPLC (Waters, USA). Estimation was performed by determining the major compound present in Ficus religiosa L. extract. The percentage entrapment efficiency (% EE) was calculated by using the following formula:
% EE ¼
Total drug content Unentrapped drug 100 Total drug content
Drug loading (%) was calculated by using the following formula:
%Drugloading¼
TotaldrugcontentFreedrug ðDrugamountusedFreedrugþWeightof lipidÞ
100
2.9. In vitro release studies Dialysis bag diffusion method was used for performing in vitro release studies of all SLN batches and Ficus religiosa L. extract suspension. 0.02 M hydrochloric acid, pH 1.2 was used as release medium for first 2 h followed by the use of phosphate buffer, pH 6.8 for the remaining time period. Dialysis membrane having molecular weight cut off 12,000 to 14,000 Da was used for holding the samples. Before using, dialysis membrane was soaked in distilled water for 10 min and tied at one end. SLN dispersion equivalent to 50 mg of lupeol was filled in the dialysis membrane bag and tied at another end and this bag was placed in a beaker containing 100 ml of release medium. Temperature and speed were maintained at 37 ± 2 C and 100 rpm, respectively using magnetic stirrer. Samples were withdrawn at predetermined time intervals and withdrawn volume was replaced with same volume of buffer to maintain sink condition. Samples were analyzed at lmax of 211 nm using RP HPLC.
Ficus religiosa L. extract and lipids interaction was observed using FTIR-8400S, Shimadzu. FTIR spectrum of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLN formulations were obtained by conventional KBr disc/pellet method. The samples were prepared by grinding with anhydrous KBr powder and compressed into pellets. FTIR spectra were measured over the range of 4000e400 cm1 with resolution of 4 cm1 for 50 scans. 2.11. Differential scanning calorimetry Thermograms of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLN formulations were obtained by using DSC25, Toledo, Mettler. Pierced aluminium pans were used to weigh the samples directly and scanned in the temperature range of 30e300 C under an atmosphere of dry nitrogen. Heating rate of 5 C/min was used. 2.12. Powder X-ray diffractometry PXRD pattern of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLN formulations were observed by using X'Pert Pro PAN with the conditions of Ni-filtered Cu-K radiation, voltage of 40 kV, and current of 1.5406 Å radiation scattered in the crystalline regions and measured with a vertical goniometer. Patterns were obtained by using a step size of 0.045 C with a detector resolution in 2q (diffraction angle) between 5 and 80 at 25 C temperature. 2.13. Stability studies The stability studies of best formulation were performed by storing at 30 ± 2 C temperature and 65% ± 5% relative humidity for 180 days and were examined at regular time intervals for changes in particle size, PDI, zeta potential and % EE. 2.14. Pharmacokinetic study 2.14.1. Animal study The study protocol was approved by Central Animal Ethical Committee (Ref. No. Dean/2014/CAEC/718), Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Twelve male Wistar rats weighing 220 ± 30 g were used and they were divided into two groups containing six each (n ¼ 6). All rats had free access to water and diet. They were housed in cages; a 12 h dark/light cycle was maintained. Room temperature of 21e24 C and relative humidity of 50e70% were maintained. General and environmental conditions were strictly monitored. Three more rats were used for calibrating lupeol in rat plasma. 2.14.2. Bioanalytical method development For the estimation of lupeol in the Ficus religiosa L. extract in rat plasma, RP HPLC method was developed. RP HPLC conditions were same as mentioned in the section 2.5. 2.14.3. Administration The rats were fasted for 12 h prior to dose administrations and for 4 h after dosing. Ficus religiosa L. extract suspension equivalent to 50 mg/kg of lupeol was administered orally into stomach of
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
group 1 rats. SLN equivalent to 50 mg/kg of lupeol was administered orally into stomach of group 2 rats. 2.14.4. Plasma sample preparation After drug administration, rats were anaesthetized with ether and heparinized capillary was inserted into retro-orbital vein of rats to collect 0.5 ml of blood at time intervals of 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h. Blood samples were centrifuged at 5000 rpm for 15 min and plasma samples were collected. It was stored immediately at 20 C until analysis. To 100 mL of plasma sample, 50 mL of methanol and 200 mL of acetonitrile were added to precipitate protein and vortexed (Remi, Cyclomixer, India) for 3 min. The denatured protein was separated by centrifugation at 15,000 rpm for 10 min at 4 C. Collected supernatant was transferred to fresh tube and filtered through 0.45 mm nylon filters. Aliquots of 20 mL were injected into the HPLC system for analysis. 2.14.5. Pharmacokinetic data analysis To access the pharmacokinetic parameters (Cmax, Tmax, AUC, and t1/2) of major compound present in Ficus religiosa L. extract, non compartmental analysis was done by using WinNonlin software (Pharsight Corp., Mountain View, CA, USA, Version 4.1). P-value less than 0.05 was considered to be significantly different using paired student's t-test. All data were presented as mean ± SD.
61
427.24 (Fig. 1). This shows that the mass to charge of the most abundant compound is 426. MS suggested the molecular formula of C30H50O. The spot corresponding to Rf value of 0.71 was scrapped from preparative TLC and it was isolated as colorless powder after several washes with organic solvents and recrystallized with ethanol. FTIR analysis of major compound (Fig. 2) showed a characteristic peak observed at 1450 cm1; this might belong to methylenic vibration, absorption frequencies observed at 3398 cm1 and 1383 cm1 might be due to O-H and C-O bond vibrations, respectively; peak observed at 976 cm1 might be due to an unsaturated out of plane C-H vibration; C¼C vibration was shown around 1649 cm1 as a weak intense band; and peaks at 2933 cm1 and 1383 cm1 might be stretching and bending vibrations. From 1H NMR (Fig. 3), a doublet at d 3.31 is due to the proton attached to secondary carbinol, two broad singlets were seen at d 4.59 and 4.65 due to the two exomethylene protons attached, hydromethine proton at d 3.19, broad singlets at 4.73 and 4.59 were indicative of olefinic protons, 2.36 d assigned to a methynic proton, a doublet at 1.97, 1.66 triplet assignable to a methyl group on double bond, a singlet at 1.23. From 13C NMR (Table 2), seven methyl groups, signals due to an exomethylene group; ten methylene, five methine and five quaternary carbons were observed. From the combined results of FTIR and NMR, the major compound was confirmed as lupeol.
2.15. Statistical analysis 3.2. RP HPLC analysis Results were given as mean ± standard deviation (SD). Mean values of nanoparticles size were compared using one-way analysis of variance (ANOVA) followed by post Tukey's test. 3. Results and discussion 3.1. Standardization of Ficus religiosa L. extract From the positive ion mode (M þ H)þ spectrum of Ficus religiosa L. extract, three peaks were obtained at charge (m/z) of 166.12, 180.10 and 427.24 and the most abundant peak was found to be
Specificity of lupeol (100 mg/ml) was determined by spiking with standard in triplicate. For the investigated compound, only one peak was observed at specified retention time ie. 9.0 min. LOD and LOQ of lupeol were found to be 23 ng/ml and 65 ng/ml, respectively. Precision (% RSD) and accuracy of lupeol in extract are shown in Table 3. The developed method was unaffected by changes in chromatographic parameters such as pH of mobile phase, mobile phase flow and stability of analytical solutions over the time period of 0, 24, 48 and 96 h. Quantity of lupeol found in the Ficus religiosa L. extract was found to be 43.67%.
Fig. 1. LCMS (positive mode) spectrum of Ficus religiosa L. extract.
62
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
Fig. 2. FTIR spectrum of marker compound.
Fig. 3. 1H NMR spectrum of marker compound.
