Preparation and Evaluation of Proniosomal Gel of Neem Seed Oil

Transcription

1 Research Paper International Journal of Pharmaceutical Sciences and Nanotechnology Volume 5 Issue 3 October December 2012 MS ID: IJPSN CHANDEL Preparation and Evaluation of Proniosomal Gel of Neem Seed Oil A. Chandel 1 *, K. Saroha 1 and S. Nanda 2 1 Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra, Haryana, India , and 2 Department of Pharmaceutical Sciences, Maharishi Dayanand University, Rohtak, Haryana, India. Received February 16, 2012; accepted April 29, 2012 ABSTRACT Niosomes are non-ionic surfactant vesicles which act as drug carrier systems. In the present study, we carried preparation and evaluation of proniosomal gel of neem seed oil. The proniosomal gel was prepared using span 40, soya lecithin and cholesterol, using neem seed oil as the active pharmaceutical ingredient. Alcohol and phosphate buffer ph 7.4 were used in formulation as pharmaceutical excipients. The resulting formulations were evaluated and observed by transmission electron microscopy. Evaluation of formulation yielded optimum results like uniform size distribution 75-80% drug entrapment and microbiological data. It is concluded that it is viable to prepare an acceptable pronisomal gel of neem seed oil for therapeutic applications. KEYWORDS: Niosomes; proniosomal gel; neem seed oil; slurry method; microbiological study. Introduction Liposomes and niosomes, which are the colloidal drug particulate carrier systems, have several advantages over common drug delivery systems. They act as drug storehouse and can be modified to show different drug release properties depending upon their composition. The non-ionic surfactant vesicles or niosomes are microscopic lamellar vesicles containing non-ionic surfactants which may or may not be incorporated with cholesterol and various other lipids. The studies have shown that niosomes in vivo behaves like liposomes but increases the circulation of entrapped drug and modifying organ distribution and metabolic stability (Azmin et al., 1995). Niosome are chemically stable, biodegradable, biocompatible systems and can encapsulate large amount of active drug in approximately less volumes of vesicles and also are cost efficient (Malhotra and Jain, 1994). These features make them an appropriate choice as drug carriers over liposomes and related systems. Niosomes prepared by thin layer evaporation method and physicochemically characterized are less toxic and provide precise control over the active availability of active drug at the stratum corneum as compared to classical formulations like stratum corneum (Handjani et al., 1979; Paolina et al., 2007). Niosomes are prepared to decrease the release of active drug which results into sustained release profile, less toxicity and drug targeting (Vyas and Khar, 2002). The size of niosomes increases on the incorporation of entrapped drug which is a result of interaction of solute with surfactant head groups, increasing charge and mutual repulsion of the surfactant bilayer and thus increasing the size of vesicles (Stafford et al., 1998). The dry product form of niosomes is known proniosomes is gaining more popularity because on hydration it yields aqueous niosome dispersion. Niosomes are advantageous in terms of physical stability as they do not aggregate or leak out or sediment (Hu and Rhodes, 1997). The proniosomes possess certain advantages like they represent a significant improvement by eliminating physical stability problems such as aggregation, or fusion of vesicles and leaking of entrapped drug during long term storage, compared to niosomes prepared by conventional methods, proniosomes derived niosomes are superior in their convenience of storage, transport and dosing (Parthasarthi et al., 1994). The proniosomes derived niosomes are at least as effective as conventional niosomes in their release characteristics and may therefore offer improved bioavailability of some drugs with poor solubility, controlled release formulation or reduced adverse effects of some drugs (Hu and Rhodes, 1997). The proniosomes on hydration by agitation with hot water for a very less amount of time yields vesicles which 1780

