Background: The dual antimicrobial and antioxidant effects of Capparis spinosa seed oil prompted us to formulate the solid lipid nanoparticles of aforesaid oil and evaluate their antiacne effect against P. acne using erythromycin as standard.
Material and methods: Capparis spinosa seed oil loaded solid lipid nanoparticles were prepared using w/o/w type double emulsification method & its drug polymer compatibility was analyzed by FT-IR & HPLC. Surface morphology (by TET) showed that the particles has evident round and homogeneous shading, the particle size of Capparis spinosa seed oil loaded SLNs ranging approximately from 290-697nm. Zeta potential of -32mV also confirmed the stability of colloidal dispersion. The G4 formulation of SLN showed maximum entrapment (83%) due to high affinity of drug with lipid matrix. During the period of storage, the formulation showed no change in colour, creaming and phase separation. The G4 formulations showed significantly less drug release over other formulations & showed significant activity at concentration of 50 ?g/mL &100 ?g/mL but on further increase no significant change in zone of inhibition was observed.
Results: Formulation of Capparis spinosa seed oil loaded solid lipid nanoparticles has good stability, no drug polymer interaction; nanoparticles size approximately 290-697nm (spherical). Test sample G4 showed sustained drug release and significant anti-acne activity against P. acne using cup-plate method.
Conclusion: The results of oil loaded SLNs showed average diameters in the narrow colloidal size range, a good loading capacity, drug release and better anti-acne activity. The oil loaded SLNs methodology and nanoemulsions developed may open a new door towards the treatment acne.

Keywords: P. acne, Oil loaded solid lipid nanoparticles, Capparis spinosa seed, Erythromycin.

INTRODUCTION

Acne vulgaris is a common skin disease, which is caused by bacteria named Propionibacterium acne (P. acne), most common in Western nations. More than 85% of adolescents suffer from this ubiquitous and psychologically unbearable disease which is moderate to severe in about 15-20 % of patients. Acne often persists into adulthood with 64% of individuals in their 20s in 43% of individuals in their 30s showing sign of visible acne (Ghodsi et al., 2009; Poli et al., 2001; Collier et al., 2008; Schafer et al., 2001; Cunliffe & Gould., 1979).
Capparis spinosa (Caper Bush or Himsra) belongs to family Capparidaceae habitat of Afghanistan, West Asia, Europe, North Africa and Australia and in India it grows from Punjab and Rajasthan to the Deccan Peninsula. The plant is considered to have very good detergent and stringent effect. Seed oil of Capparis spinosa has also antimicrobial (Mu¨ller et al., 1995; Vringer de and de Ronde,1995) and antioxidant(Mu¨ller et al., 1998; Schu¨tt et al., 1998) properties and therefore widely used in the treatment of skin disease. Its seed oil is reported to contain linoleic acid (24.6-50.5%), tocopherols (127.3-2228.2 mg/100gm), and sterols (10%) as well as the content of glucosinolates (34.5-84.6 ?mol/g)( Mukherjee et al., 2009).
Solid lipid nanoparticles (SLNTM) introduced in 1991 represent an alternative carrier system to traditional colloidal carriers such as emulsions, liposomes and polymeric micro and nanoparticles. In monodispersed systems, SLNs are the new generation of nanoparticulated active substance carriers and are attracting major attention as novel colloidal drug carriers for topical use. The small particle size ensures close contact to the stratum corneum and increase penetrating into the viable skin. Due to their solid matrix, sustained drug release is possible. Sustained release becomes important with actives that are irritating at high concentration to supply the skin over a prolonged period of time with a drug and to reduce systemic absorption (Matthäus and Ozcan, 2005; Ali-Shtayeh and Abu Ghdeib, 1999; Mahasneh, 2002; Tesoriere et al., 2007; Germano, 2002). Based on the above facts, we investigated the anti-acne effect of solid lipid nanoparticle loaded Capparis spinosa seed oil.

MATERIALS AND METHODS

Soya lecithin was purchased from Delhi Petro Chemicals Co. Delhi, India. Capparis spinosa seed oil purchased from La-Medicca India Pvt. (Gurgaon) and PVA, Tween 80 and Dichloro methane were purchased from E. Merck (Darmstadt, Germany) and S.D. Fine Chemicals (Mumbai, India) and all other chemicals used were of analytical grade.