3.3. Preparation of Ficus religiosa L. extract loaded solid lipid nanoparticles Ficus religiosa L. extract loaded SLN were prepared by using hot homogenization followed by ultrasonication method. The followed method was found to be reliable, simple, and reproducible. Prepared SLN formulations were found to be uniform and homogenous in appearance.
3.4. Particle size, PDI, zeta potential and surface morphology Results of particle size, PDI and zeta potential of all formulations are given in Table 4. It was observed that SLN prepared by using glyceryl monostearate had particle size in the range of 154e195 nm and SLN prepared by using compritol ATO 888 had particle size in the range of 202e287 nm. Increase in lipid concentration increased particle size which is independent of lipid type. Formulations
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67 Table 2 13 C NMR signals of marker compound.
63
Table 4 Size and charge analysis of all SLN batches.
d (ppm)
Type of carbon
d (ppm)
Type of carbon
Formulations
Particle size (nm)
15.6 16.2 16.3 18.1 19.0 19.3 21.3 24.9 27.9 28.0 30.3 34.5 36.2 37.3
-CH3 -CH3 -CH3 -CH3 ¼CH2 -CH3 ¼CH2 ¼CH2 ¼¼CH2 -CH3 ¼CH2 ¼CH2 ¼CH2 C
38.2 38.5 39.8 40.3 41.4 42.8 43.7 47.8 48.5 50.9 56.7 79.3 110.6 151.6
^C-H ¼CH2 C ¼CH2 C C C ^C-H ^C-H ^C-H ^C-H ^C-H ¼CH2 C
SLN SLN SLN SLN SLN SLN
154.0 183.4 195.4 202.7 256.3 287.1
1 2 3 4 5 6
± ± ± ± ± ±
27.61 30.64 45.01 33.47 29.36 41.89
PDI 0.301 0.312 0.379 0.457 0.547 0.599
Zeta potential ± ± ± ± ± ±
0.03 1.01 0.74 0.64 0.90 0.41
48.47 48.57 49.76 47.63 43.21 47.81
± ± ± ± ± ±
2.18 5.64 1.17 3.48 4.09 3.68
Mean Values ± SD; n ¼ 3.
containing compritol ATO 888 (SLN 4e6) had higher particle size than formulations containing glyceryl monostearate (SLN 1e3). This increase in particle size of SLN prepared by using compritol ATO 888 can be correlated with its higher melting point ie. 72 C than GMS (59 C). During destabilization of SLN, metastable form (b/) of compritol ATO 888 changes to more stable forms (bi or b). This increases particle size of SLN prepared using compritol ATO 888 than lipids with lower melting point [12]. Hence from the results, it was observed that higher the melting points of lipids, higher the particle size [13]. Increase in lipid concentration (in both types) increased the PDI and had no effect on zeta potential. The SEM image of best formulation is shown in Fig. 4. SLN particles diameter were found to be in nanometer range and spherical in shape. From the image of AFM (Fig. 5), spherical shape of particles was further confirmed and the particles diameter were found to be around 200 nm which was well correlated well with the size measurement observed by particle size analyzer. Average roughness was found to be 11.36 nm which indicates the surface smoothness of SLN.
Fig. 4. SEM image of best formulation.
3.5. Entrapment efficiency and drug loading From the results of EE, it was observed that increasing the lipid concentration increased the EE of both types of lipid [Table 5]. Higher concentration of lipid is believed to provide more number of molecules to cover the drug molecules and also prevents drug leaching into external phase, thus, ensuring highest EE [14]. There was no significant change in EE of batches containing glyceryl monostearate and compritol ATO 888. Drug loading of all formulations is shown in Table 5. Of all the formulations, SLN 3 had higher drug loading. Fig. 5. AFM image of best formulation.
3.6. In vitro release studies The release of lupeol from Ficus religiosa L. extract suspension was much faster with nearly 95% of lupeol diffused into the release medium at 3rd hour. SLN containing two different lipids displayed a
similar biphasic drug release pattern with a burst release within 30 min followed by sustained release afterwards (Fig. 6). The initial burst effect of the formulations containing glyceryl monostearate
Table 3 Precision and accuracy for the determination of lupeol in extract. Compound and Concentration (mg/ml)
Lupeol 50 100 150
Intraday assay
Interday assay
Mean (mg/ml)
Precision (%RSD)
Accuracy (%)
Mean (mg/ml)
Precision (%RSD)
Accuracy (%)
49.65 100.12 148.73
0.365 0.456 0.321
99.38 100.12 99.15
50.01 99.98 149.64
0.145 0.654 1.687
100.02 99.98 99.76
64
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
Table 5 Entrapment efficiency and drug loading of all formulations. Formulations
Entrapment efficiency (%)
SLN SLN SLN SLN SLN SLN
43.14 56.13 56.98 43.65 55.14 56.17
1 2 3 4 5 6
± ± ± ± ± ±
1.34 1.64 2.11 2.45 1.97 2.04
Drug loading (%) 8.25 9.68 9.78 8.34 9.55 9.72
± ± ± ± ± ±
1.23 1.82 2.35 2.18 0.97 0.79
Mean ± SD; n ¼ 3.
lupeol on the surface of nanoparticles. From the results, it was observed that higher the lipid ratio, higher the initial burst release. The sustained release of lupeol from the formulations containing glyceryl monostearate were found to be 59.1%, 56% and 41.6% for SLN 1, 2 and 3, respectively; and compritol containing formulations (SLN 4, 5 and 6) showed 52.1%, 44.3%, and 28.1%, respectively, of drug release over 24 h. The results revealed that the release was chiefly dependent on type and concentration of the lipids used ie. increase in lipid concentration sustained the release rate which is independent of the lipids type. This might be due to increased viscosity of formulations by increase in lipid concentration which slows the drug release from the lipid matrix (Muller et al., 2000). Among the lipids used, compritol showed sustained release than glyceryl monostearate due to its longer carbon chain length. All SLN formulations had higher linearity for Higuchi model. From the results of in vitro characterization of all SLN batches, batch SLN 2 was selected for bioavailability studies based on lesser particle size and higher entrapment efficiency. 3.7. Fourier transform infrared spectroscopy
Fig. 6. In vitro release profile of Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN batches.
FTIR spectra of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLNs are shown in Fig. 7. Ficus religiosa L. extract showed characteristic peaks at 3398 (O-H stretching), 2926 and 1383 (C-O bond vibrations, C-H stretching), 1629 (-C¼C- vibration), 1157 (C-N stretching) and 898 (-C¼C-H stretching). All these peaks are characteristic peaks of lupeol and all were present in Ficus religiosa L. extract loaded SLN and there was no absence of any functional peaks in all spectra. Thus, it revealed that there was no significant physicochemical interaction between drug and lipid. 3.8. Differential scanning calorimetry
varied from 7.5% to 21.9% (SLN 1e3); and for compritol 9.3e16.3% (SLN 4e6) and the burst release might be due to association of
DSC studies revealed that in the thermograms of SLN (using both lipids), Ficus religiosa L. extract peak was reduced and
Fig. 7. FTIR spectra: A - Extract, B e Glyceryl monostearate, C e Compritol ATO 888, D e SLN 2 and E SLN 5.