2 Chandel et al: Preparation and Evaluation of Proniosomal Gel of Neem Seed Oil 1781 offer potential drug delivery through the transdermal route. The aim of the present study is to investigate the proniosomal gel of neem seed oil for topical drug delivery system. Neem seed oil has been used medicinally and cosmetically for hundreds of years. Its long term use has made it one of the oldest medicines available today. Neem seed oil contains several terpenoids, steroids, alkaloids, flavonoids and glycosides. The isolated constituents are: margosic acid, nimbin, nimbidin, nimbinin, kaempeerol, azadirone, quercursertin, betasitosterol, praisine and nimbicetin. Neem is antibacterial, antiviral, antifungal, antiseptic, and antiparasitic. The oil has moisturizing and regenerative properties, contains vitamin E, and has essential fatty acids. Scientific research today validates many of the traditional uses of neem seed oil, it is used to treat bacterial, fungal, and viral infections, boost the immune system, and for many specific health problems. Topically proniosomal gel of neem seed oil can be used for therapeutic and cosmetic purposes and used in conditions like acne, pimples, burns, cuts, and wounds. In this study, we formulated various need seed oil containing proniosomal gel and evaluated their properties as topical drug delivery systems. Materials and Methods Drugs and Chemicals Cholesterol, span 40, span 60, span 80, potassium dihydrogen phosphate and toluene were purchased from Loba Chemie Pvt. Ltd. (Mumbai, India). Soya lecithin powder was supplies from Titan Biotech Ltd. (Bhiwadi, Rajasthan). Sodium hydroxide and sodium chloride were obtained from S.D. Fine Chemicals (Mumbai, India). Agar was supplied by HiMedia Labs. Neem seed oil was obtained from the pharmacognosy lab of Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra, India. Preparation of Proniosomes Proniosomal gel was prepared by slurry method using span 40, span 60 and span 80, cholesterol and lecithin. The compositions of different prioniosomal formulation are listed in Table 1. Using a wide mouth glass bottles, 100 mg equivalent i.e ml neem seed oil with surfactant, lecithin, cholesterol and 2.5 ml of alcohol. Then the open end of the glass tube was covered with a lid and the bottle was warmed in a water bath at 65 o C ± 3 o C for 5 minutes. Then 1.6 ml of ph 7.4 phosphate buffer was added and the mixture was further warmed in water bath for 2 minutes so that a uniform solution is obtained. The mixture was allowed to cool at room temperature until the dispersion was converted to proniosomal gel (Alsarra et al., 2005). The composition of different formulations prepared is given in Table 1. TABLE 1 Composition of various proniosomal formulations of neem seed oil. Code Surfactant Quantity Lecithin Cholesterol N1 Span N2 Span N3 Span N4 Span N5 Span N6 Span N7 Span N8 Span N9 Span Size Distribution Evaluation of vesicle diameter and the particle size range was done using the optical microscope. The mean size and size distribution of drug loaded niosomes were determined using ocular and stage micrometer at a magnification of 1000X. Average particle size was determined along with standard deviation. Percentage Drug Entrapment (PDE) of Neem Seed Oil For determining the PDE of proniosomal gel (25 mg) of neem seed oil was hydrated in 80 C distilled water and vortexed for 2 minutes. The niosome dispersion so obtained was centrifuged at rpm for 40 minutes at 5 C (Remi CPR-24 centrifuge). The clear fraction was used for the determination of free drug at 310 nm spectrophotometrically (systronic). The percentage encapsulation efficiency was calculated by using formula PDE = [1 (unecapsulated drug/total drug)] 100 Where PDE = Percentage Drug Entrapment Morphology The morphology of hydrated niosome dispersion prepared from proniosome was also determined using transmission electron microscope (Hu and Rhodes, 1999). A drop of niosome dispersion was applied to a carboncoated 300 mesh copper grid and left for 1 minute to allow some of the niosomes to adhere to the carbon substrate. The remaining dispersion was removed by absorbing the drop with the corner of a piece of filter paper. A drop of 2% aqueous solution of uranyl acetate was applied for 35 seconds. The remaining solution was removed by absorbing the liquid with the tip of a piece of filter paper and the sample was air dried. The sample was observed with a Fei-Philips morgagni 268D transmission electron microscope at 100 KV. Microbiological Activity of Formulation The antibacterial activities of formulations were evaluated by agar well diffusion method (Ahmad and Beg, 2001; Andrews, 2001). All the microbial cultures

3 1782 Int J Pharm Sci Nanotech Vol 5; Issue 3 October December 2012 were adjusted to 0.5 McFarland standard, which is visually comparable to a microbial suspension of approximately cfu/ml. 20 ml of Mueller Hinton agar media was poured into each Petri plate and plates were swabbed with 100 µl inocula of the test microorganisms and kept for 15 minutes for adsorption. Using sterile cork borer of 8mm diameter, wells were bored into the seeded agar plates and these were loaded with a 100 µl volume with test compound with concentration of 25 mg reconstituted in 10 ml. All the plates were incubated at 37 o C for 24 hours. Antibacterial activity of formulations was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (HiAntibiotic zone scale). DMSO as solvent was used as a negative control whereas ciprofloxacin was used as positive control. The experiments were performed in triplicates. Stability Studies Proniosomal gel formulations and niosomal suspensions were tested for stability. Three batches were prepared and were kept in vials covered with a lid and stored at: (i) room temperature, (ii) 4 C-8 C, and (iii) 45 C ± 2 C. The samples were evaluated for the ability of vesicles to retain the drug. The samples were visually verified from time point during 1 month, and various factors like aggregation, flocculation, and loss of solvent were observed. Results and Discussion We designed several formulations and prepared them using standard methods. These formulations were evaluated as per the physical criteria shown in Table 2. Transmission electron microscope was used to evaluate morphology of niosomes derived from niosomal dispersion of formulation code N1 and N2 of neem seed oil. Niosomes prepared from proniosomal gel were oval and more uniform in shape. The morphological aspects are shown in Figures 1 and 2. TABLE 2 Physical observation of proniosomal gel formulations. Sr. No. Formulation Code Surfactant Quantity Lecithin Cholesterol Observation 1 N1 Span Brownish gel, stable 2 N2 Span Light brown gel, stable 3 N3 Span Brownish semi solid, Slightly unstable 4 N4 Span Brownish, Phase separated 5 N5 Span Light brown liquid, stable 6 N6 Span Brownish semi-solid, stable 7 N7 Span Brownish semi-solid, unstable 8 N8 Span Light brownish semi-solid, unstable 9 N9 Span Brownish liquid,unstable Fig. 1. TEM image of formulation N1. Fig. 2. TEM image of formulation N2.