Method of preparation of solid lipid nanoparticles loaded Capparis spinosa seed oil

Solid lipid nanoparticles of oils were prepared by w/o/w type double emulsification method (Trotta et al., 2005). Required quantity of oils was dispersed in aqueous mixture of methanol (75% v/v) and required quantity of Lecithin & Cholesterol was dissolved in dichloromethane. The oils solution was added slowly to lipid mixture and homogenized for 15 min at 15000 rpm in ultra probe sonicator (Orchid Scientific, India) to produced white cloudy primary emulsion. The resultant primary emulsion was poured in to 2% w/v of PVA solution and homogenized for additional 10 min at15 000 rpm. The resultant w/o/w type emulsion was stored at room temperature. The solvent was evaporated in Rota evaporator at 45°C. The stable emulsion was freeze dried at -20°C under reduced pressure to get dried powder of solid lipid nanoparticles.

Drug polymer compatibility

The samples of pure oils and mixture of oils with lipids were mixed with 100 mg of potassium Bromide (KBr). The samples were compressed to disc by applying pressure of 5 tons for 3 min in a hydraulic press. The prepared pellets were placed in the sample cell and the spectrums were analysed in the region of 4000-400 cm-1. By comparing the spectrums of oils and oils with lipids, the compatibility study was performed (Liu et al., 2004).

HPLC analysis of oil

Oils were analyzed by reversed phase HPLC (LC-20AD, SPD-20A), Simadzu. The HPLC system consists of quaternary pump, an autosampler, a diode array detector (DAD detector) and a workstation. The column temperature was set at 40°C. The mobile phase was a methanol–water–glacial acetic acid (79:20:1 v/v) mixture with a flow rate of 2.0 ml/min. The detection wavelength was set at 356 nm. All samples were filtered through an aqueous 0.45 ?m pore size filter membrane in order to protect the column.

Surface morphology

TEM (JEM-100CXII, Japan) was a method of probing the microstructure of rather delicate systems such as micelles, liquid crystalline phases, vesicles, emulsions and also nanoparticles. Without surfactants the lyophilized SLNs were dispersed directly into the tristilled water. Then Cu grid coated with C film was put into the above solution several times. After being stained by 2% phosphotungstic acid (PTA) solution and dried under room temperature, the sample was ready for the TEM investigation(Bouwstra and Honeywell-Nguyen, 2002).

Zeta potential

Zeta potentials were measured in folded capillary cells using the Nano ZS90 zetasizer. Measurements were performed in distilled water adjusted with a solution of 0.1mmol/l sodium chloride to a conductivity of 50 ?S/cm at 25°C.

Polydispersity index

Poly dispersity index (PDI) value was used to characterize the mono dispersed a polydispersed nature of nanoparticles. Higher the Polydispersity index values indicate the high level of non uniformity.

Quantification of drug entrapment efficiency

The entrapment efficiency was calculated by using 100 mg of solid lipid nanoparticles dissolved in 20 mL of dichloromethane and the solution was centrifuged at 12000 rpm. The supernated fluid was collected and passed through membrane filter. The quantity of drug in the solution was measured by ultra violet spectroscopy at 263 nm (Anandrao et al., 1999).
Drug entrapment (%) = Quantity of drug in nanoparticles/ Mass of drug in the formulation×100

Stability studies

For evaluation of stability selected formulation (G4) containing 10:1 ratio of Lecithin & Cholesterol was stored at 25°C, 45°C and room temperature for 3 weeks. The entrapment efficiency and particles size measurement were evaluated after storage period of 3 week at 25 °C and 45°C and room temperature in the dark condition (Lim and Kim, 2002).

In-vitro release study

In-vitro release of solid lipid nanoparticles loaded oils were carried out by using modified Franz diffusion apparatus (Wissing and Müller, 2002). About 10 mg oils equivalent solid lipid nanoparticles were placed in donor compartment containing phosphate buffer (pH 7.4) at (37±2) °C. Drug release was assessed by intermittently sampling the receptor medium (5 mL) and fresh phosphate buffer saline solution was replaced. The samples were filtered in membrane filter (0.22 ?m) and the amount of drug released was quantified by a U.V. Spectrophotometer at 263 nm.