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
65
Fig. 8. DSC thermograms of A - Extract, B e Glyceryl monostearate, C e Compritol ATO 888, D e SLN 2 and E SLN 5.
broadened but no change was observed in lipids peak (Fig. 8). The broadening of Ficus religiosa L. extract peak in nanoparticles might be due to conversion of crystalline form to amorphous form. 3.9. Powder X-ray diffractometry PXRD spectra of standardized extract, glyceryl monostearate, compritol ATO 888 and SLN are shown in Fig. 9. The diffraction spectrum of Ficus religiosa L. extract showed characteristic peaks at 2q of 13.64, 14.74, 15.97, 19.37, 21.20, 21.57, 22.90, and 24.32 indicating crystalline nature of the Ficus religiosa L. extract. The crystalline peaks of Ficus religiosa L. extract were absent in SLN
formulations indicating that Ficus religiosa L. extract was not in crystalline form. Intensity of lipid peaks (glyceryl monostearate and compritol ATO 888) was also decreased in the SLN formulation. This reduced intensity confirms the decreased crystallinity of lipids in SLN formulations. 3.10. Stability studies After 180 days of storage, there were no significant changes in particle size, PDI, zeta potential and % EE of optimized formulation which revealed that the best formulation was stable over 6 months (Table 6).
Fig. 9. PXRD spectra of A - Extract, B e Glyceryl monostearate, C e Compritol ATO 888, D e SLN 2 and E SLN 5.
66
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
Table 6 Stability studies data. S. No.
Stability parameters
0 day
180 days
1 2 3 4
Particle size PDI Zeta potential % Entrapment efficiency
180 nm ± 21.67 0.347 ± 1.31 56.17 ± 5.64 59.14% ± 7.19
197 nm ± 32.14 0.314 ± 1.78 53.47 ± 7.63 58.67% ± 2.64
Mean values ± SD; n ¼ 3.
3.11. Pharmacokinetic study The HPLC peak of lupeol standard was free of interfering peaks of plasma at the retention time of 10.3 min. Linearity was obtained in the concentration range of 20e100 ng/ml with correlation coefficient (r2) values > 0.998. The inter- and intra-day precision of lupeol was within 2% relative standard deviation (RSD). Accuracy was between 98.24 and 99.15%. LOD and LOQ for lupeol were found to be 35.90 ng/ml and 81.64 ng/ml, respectively. The extraction efficacies in case of spiked plasma samples were 67 ± 17.4% with RSD values less than 6.2%. The mean plasma concentration vs. time curve profiles of lupeol from Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN are illustrated in Fig. 10. Pharmacokinetic parameters of lupeol in Ficus religiosa L. extract and SLN are shown in Table 7. The mean plasma AUC0e24 of lupeol in animals treated with SLN formulation was 9829.83 ± 56.12ng hr/ml whereas in animals treated with Ficus religiosa L. extract suspension, AUC0e24 of lupeol was 1068.46 ± 96.4 ng hr/ml. This increase in AUC0e24 for SLN might be due to the avoidance of first pass metabolism by lymphatic transport. The peak plasma concentration (Cmax) of lupeol in Ficus religiosa L. extract suspension was found to be 178.61 ± 24.6 ng/ml and in SLN formulation was found to be 696.57 ± 34.83 ng/ml. Time to reach plasma concentration (tmax) in Ficus religiosa L. extract suspension was found to be 6 ± 1.1 h and in SLN formulation was found to be 2 ± 0.11 h t1/2 of lupeol was found to be 7.3 ± 1.0 h in Ficus religiosa L. extract and 15.3 ± 1.3 h in SLN. From these results, it
clearly suggested that the pharmacokinetic profiles of lupeol have been improved in SLN form than Ficus religiosa L. extract after oral administration. 4. Conclusion Ficus religiosa L. extract loaded SLN were prepared successfully by optimizing lipids type and concentration. Best formulation was selected based on in vitro characterization of developed SLN. Pharmacokinetic studies of Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN showed greater increase in pharmacokinetic parameters of lupeol in SLN form than Ficus religiosa L. extract at the same dose of lupeol i.e. 50 mg/kg body weight. This study illustrates the pharmacokinetic parameters of lupeol in Ficus religiosa L. extract which is not reported before and its bioavailability enhancement through SLN. Conflict of interest The authors declare no conflicts of interest in this work. Acknowledgements The first author is grateful to the Indian Institute of Technology (Banaras Hindu University), Varanasi for providing financial support in the form of Teaching Assistantship funded by Ministry of Human Resource Development, Government of India. Authors acknowledge Special Assistance Programme of University Grants Commission, New Delhi, India for instrumentation facility (Particle size analyzer). The authors are thankful to Prof. O. N. Srivastava and Prof. R. K. Singh, Dept. of Physics, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India for carrying out for PXRD and DSC studies, respectively. The authors extend their sincere thanks to The Head, Dept. of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India for carrying out AFM studies and to Mr. Vinay K Ahire, Consortium for Scientific Research, Indore, Madhya Pradesh, India for carrying out SEM study. References
Fig. 10. Plasma concentration vs. time curve of lupeol in Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN.
Table 7 Pharmacokinetic parameters of lupeol in rat plasma. Parameters
Ficus religiosa L. extract
SLN
Cmax (ng/ml) Tmax (hr) AUC0-24 (ng hr/ml) t1/2 (hr)
178.61 ± 24.6 6 ± 1.1 1068.46 ± 96.4 7.3 ± 1.0
696.57 ± 34.83a 2 ± 0.11a 9829.83 ± 56.12a 15.3 ± 1.3a
Mean ± SD; n ¼ 6. a p< 0.05, significance difference compared to Ficus religiosa L. extract.
[1] C.S. Beevers, S. Huang, Pharmacological and clinical properties of curcumin, Bot. Targets Ther. (2011) 15e18. https://doi.org/10.2147/BTAT.S17244. [2] P.R. Vuddanda, S. Chakraborty, S. Singh, Berberine: a potential phytochemical with multispectrum therapeutic activities, Expert Opin. Investig. Drugs 19 (2010) 1297e1307. http://dx.doi.org/10.1517/13543784.2010.517745. ska, Pharmacological action of paclitaxel, Pol. Merkur [3] P. Potemski, A. Płuzan Lek. 6 (1999) 27e29. [4] S.M. Wittemer, M. Ploch, T. Windeck, S.C. Muller, B. Drewelow, et al., Bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of Artichoke leaf extracts in humans, Phytomedicine 12 (2005) 28e38. https://doi.org/10.1016/j.phymed.2003.11.002. [5] Y.Y. Gai, W.H. Liu, C.J. Sha, Y.L. Wang, Y.T. Sun, et al., Pharmacokinetics and bioavailability of cimicifugosides after oral administration of Cimicifuga foetida L. extract to rats, J. Ethnopharmacol. 143 (2012) 249e255. https://doi.org/10. 1016/j.jep.2012.06.031. [6] N. Sermkaew, W. Ketjinda, P. Boonme, N. Phadoongsombut, R. Wiwattanapatapee, Liquid and solid self-microemulsifying drug delivery systems for improving the oral bioavailability of andrographolide from crude extract of Andrographis paniculata, Eur. J. Pharm. Sci. 50 (2013) 459e466. https://doi.org/10.1016/j.ejps.2013.08.006. [7] Z.P. Chen, J. Sun, H.X. Chen, Y.Y. Xiao, D. Liu, et al., Comparative pharmacokinetics and bioavailability studies of quercetin, kaempferol and isorhamnetin after oral administration of Ginkgo biloba extracts, Ginkgo biloba extract phospholipid complexes and Ginkgo biloba extract solid dispersions in rats, Fitoterapia 81 (2010) 1045e1052. https://doi.org/10.1016/j.fitote.2010.06. 028. [8] R. Pandit, A. Phadke, A. Jagtap, Antidiabetic effect of Ficus religiosa extract in streptozotocin-induced diabetic rats, J. Ethnopharmacol. 128 (2010) 462e466. https://doi.org/10.1016/j.jep.2010.01.025. [9] M. Gregory, B. Divya, R.A. Mary, et al., Anti-ulcer activity of Ficus religiosa leaf ethanolic extract, Asian pac. J. Trop. Biomed. 3 (2013) 554e556. https://doi.