Anti acne activity by using Cup-Plate method

The nutrient agar medium was prepared and autoclaved at 15.1 lbs pressure for 20 minutes. This media was poured into petri plates and allowed to solidify. On the surface of media microbial suspension was spread with the help of sterilized cotton swab. Cups were made by boring into agar surface with a previously sterilized cork borer and scooping out the punched part of agar. Five cups were made in each petri plate and into these cups was added the concentration (50 ?g/mL, 100 ?g/mL, 200 ?g/mL) of the test samples, forth was filled by standard and fifth was filled by the control (DMSO).
The plates were kept in cold for one hour to allow the diffusion of test samples and then incubated at 37±0.5°C for 24h for anti-Acne activity. The zone of inhibition formed around the cups after respective incubation was measured and percentage inhibition of the compounds were calculated.

RESULTS

Preparation of solid lipid nanoparticles

In the present study, the effect of concentration of lipid mixture (lecithin: cholesterol) and surfactant on preparation of Capparis spinosa seed oil loaded solid lipid nanoparticles were evaluated. In these preparations, lecithin and cholesterol were used as lipid and Tween 80 as surfactant. SLNs loaded Capparis spinosa seed oil was prepared by double emulsification (w/o/w) using high speed ultra probe sonicator. The aqueous phase of methanolic solution containing Capparis spinosa seed oil was poured to organic phase containing lipid at high homogenization speed of 15000 rpm for 15 min with addition of Tween 80. The Tween 80 solution reduced the interfacial tension between the phases. The uniform size globules were formed as w/o type emulsion. This emulsion was further homogenized with 2% w/v PVA solution as co-surfactant to form double emulsion (w/o/w). The formed emulsion was stabilized and stability was maintained by hardening properties of co-emulsifier. The solid lipid nanoparticles were prepared by using increasing concentration of lipid and surfactant.

Table 1. Preparation of Capparis spinosa seed oil loaded solid lipid nanoparticles. Click here to view full image

Drug polymer compatibility

FT-IR spectrum of pure oil and mixture of oil and lipid. Form the spectral study it was observed that there was no significant change in the peaks of pure oil and oil lipid mixture. Hence, no specific interaction was observed between the drug and the lipid used in the formulations.

HPLC analysis

The assay was linear in the concentration range of 0.2–10.0?g/ml. The recovery rate ranged from 97.6% to 100.2%. The R.S.D. value for precision is below 2%. No interference from the formulation was observed. All samples were filtered through an aqueous 0.45 ?m pore size filter membrane in order to protect the column.

Surface morphology

Surface morphology of solid lipid nanoparticles loaded of oils was observed by Transmission electron microscope (TEM). Figure 1a shows the shape of the nanoparticles entrapping with the oils. It was evident that the particles investigated revealed round and homogeneous shading, the particle size of Capparis spinosa seed oil loaded SLNs ranging approximately from 290-697, nm. However, in order to obtain more precise information on the size distribution.

Figure 1a.TEM solid lipid Capparis spinosa seed oil loaded nanoparticles . Click here to view full image

The distribution of zeta potentials of SLNs were shown in Figure 1b. The zeta potentials of Capparis spinosa seed oil loaded solid lipid nanoparticles were -32 mV respectively. Zeta potential can make a prediction about the stability of colloid dispersions. A high zeta potential (>|30| mV) can provide an electric repulsion to avoid the aggregation of particles.

Figure 1b.Zeta potential of Capparis spinosa seed oil loaded solid lipid nanoparticles. Click here to view full image

Quantification of drug entrapment efficiency & Polydispersity index

The entrapment efficiency (%) was directly proportional to concentration of Lecithin and cholesterol. Normally low entrapment efficiency was observed at low affinity of drug between the solvents (organic and aqueous). The G4 formulation of SLN has showed maximum entrapment (83%) due to high affinity of drug with lipid matrix. During homogenization of SLN, the aggregation was reduced by reducing interfacial tension and stabilized spherical shape particles were formed by addition of tween 80. On increasing the concentration of surfactant, mean particle size of SLNs was reduced (Table 2).

Table 2. Evaluation of solid lipid nanoparticles of Capparis spinosa seed oil formulation. Click here to view full image

Stability studies

During the period of storage, the formulation showed no change in colour, creaming and phase separation. The particle size increased significantly, after storage of 3 weeks at 45°C. But there was no significant increase in particle size of SLNs stored at 5°C and 25°C after 3 weeks. The entrapment efficiency was reduced at higher temperature (45°C) than other storage conditions.