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67 org/10.1016/S2221-1691(13)60112-4. [10] D. Singh, B. Singh, R.K. Goel, Traditional uses, phytochemistry and pharmacology of Ficus religiosa: a review, J. Ethnopharmacol. 134 (2011) 565e583. https://doi.org/10.1016/j.jep.2011.01.046. [11] A.K. Kushwaha, P.R. Vuddanda, P. Karunanidhi, S.K. Singh, S. Singh, Development and evaluation of solid lipid nanoparticles of raloxifene hydrochloride for enhanced bioavailability, BioMed Res. Int. 584549 (2013), 10.1155/2013/ 584549. [12] M.H. Aburahma, S.M. Badr-Eldin, Compritol 888 ATO: a multifunctional lipid
67
excipient in drug delivery systems and nanopharmaceuticals, Expert Opin. Drug Deliv. 11 (2014) 1865e1883. [13] K. Priyanka, A.A. Sathali, Preparation and evaluation of montelukast sodium loaded solid lipid nanoparticles, J. young Pharm. 4 (2012) 129e137. https:// doi.org/10.4103/0975-1483.100016. [14] R.H. Muller, K. Mader, S. Gohla, Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art, Eur. J. Pharm. Biopharm. 50 (2000) 161e177, http://dx.doi.org/10.1016/S0939-6411(00)00087e4.
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Assessment of pharmacokinetic parameters of lupeol in Ficus religiosa L. extract after oral administration of suspension and solid lipid nanoparticles to Wistar rats Karunanidhi Priyanka a, Ramoji Kosuru a, Raju Prasad Sharma a, Puran Lal Sahu b, Sanjay Singh a, * a b
Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005, India Indian Pharmacopoeia Commission, Ministry of Health and Family Welfare, Government of India, Sector-23, Raj Nagar, Ghaziabad, 201002, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 April 2017 Received in revised form 24 June 2017 Accepted 25 June 2017 Available online 29 June 2017
In spite of wide applications of herbal drugs as phytochemicals or extracts, their application is limited because of their poor bioavailability. The aim of the present work was to enhance the poor bioavailability of lupeol present in Ficus religiosa L. extract through solid lipid nanoparticles (SLN). Ficus religiosa L. extract loaded SLN were prepared and administered orally to male Wistar rats for bioavailability studies. Developed SLN were characterized for particle size, PDI, zeta potential, entrapment efficiency, in vitro drug release and stability studies. Further, interaction studies were carried out. Bioavailability studies were performed for Ficus religiosa L. extract in suspension form and SLN form. AUC of lupeol was increased by 9.2 - folds in rats treated with SLN compared to suspension. Also, Cmax of lupeol was increased by 3.9 - folds in rats treated with SLN compared to suspension. t1/2 of lupeol was found to be 7.3 ± 1.0 h in suspension and 15.3 ± 1.3 h in SLN. SLN enhanced AUC and Cmax and prolonged t1/2 of lupeol in Ficus religiosa L. extract which in turn may lead to dose reduction, prolonged duration of action and also enhanced therapeutic efficacy. © 2017 Elsevier B.V. All rights reserved.
Chemical compound studied in this article: Lupeol (PubChem CID: 259846) Keywords: Ficus religiosa L. Bioavailability Lupeol Solid lipid nanoparticles
1. Introduction Herbal drugs as single phytochemical or as extracts are known for their mixed pharmacological actions with the benefit of no or less harmful side effects. Phytochemicals such as curcumin [1], berberine [2] and paclitaxel [3] are well known for their effective pharmacological actions without or with less side effects and extracts such as Artichoke leaf extract [4], Cimicifuga foetida extract [5], Andrographis paniculata extract [6] and Ginkgo biloba extract [7] are widely used in the treatment of various diseases. The availability of synthetic drugs is more in the market than herbal drugs. The presence of several phytochemicals in a single plant or extract causes difficulty in carrying out qualitative and quantitative analyses. For qualitative and quantitative analyses, there is a need of reference compounds for each phytochemical present which results in higher cost of the experiment. Also, due to unavailability of
* Corresponding author. E-mail addresses: [email protected] (P.L. Sahu), [email protected], [email protected] (S. Singh). http://dx.doi.org/10.1016/j.jddst.2017.06.019 1773-2247/© 2017 Elsevier B.V. All rights reserved.
pharmacokinetic parameters of most of the phytochemicals in the current scenario limits the use of natural products. Ficus religiosa L. possesses strong anti-oxidant activity and it is included in several ayurvedic formulations for the treatment of diabetes, epilepsy, inflammatory conditions, microbials, gout, stomatitis, leucorrhea, ulcers and against several microbes [8,9]. Phytoconstituents present in the bark, leaves, fruits, latex and decoction of Ficus religiosa L. are responsible for its pharmacological actions. Alcoholic extracts (ethanolic and methanolic extracts) of Ficus religiosa L. bark were reported to contain phytosterolins such as lupeol, b-sitosterol, b-sitosterol-d-glucoside, stigmasterol, lanosterol and campesterol. Octacosanol, methyl oleonate and lupen-3one were reported in petroleum ether extract of the bark. In the benzene extract of bark, furanocoumarins (bergapten and bergaptol) were reported [10]. Even though several marketed ayurvedic formulations of Ficus religiosa L. are available, there is very less information about its qualitative and quantitative estimations and there is no reported pharmacokinetics data. In this context, we aimed to perform qualitative and quantitative estimations to find out the major
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
compound present in Ficus religiosa L. extract and proceeded to estimate the bioavailability of the major compound and developed a drug delivery system which enhances the bioavailability of the major compound. Solid lipid nanoparticles (SLN) are well known for enhancing bioavailability of poorly water soluble drugs by lymphatic uptake [11]. In the present study, pharmacokinetic parameters of Ficus religiosa L. extract were estimated. To avoid the complexity associated with analyzing each phytochemical present in the extract, this study focused on major phytochemical present in the ethanolic extract of Ficus religiosa L. Further, Ficus religiosa L. extract loaded SLN were developed. Comparison of pharmacokinetic profile of Ficus religiosa L. extract suspension with developed Ficus religiosa L. extract loaded SLN has been carried out and found that the major compound was found to have increased bioavailability in SLN form. Briefly, Ficus religiosa L. extract loaded SLN were prepared by using two lipids separately (glyceryl monostearate and compritol ATO 888) by hot homogenization followed by ultrasonication method using binary surfactant mixture (poloxamer 188 and sodium deoxycholate in the ratio of 25:75). Developed Ficus religiosa L. extract loaded SLN were characterized by particle size and PDI, zeta potential, entrapment efficiency, in vitro drug release and kinetics, fourier transform infra-red spectroscopy (FTIR), differential scanning calorimetry (DSC), powder Xray diffractrometry (PXRD) and stability studies. Based on in vitro characterization, best formulation was selected and bioavailability studies were conducted for Ficus religiosa L. extract suspension and SLN. From the results, it was found that the major compound present in the Ficus religiosa L. has very less bioavailability and the poor bioavailability was tremendously increased by SLN. We strongly anticipate that this study may provide pharmacokinetics parameters of lupeol present in Ficus religiosa L. extract and its bioavailability enhancement through SLN. 2. Materials and methods 2.1. Materials Glyceryl monostearate, poloxamer 188 and sodium deoxycholate were obtained as gift samples from Hospira Pvt. Ltd, Chennai, India. Dialysis membrane (cutoff MW 12,000e14,000 Da) was procured from HiMedia, India. Standard, lupeol (with the purity of 95% by HPLC) was procured from Sigma Aldrich, India. Water used in all experiments was purified by Milli-Q-plus system (Millipore, India). All other chemicals and solvents were of analytical grade. 2.2. Plant studied Plant studied in this study was Ficus religiosa L. Local name of Ficus religiosa L. is peepal tree. The plant name has been checked with www.theplantlist.org mentioning the data of accessing that website. The stem barks of Ficus religiosa L. were collected in the month of November from the locality of Banaras Hindu University, Varanasi, India and authenticated by Prof. R. S. Upadhyay, Department of Botany, Banaras Hindu University, Varanasi, India.
59
completely removed at 70 C using rotary evaporator (IKA RV 10) by nitrogen gas purging. Dried ethanolic extract was used for the study. 2.4. Standardization of Ficus religiosa L. extract For qualitative analysis, LCMS was carried out by using quadrapole time-of-flight spectrometer (G6520B, Agilent technologies) equipped with a electrospray ionisation source in both positive and negative modes. Minimum range and maximum range of 50 and 1500, respectively were used. Other parameters such as scan rate of 1, gas temperature of 300 C, gas flow of 10.0 l/minute and nebulizer pressure of 32 psi were used. Thin layer chromatography (TLC) was performed by using Silicagel G as adsorbent and n-butanol: acetic acid: water (4:0.5:5) as mobile phase. Plate was prepared by pouring silica gel slurry on glass plate and activated by heating at 110 C for 30 min. The spots were detected using vanillin reagent in sulphuric acid and Rf values were calculated. Preparative TLC using silica gel 60F254 precoated TLC plates (Merck) 20 20 cm was carried out by partitioning 25 g of ethanolic extract with 50 ml of petroleum ether. This extract was then concentrated and used for isolating major compound. FTIR analysis of major compound was done by using FTIR-8400S, Shimadzu by conventional KBr disc/ pellet method. An FTIR spectrum was measured over the range of 4000e400 cm1 with resolution of 4 cm1 for 50 scans. Structural elucidation of major compound was obtained from 1H NMR and 13C NMR analyses by using Bruker 400 Avance Spectrometer operating at 400 MHz using tetramethylsilane as internal standard. 2.5. RP HPLC analysis For the quantification of major compound present in Ficus religiosa L. extract, RP HPLC was carried out. RP HPLC setup (Waters, USA) comprising of binary pumps and PDA 2998 detector was used. Spherisorb ODS2 C18 column with dimensions of 250 4.6 mm was used. Acetonitrile: methanol: water combination was used as mobile phase in the ratio of 40:40:20. The mobile phase was filtered through 0.45 mm nylon filters (Millipore, USA). 20 mL volume samples were injected at the flow rate of 1 ml/min and analyzed at lmax of 211 nm. 2.6. Preparation of Ficus religiosa L. extract loaded SLN SLN were prepared by the combined method of hot homogenization and ultrasonication. Composition of all SLN formulations is shown in Table 1. Lipid (either glyceryl monosterate or compritol ATO 888) was melted at 5e10 C above the melting point of lipid and Ficus religiosa L. extract was dispersed into the melted lipid phase. Aqueous phase containing 0.5% concentration of surfactant (combination of poloxamer 188 and sodium deoxycholate in the ratio of 25:75) was poured into the lipid phase which was maintained at the same temperature of lipid phase. Then, homogenized at 12,000 rpm for 30 min using high speed homogenizer (T25, UT, IKA). SLN suspension was then allowed to cool at room temperature. Sonication of formed suspension was performed using
2.3. Preparation of ethanolic extract of Ficus religiosa L. Stem barks of Ficus religiosa L. were dried under sun for 15 days and powdered finely. 50 g of this powder was taken into the porous container of soxhlet apparatus. 500 ml of ethanol was used for extraction; 250 ml of ethanol was taken in a distilling pot and remaining ethanol was poured into porous container. Temperature of 40 C was maintained and soxhleted for 48 h. Further, solvent was recovered and dried the extract. Traces of organic solvent were
Table 1 Composition of SLN formulations. Ingredients
SLN 1
SLN 2
SLN 3
SLN 4
SLN 5
SLN 6
Drug (%) Glycerylmonostearate (%) Compritol ATO 888 (%) Surfactant (%) Distilled water (ml)
0.5 1 e 0.5 50
0.5 2 e 0.5 50
0.5 3 e 0.5 50
0.5 e 1 0.5 50
0.5 e 2 0.5 50
0.5 e 3 0.5 50
60
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
ultrasonic processor (200H, Hielscher) at 50% amplitude for 2.5 min. Then the final SLN suspension was lyophilized using lyophilizer (Decibel Digital Technologies, India) for 36 h at - 60 C temperature and pressure below 15 Pa.
Cumulative percentage drug release was then calculated from the amount of drug released. The results of in vitro release studies were fitted into different kinetic equations. 2.10. Fourier transform infrared studies
2.7. Particle size, polydispersity index, zeta potential and surface morphology Particle size, polydispersity index (PDI) and zeta potential of SLN formulations were measured by using particle size analyzer (Delsa™ Nano C, Beckman coulter). Fixed angle of 165 and temperature of 25 C were used for particle size and PDI measurements. Zeta potential of SLN was measured at 25 C. Analyses were performed in triplicate. Surface morphology of best formulation was assessed by using scanning electron microscope (SEM) (JSM 5600, JEOL) and atomic force microscope (AFM) (NT-MDT Solver NEXT, Russia). For SEM analysis, samples were stained with 2% (w/v) phosphotungstic acid and placed on copper grids for observation and for AFM analysis, few drops of SLN dispersion were placed on clean glass slide to form a film and it was used for observation in noncontact mode at a scanning rate of 0.5 Hz. 2.8. Entrapment efficiency and drug loading Entrapment efficiency (EE) was determined by measuring the concentration of unentrapped drug in aqueous medium. From the unentrapped drug concentration, entrapped drug concentration was determined. Briefly, 1 ml of SLN dispersion was taken in eppendroff tubes and centrifuged in a high speed cooling centrifuge (C-24, Remi) at 18,000 rpm for 45 min at 4 C. After centrifugation, supernatant was separated. The amount of unentrapped drug in supernatant was determined by using RP HPLC (Waters, USA). Estimation was performed by determining the major compound present in Ficus religiosa L. extract. The percentage entrapment efficiency (% EE) was calculated by using the following formula:
% EE ¼
Total drug content Unentrapped drug 100 Total drug content
Drug loading (%) was calculated by using the following formula:
%Drugloading¼
TotaldrugcontentFreedrug ðDrugamountusedFreedrugþWeightof lipidÞ
100
2.9. In vitro release studies Dialysis bag diffusion method was used for performing in vitro release studies of all SLN batches and Ficus religiosa L. extract suspension. 0.02 M hydrochloric acid, pH 1.2 was used as release medium for first 2 h followed by the use of phosphate buffer, pH 6.8 for the remaining time period. Dialysis membrane having molecular weight cut off 12,000 to 14,000 Da was used for holding the samples. Before using, dialysis membrane was soaked in distilled water for 10 min and tied at one end. SLN dispersion equivalent to 50 mg of lupeol was filled in the dialysis membrane bag and tied at another end and this bag was placed in a beaker containing 100 ml of release medium. Temperature and speed were maintained at 37 ± 2 C and 100 rpm, respectively using magnetic stirrer. Samples were withdrawn at predetermined time intervals and withdrawn volume was replaced with same volume of buffer to maintain sink condition. Samples were analyzed at lmax of 211 nm using RP HPLC.