In-vitro release study

The in vitro release studies of Solid lipid nanoparticles loaded Capparis spinosa seed.

Figure 2a.In vitro drug release study of solid lipid nanoparticles loaded Capparis spinosa seed oil. Click here to view full image

Anti–acne activity by using cup-plate method

All oils based solid lipids nanoparticles samples (G1-G4) were screened for anti-acne activity against P.acne strain. All test samples showed moderate to good anti-acne activity against P.acne bacteria. The zone of inhibitions of test samples at the concentration of 50 ?g/mL were 6-12 mm (G1-G4), at 100 ?g/mL were 7-16mm and 200?g/mL were 8-16mm. The test samples showed even better inhibition when tested at higher concentration (100?g/mL & 200?g/mL) comparative to the standard drug Erythromycin. G4 showed highly significant activity at concentration of 50 ?g/mL &100 ?g/mL but the concentration further increased no significant change in zone of inhibition was observed (Table 3 & Figure 2b).

Figure 2b.Anti Acne activity of oils loaded solid lipid nanoparticles (G1-G4) against P. acne. Click here to view full image

Test sample G4 showed highly significant activity aginst P. acne at concentration of 50 ?g/mL &100 ?g/mL as compared to erythromycin.

DISCUSSION

The Capparis spinosa seed oil loaded solid lipid nanoparticles were prepared by double emulsification technique. The Tween 80 solution reduced the interfacial tension between the phases. The uniform size globules were formed as w/o type. The uniform size globules were formed as w/o type emulsion. The formed emulsions were stabilized and stability was maintained by hardening properties of co-emulsifier (PVA). The solid lipid nanoparticles were prepared by using increasing concentration of lipid and surfactant.
The solid lipid nanoparticles were prepared by using increasing concentration of lipid and surfactant. The FTIR spectrums reveal that drug incompatibles with lipids. HPLC assay, the recovery rate ranged from 97.6% to 100.2%. The R.S.D. value for precision is below 2%. No interference from the formulation was observed. The particle size of Capparis spinosa seed oil loaded SLNs ranging approximately from 290-697, nm. Zeta potential prediction indicates the stability of colloid dispersions. The entrapment efficiency (%) was directly proportional to concentration of Lecithin and cholesterol. Normally low entrapment efficiency was observed at low affinity of drug between the solvents (organic and aqueous). The G4 formulation of SLN has showed maximum entrapment (83.2%) due to high affinity of drug with lipid matrix.
During homogenization of SLN, the aggregation was reduced by reducing interfacial tension and stabilized spherical shape particles were formed by addition of Tween 80. On increasing the concentration of surfactant, mean particle size of SLNs was reduced.
The optimized formulation showed stability at room temperature and 5°C but at 45°C, particles were aggregated, drug molecule was leaked out from lipid matrix. It was assumed that the high temperature (45°C) increased the kinetic energy of system, which could accelerate the collision of particles which consequently increase the possibility of aggregation.

Table 3. Anti-acne activity of oils loaded solid lipid nanoparticles against Propionibacterium acne. Click here to view full image

The organic solvent used in these formulations rapidly partitioned into the continuous aqueous medium and the lipid precipitated around the drug. The simultaneous evaporation of the entrapped solvent lead to the formation of spherical shaped solid lipid nanoparticles. From the four formulations, G4 showed significantly less drug release over other formulations which clearly indicated that they can provide satisfied result for acne treatment over prolonged period of time.

CONCLUSION

In this study the potential of SLNs dispersions as carriers for delivery of Capparis spinosa seed oil was developed. Solid lipid nanoparticles were prepared by the w/o/w type double emulsification method by using bio-acceptable lipids such as cholesterol and lecithin and tween 80 as emulsifier. Oil loaded SLNs showed average diameters in the narrow colloidal size range, a good loading capacity, drug release and better anti-acne activity. The oil loaded SLNs methodology and nanoemulsions developed may open a new door towards the treatment anti-acne.

ACKNOLEDGEMENT

We are thankful to SunRise Univesity, Alwar and Department of Biochemistry, Alwar Pharmacy College, Alwar (India), for providing research facilities.

CONFLICT OF INTEREST
None declared.

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