Ficus religiosa L. extract and lipids interaction was observed using FTIR-8400S, Shimadzu. FTIR spectrum of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLN formulations were obtained by conventional KBr disc/pellet method. The samples were prepared by grinding with anhydrous KBr powder and compressed into pellets. FTIR spectra were measured over the range of 4000e400 cm1 with resolution of 4 cm1 for 50 scans. 2.11. Differential scanning calorimetry Thermograms of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLN formulations were obtained by using DSC25, Toledo, Mettler. Pierced aluminium pans were used to weigh the samples directly and scanned in the temperature range of 30e300 C under an atmosphere of dry nitrogen. Heating rate of 5 C/min was used. 2.12. Powder X-ray diffractometry PXRD pattern of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLN formulations were observed by using X'Pert Pro PAN with the conditions of Ni-filtered Cu-K radiation, voltage of 40 kV, and current of 1.5406 Å radiation scattered in the crystalline regions and measured with a vertical goniometer. Patterns were obtained by using a step size of 0.045 C with a detector resolution in 2q (diffraction angle) between 5 and 80 at 25 C temperature. 2.13. Stability studies The stability studies of best formulation were performed by storing at 30 ± 2 C temperature and 65% ± 5% relative humidity for 180 days and were examined at regular time intervals for changes in particle size, PDI, zeta potential and % EE. 2.14. Pharmacokinetic study 2.14.1. Animal study The study protocol was approved by Central Animal Ethical Committee (Ref. No. Dean/2014/CAEC/718), Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Twelve male Wistar rats weighing 220 ± 30 g were used and they were divided into two groups containing six each (n ¼ 6). All rats had free access to water and diet. They were housed in cages; a 12 h dark/light cycle was maintained. Room temperature of 21e24 C and relative humidity of 50e70% were maintained. General and environmental conditions were strictly monitored. Three more rats were used for calibrating lupeol in rat plasma. 2.14.2. Bioanalytical method development For the estimation of lupeol in the Ficus religiosa L. extract in rat plasma, RP HPLC method was developed. RP HPLC conditions were same as mentioned in the section 2.5. 2.14.3. Administration The rats were fasted for 12 h prior to dose administrations and for 4 h after dosing. Ficus religiosa L. extract suspension equivalent to 50 mg/kg of lupeol was administered orally into stomach of
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
group 1 rats. SLN equivalent to 50 mg/kg of lupeol was administered orally into stomach of group 2 rats. 2.14.4. Plasma sample preparation After drug administration, rats were anaesthetized with ether and heparinized capillary was inserted into retro-orbital vein of rats to collect 0.5 ml of blood at time intervals of 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h. Blood samples were centrifuged at 5000 rpm for 15 min and plasma samples were collected. It was stored immediately at 20 C until analysis. To 100 mL of plasma sample, 50 mL of methanol and 200 mL of acetonitrile were added to precipitate protein and vortexed (Remi, Cyclomixer, India) for 3 min. The denatured protein was separated by centrifugation at 15,000 rpm for 10 min at 4 C. Collected supernatant was transferred to fresh tube and filtered through 0.45 mm nylon filters. Aliquots of 20 mL were injected into the HPLC system for analysis. 2.14.5. Pharmacokinetic data analysis To access the pharmacokinetic parameters (Cmax, Tmax, AUC, and t1/2) of major compound present in Ficus religiosa L. extract, non compartmental analysis was done by using WinNonlin software (Pharsight Corp., Mountain View, CA, USA, Version 4.1). P-value less than 0.05 was considered to be significantly different using paired student's t-test. All data were presented as mean ± SD.
61
427.24 (Fig. 1). This shows that the mass to charge of the most abundant compound is 426. MS suggested the molecular formula of C30H50O. The spot corresponding to Rf value of 0.71 was scrapped from preparative TLC and it was isolated as colorless powder after several washes with organic solvents and recrystallized with ethanol. FTIR analysis of major compound (Fig. 2) showed a characteristic peak observed at 1450 cm1; this might belong to methylenic vibration, absorption frequencies observed at 3398 cm1 and 1383 cm1 might be due to O-H and C-O bond vibrations, respectively; peak observed at 976 cm1 might be due to an unsaturated out of plane C-H vibration; C¼C vibration was shown around 1649 cm1 as a weak intense band; and peaks at 2933 cm1 and 1383 cm1 might be stretching and bending vibrations. From 1H NMR (Fig. 3), a doublet at d 3.31 is due to the proton attached to secondary carbinol, two broad singlets were seen at d 4.59 and 4.65 due to the two exomethylene protons attached, hydromethine proton at d 3.19, broad singlets at 4.73 and 4.59 were indicative of olefinic protons, 2.36 d assigned to a methynic proton, a doublet at 1.97, 1.66 triplet assignable to a methyl group on double bond, a singlet at 1.23. From 13C NMR (Table 2), seven methyl groups, signals due to an exomethylene group; ten methylene, five methine and five quaternary carbons were observed. From the combined results of FTIR and NMR, the major compound was confirmed as lupeol.
2.15. Statistical analysis 3.2. RP HPLC analysis Results were given as mean ± standard deviation (SD). Mean values of nanoparticles size were compared using one-way analysis of variance (ANOVA) followed by post Tukey's test. 3. Results and discussion 3.1. Standardization of Ficus religiosa L. extract From the positive ion mode (M þ H)þ spectrum of Ficus religiosa L. extract, three peaks were obtained at charge (m/z) of 166.12, 180.10 and 427.24 and the most abundant peak was found to be
Specificity of lupeol (100 mg/ml) was determined by spiking with standard in triplicate. For the investigated compound, only one peak was observed at specified retention time ie. 9.0 min. LOD and LOQ of lupeol were found to be 23 ng/ml and 65 ng/ml, respectively. Precision (% RSD) and accuracy of lupeol in extract are shown in Table 3. The developed method was unaffected by changes in chromatographic parameters such as pH of mobile phase, mobile phase flow and stability of analytical solutions over the time period of 0, 24, 48 and 96 h. Quantity of lupeol found in the Ficus religiosa L. extract was found to be 43.67%.
Fig. 1. LCMS (positive mode) spectrum of Ficus religiosa L. extract.
62
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
Fig. 2. FTIR spectrum of marker compound.
Fig. 3. 1H NMR spectrum of marker compound.
3.3. Preparation of Ficus religiosa L. extract loaded solid lipid nanoparticles Ficus religiosa L. extract loaded SLN were prepared by using hot homogenization followed by ultrasonication method. The followed method was found to be reliable, simple, and reproducible. Prepared SLN formulations were found to be uniform and homogenous in appearance.
3.4. Particle size, PDI, zeta potential and surface morphology Results of particle size, PDI and zeta potential of all formulations are given in Table 4. It was observed that SLN prepared by using glyceryl monostearate had particle size in the range of 154e195 nm and SLN prepared by using compritol ATO 888 had particle size in the range of 202e287 nm. Increase in lipid concentration increased particle size which is independent of lipid type. Formulations
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67 Table 2 13 C NMR signals of marker compound.
63
Table 4 Size and charge analysis of all SLN batches.
d (ppm)
Type of carbon
d (ppm)
Type of carbon
Formulations
Particle size (nm)
15.6 16.2 16.3 18.1 19.0 19.3 21.3 24.9 27.9 28.0 30.3 34.5 36.2 37.3
-CH3 -CH3 -CH3 -CH3 ¼CH2 -CH3 ¼CH2 ¼CH2 ¼¼CH2 -CH3 ¼CH2 ¼CH2 ¼CH2 C
38.2 38.5 39.8 40.3 41.4 42.8 43.7 47.8 48.5 50.9 56.7 79.3 110.6 151.6
^C-H ¼CH2 C ¼CH2 C C C ^C-H ^C-H ^C-H ^C-H ^C-H ¼CH2 C
SLN SLN SLN SLN SLN SLN
154.0 183.4 195.4 202.7 256.3 287.1
1 2 3 4 5 6
± ± ± ± ± ±
27.61 30.64 45.01 33.47 29.36 41.89
PDI 0.301 0.312 0.379 0.457 0.547 0.599
Zeta potential ± ± ± ± ± ±
0.03 1.01 0.74 0.64 0.90 0.41
48.47 48.57 49.76 47.63 43.21 47.81
± ± ± ± ± ±
2.18 5.64 1.17 3.48 4.09 3.68
Mean Values ± SD; n ¼ 3.
containing compritol ATO 888 (SLN 4e6) had higher particle size than formulations containing glyceryl monostearate (SLN 1e3). This increase in particle size of SLN prepared by using compritol ATO 888 can be correlated with its higher melting point ie. 72 C than GMS (59 C). During destabilization of SLN, metastable form (b/) of compritol ATO 888 changes to more stable forms (bi or b). This increases particle size of SLN prepared using compritol ATO 888 than lipids with lower melting point [12]. Hence from the results, it was observed that higher the melting points of lipids, higher the particle size [13]. Increase in lipid concentration (in both types) increased the PDI and had no effect on zeta potential. The SEM image of best formulation is shown in Fig. 4. SLN particles diameter were found to be in nanometer range and spherical in shape. From the image of AFM (Fig. 5), spherical shape of particles was further confirmed and the particles diameter were found to be around 200 nm which was well correlated well with the size measurement observed by particle size analyzer. Average roughness was found to be 11.36 nm which indicates the surface smoothness of SLN.
Fig. 4. SEM image of best formulation.
3.5. Entrapment efficiency and drug loading From the results of EE, it was observed that increasing the lipid concentration increased the EE of both types of lipid [Table 5]. Higher concentration of lipid is believed to provide more number of molecules to cover the drug molecules and also prevents drug leaching into external phase, thus, ensuring highest EE [14]. There was no significant change in EE of batches containing glyceryl monostearate and compritol ATO 888. Drug loading of all formulations is shown in Table 5. Of all the formulations, SLN 3 had higher drug loading. Fig. 5. AFM image of best formulation.
3.6. In vitro release studies The release of lupeol from Ficus religiosa L. extract suspension was much faster with nearly 95% of lupeol diffused into the release medium at 3rd hour. SLN containing two different lipids displayed a
similar biphasic drug release pattern with a burst release within 30 min followed by sustained release afterwards (Fig. 6). The initial burst effect of the formulations containing glyceryl monostearate
Table 3 Precision and accuracy for the determination of lupeol in extract. Compound and Concentration (mg/ml)
Lupeol 50 100 150
Intraday assay
Interday assay
Mean (mg/ml)
Precision (%RSD)
Accuracy (%)
Mean (mg/ml)
Precision (%RSD)
Accuracy (%)
49.65 100.12 148.73
0.365 0.456 0.321
99.38 100.12 99.15
50.01 99.98 149.64
0.145 0.654 1.687
100.02 99.98 99.76
64
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
Table 5 Entrapment efficiency and drug loading of all formulations. Formulations
Entrapment efficiency (%)
SLN SLN SLN SLN SLN SLN
43.14 56.13 56.98 43.65 55.14 56.17
1 2 3 4 5 6
± ± ± ± ± ±
1.34 1.64 2.11 2.45 1.97 2.04
Drug loading (%) 8.25 9.68 9.78 8.34 9.55 9.72
± ± ± ± ± ±
1.23 1.82 2.35 2.18 0.97 0.79
Mean ± SD; n ¼ 3.
lupeol on the surface of nanoparticles. From the results, it was observed that higher the lipid ratio, higher the initial burst release. The sustained release of lupeol from the formulations containing glyceryl monostearate were found to be 59.1%, 56% and 41.6% for SLN 1, 2 and 3, respectively; and compritol containing formulations (SLN 4, 5 and 6) showed 52.1%, 44.3%, and 28.1%, respectively, of drug release over 24 h. The results revealed that the release was chiefly dependent on type and concentration of the lipids used ie. increase in lipid concentration sustained the release rate which is independent of the lipids type. This might be due to increased viscosity of formulations by increase in lipid concentration which slows the drug release from the lipid matrix (Muller et al., 2000). Among the lipids used, compritol showed sustained release than glyceryl monostearate due to its longer carbon chain length. All SLN formulations had higher linearity for Higuchi model. From the results of in vitro characterization of all SLN batches, batch SLN 2 was selected for bioavailability studies based on lesser particle size and higher entrapment efficiency. 3.7. Fourier transform infrared spectroscopy
Fig. 6. In vitro release profile of Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN batches.
FTIR spectra of Ficus religiosa L. extract, glyceryl monostearate, compritol ATO 888 and SLNs are shown in Fig. 7. Ficus religiosa L. extract showed characteristic peaks at 3398 (O-H stretching), 2926 and 1383 (C-O bond vibrations, C-H stretching), 1629 (-C¼C- vibration), 1157 (C-N stretching) and 898 (-C¼C-H stretching). All these peaks are characteristic peaks of lupeol and all were present in Ficus religiosa L. extract loaded SLN and there was no absence of any functional peaks in all spectra. Thus, it revealed that there was no significant physicochemical interaction between drug and lipid. 3.8. Differential scanning calorimetry
varied from 7.5% to 21.9% (SLN 1e3); and for compritol 9.3e16.3% (SLN 4e6) and the burst release might be due to association of
DSC studies revealed that in the thermograms of SLN (using both lipids), Ficus religiosa L. extract peak was reduced and
Fig. 7. FTIR spectra: A - Extract, B e Glyceryl monostearate, C e Compritol ATO 888, D e SLN 2 and E SLN 5.
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
65
Fig. 8. DSC thermograms of A - Extract, B e Glyceryl monostearate, C e Compritol ATO 888, D e SLN 2 and E SLN 5.
broadened but no change was observed in lipids peak (Fig. 8). The broadening of Ficus religiosa L. extract peak in nanoparticles might be due to conversion of crystalline form to amorphous form. 3.9. Powder X-ray diffractometry PXRD spectra of standardized extract, glyceryl monostearate, compritol ATO 888 and SLN are shown in Fig. 9. The diffraction spectrum of Ficus religiosa L. extract showed characteristic peaks at 2q of 13.64, 14.74, 15.97, 19.37, 21.20, 21.57, 22.90, and 24.32 indicating crystalline nature of the Ficus religiosa L. extract. The crystalline peaks of Ficus religiosa L. extract were absent in SLN
formulations indicating that Ficus religiosa L. extract was not in crystalline form. Intensity of lipid peaks (glyceryl monostearate and compritol ATO 888) was also decreased in the SLN formulation. This reduced intensity confirms the decreased crystallinity of lipids in SLN formulations. 3.10. Stability studies After 180 days of storage, there were no significant changes in particle size, PDI, zeta potential and % EE of optimized formulation which revealed that the best formulation was stable over 6 months (Table 6).
Fig. 9. PXRD spectra of A - Extract, B e Glyceryl monostearate, C e Compritol ATO 888, D e SLN 2 and E SLN 5.
66
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67
Table 6 Stability studies data. S. No.
Stability parameters
0 day
180 days
1 2 3 4
Particle size PDI Zeta potential % Entrapment efficiency
180 nm ± 21.67 0.347 ± 1.31 56.17 ± 5.64 59.14% ± 7.19
197 nm ± 32.14 0.314 ± 1.78 53.47 ± 7.63 58.67% ± 2.64
Mean values ± SD; n ¼ 3.
3.11. Pharmacokinetic study The HPLC peak of lupeol standard was free of interfering peaks of plasma at the retention time of 10.3 min. Linearity was obtained in the concentration range of 20e100 ng/ml with correlation coefficient (r2) values > 0.998. The inter- and intra-day precision of lupeol was within 2% relative standard deviation (RSD). Accuracy was between 98.24 and 99.15%. LOD and LOQ for lupeol were found to be 35.90 ng/ml and 81.64 ng/ml, respectively. The extraction efficacies in case of spiked plasma samples were 67 ± 17.4% with RSD values less than 6.2%. The mean plasma concentration vs. time curve profiles of lupeol from Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN are illustrated in Fig. 10. Pharmacokinetic parameters of lupeol in Ficus religiosa L. extract and SLN are shown in Table 7. The mean plasma AUC0e24 of lupeol in animals treated with SLN formulation was 9829.83 ± 56.12ng hr/ml whereas in animals treated with Ficus religiosa L. extract suspension, AUC0e24 of lupeol was 1068.46 ± 96.4 ng hr/ml. This increase in AUC0e24 for SLN might be due to the avoidance of first pass metabolism by lymphatic transport. The peak plasma concentration (Cmax) of lupeol in Ficus religiosa L. extract suspension was found to be 178.61 ± 24.6 ng/ml and in SLN formulation was found to be 696.57 ± 34.83 ng/ml. Time to reach plasma concentration (tmax) in Ficus religiosa L. extract suspension was found to be 6 ± 1.1 h and in SLN formulation was found to be 2 ± 0.11 h t1/2 of lupeol was found to be 7.3 ± 1.0 h in Ficus religiosa L. extract and 15.3 ± 1.3 h in SLN. From these results, it
clearly suggested that the pharmacokinetic profiles of lupeol have been improved in SLN form than Ficus religiosa L. extract after oral administration. 4. Conclusion Ficus religiosa L. extract loaded SLN were prepared successfully by optimizing lipids type and concentration. Best formulation was selected based on in vitro characterization of developed SLN. Pharmacokinetic studies of Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN showed greater increase in pharmacokinetic parameters of lupeol in SLN form than Ficus religiosa L. extract at the same dose of lupeol i.e. 50 mg/kg body weight. This study illustrates the pharmacokinetic parameters of lupeol in Ficus religiosa L. extract which is not reported before and its bioavailability enhancement through SLN. Conflict of interest The authors declare no conflicts of interest in this work. Acknowledgements The first author is grateful to the Indian Institute of Technology (Banaras Hindu University), Varanasi for providing financial support in the form of Teaching Assistantship funded by Ministry of Human Resource Development, Government of India. Authors acknowledge Special Assistance Programme of University Grants Commission, New Delhi, India for instrumentation facility (Particle size analyzer). The authors are thankful to Prof. O. N. Srivastava and Prof. R. K. Singh, Dept. of Physics, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India for carrying out for PXRD and DSC studies, respectively. The authors extend their sincere thanks to The Head, Dept. of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India for carrying out AFM studies and to Mr. Vinay K Ahire, Consortium for Scientific Research, Indore, Madhya Pradesh, India for carrying out SEM study. References
Fig. 10. Plasma concentration vs. time curve of lupeol in Ficus religiosa L. extract suspension and Ficus religiosa L. extract loaded SLN.
Table 7 Pharmacokinetic parameters of lupeol in rat plasma. Parameters
Ficus religiosa L. extract
SLN
Cmax (ng/ml) Tmax (hr) AUC0-24 (ng hr/ml) t1/2 (hr)
178.61 ± 24.6 6 ± 1.1 1068.46 ± 96.4 7.3 ± 1.0
696.57 ± 34.83a 2 ± 0.11a 9829.83 ± 56.12a 15.3 ± 1.3a
Mean ± SD; n ¼ 6. a p< 0.05, significance difference compared to Ficus religiosa L. extract.
[1] C.S. Beevers, S. Huang, Pharmacological and clinical properties of curcumin, Bot. Targets Ther. (2011) 15e18. https://doi.org/10.2147/BTAT.S17244. [2] P.R. Vuddanda, S. Chakraborty, S. Singh, Berberine: a potential phytochemical with multispectrum therapeutic activities, Expert Opin. Investig. Drugs 19 (2010) 1297e1307. http://dx.doi.org/10.1517/13543784.2010.517745. ska, Pharmacological action of paclitaxel, Pol. Merkur [3] P. Potemski, A. Płuzan Lek. 6 (1999) 27e29. [4] S.M. Wittemer, M. Ploch, T. Windeck, S.C. Muller, B. Drewelow, et al., Bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of Artichoke leaf extracts in humans, Phytomedicine 12 (2005) 28e38. https://doi.org/10.1016/j.phymed.2003.11.002. [5] Y.Y. Gai, W.H. Liu, C.J. Sha, Y.L. Wang, Y.T. Sun, et al., Pharmacokinetics and bioavailability of cimicifugosides after oral administration of Cimicifuga foetida L. extract to rats, J. Ethnopharmacol. 143 (2012) 249e255. https://doi.org/10. 1016/j.jep.2012.06.031. [6] N. Sermkaew, W. Ketjinda, P. Boonme, N. Phadoongsombut, R. Wiwattanapatapee, Liquid and solid self-microemulsifying drug delivery systems for improving the oral bioavailability of andrographolide from crude extract of Andrographis paniculata, Eur. J. Pharm. Sci. 50 (2013) 459e466. https://doi.org/10.1016/j.ejps.2013.08.006. [7] Z.P. Chen, J. Sun, H.X. Chen, Y.Y. Xiao, D. Liu, et al., Comparative pharmacokinetics and bioavailability studies of quercetin, kaempferol and isorhamnetin after oral administration of Ginkgo biloba extracts, Ginkgo biloba extract phospholipid complexes and Ginkgo biloba extract solid dispersions in rats, Fitoterapia 81 (2010) 1045e1052. https://doi.org/10.1016/j.fitote.2010.06. 028. [8] R. Pandit, A. Phadke, A. Jagtap, Antidiabetic effect of Ficus religiosa extract in streptozotocin-induced diabetic rats, J. Ethnopharmacol. 128 (2010) 462e466. https://doi.org/10.1016/j.jep.2010.01.025. [9] M. Gregory, B. Divya, R.A. Mary, et al., Anti-ulcer activity of Ficus religiosa leaf ethanolic extract, Asian pac. J. Trop. Biomed. 3 (2013) 554e556. https://doi.
K. Priyanka et al. / Journal of Drug Delivery Science and Technology 41 (2017) 58e67 org/10.1016/S2221-1691(13)60112-4. [10] D. Singh, B. Singh, R.K. Goel, Traditional uses, phytochemistry and pharmacology of Ficus religiosa: a review, J. Ethnopharmacol. 134 (2011) 565e583. https://doi.org/10.1016/j.jep.2011.01.046. [11] A.K. Kushwaha, P.R. Vuddanda, P. Karunanidhi, S.K. Singh, S. Singh, Development and evaluation of solid lipid nanoparticles of raloxifene hydrochloride for enhanced bioavailability, BioMed Res. Int. 584549 (2013), 10.1155/2013/ 584549. [12] M.H. Aburahma, S.M. Badr-Eldin, Compritol 888 ATO: a multifunctional lipid
67
excipient in drug delivery systems and nanopharmaceuticals, Expert Opin. Drug Deliv. 11 (2014) 1865e1883. [13] K. Priyanka, A.A. Sathali, Preparation and evaluation of montelukast sodium loaded solid lipid nanoparticles, J. young Pharm. 4 (2012) 129e137. https:// doi.org/10.4103/0975-1483.100016. [14] R.H. Muller, K. Mader, S. Gohla, Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art, Eur. J. Pharm. Biopharm. 50 (2000) 161e177, http://dx.doi.org/10.1016/S0939-6411(00)00087e4.