4 Chandel et al: Preparation and Evaluation of Proniosomal Gel of Neem Seed Oil 1783 Particle size analysis of niosome preparations showed that the size distributions of niosomes based on neem seed oil were approximately the same, the average particle size of neem seed oil niosomes were in the range of 152 nm nm (Table 3). TABLE 3 Average particle size of niosomes. Sr.no. Particle size N1 (nm) Particle size N2 (nm) Mean For niosomes derived proniosomal gel based on neem seed oil, percentage drug entrapment of neem seed oil formulation N1 was found to be approximately 80% while the entrapment efficiency of formulation of N2 was found to be approximately 72-76%. The comparison is shown is Figure 3. Higher entrapment in case of niosomes from formulation N1 may be because of less amount of lecithin used therefore thinner surfactant film on surface. In microbiological study, 6 formulations and neem seed oil were evaluated against Gram-positive and Gram-negative bacteria. Standard antibiotic namely ciprofloxacin was used for comparison with antibacterial activities shown by compounds. In case of Gram-positive bacteria, these compounds showed activity against both bacterium, that is, S. aureus and B. subtilis. However in case of Gram-negative bacteria, these compounds showed activity only against one bacterium, E. coli but did not exhibit any activity against P. aeruginosa (Table 4). The graphical representation of main results is shown in Figure 4, 5 and 6. Based on this finding, it may be suggested that the compounds used and oil may be used as source for formulating antimicrobial agents as they are active against both Gram-positive and negative bacteria. Fig. 4. Comparison between microbiological activity of different formulations Staphylococcus aureus. Fig. 5. Comparison between microbiological activity of different formulations Bacillus subtilis. Fig. 3. Comparative entrapment efficiency. Fig. 6. Comparison between microbiological activity of different formulations Escherichia coli.

5 1784 Int J Pharm Sci Nanotech Vol 5; Issue 3 October December 2012 Table 4 In vitro antimicrobial activity of compounds through agar well diffusion method. Compounds Diameter of growth of inhibition zone (mm) a Bacillus Escherichia subtilis coli Staphylococcus aureus Pseudomonas aeruginosa N N N N N N Neem seed Oil Ciprofloxacin DMSO No activity, mm- millimeter The results of stability study showed that proniosomal gel formulation were quite stable at refrigeration (4 o C-8 o C) and at some extent at room temperature as much leakage was not found at these temperatures. Percent drug retained at 45 o C might have decreased due to melting of the surfactant and lipid present in the formulation. Therefore, the proniosomal gel formulation of neem seed oil should be preferably stored at refrigeration or in other case at room temperature. From this study, it is observed that neem seed oil can be well utilized for various topical treatments when prepared as proniosome/niosomes, as these vesicular systems have the advantages of controlled and sustained action against dermal infections and also are more stable and versatile than liposomes. Proniosomal drug delivery, without doubt has immense scope in future, especially in the area of dermatitis, periodontitis and cosmetics. Conclusions Based on the results of formulation and evaluation studies, it is concluded that it is viable to prepare an acceptable pronisomal gel of neem seed oil for therapeutic applications. References Ahmad I and Beg AJ (2001). Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multidrug resistant human pathogens. J Ethnopharmacol 74: Alsarra IA, Bosela AA, Ahmed SM and Mahrous GM (2005). Proniosomes as a carrier for transdermal delivery of Ketorolac. Eur J Pharm Biopharm. 59: Andrews JM (2001). Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48: Azmin MN, Florence AT, Handjani, Vila RM, Stuart JFB, Vanlerberghe G and Whittaker JS (1995). The effect of non-ionic surfactant vesicle (niosome) entrapment on the absorption and distribution of methotrexate in mice. J Pharm Pharmacol 37: Handjani, Vila RM, Ribier A, Rondot B and Vanlerberghe G (1979). Dispersions of lamellar phases of non-ionic lipids in cosmetic products. Int J Cosmetic Sci 1: Hu C and Rhodes DG (1999). Proniosomes: A novel drug carrier preparation. Int J Pharm 185: Malhotra M and Jain NK (1994). Niosomes as Drug Carriers. Indian Drugs 31(3): Paolina D, Muzzalupo R, Ricciardi A, Celina C, Picci N and Fresta M (2007). In vitro and in vivo evaluation of Bola-surfactant containing niosome for transdermal delivery. Biomedical Microdevices (4): Parthasarthi G, Udupa N and Pillai GK (1994). Formulation and in vitro evaluation of vincristine encapsulated niosomes. Indian J Pharm Sci 56(7): Stafford S, Baillie AJ and Florence AT (1988). Drug effects on the size of chemically defined non-ionic surfactant vesicles. J Pharm Pharmacol 40 (suppl.): 26p. Vyas SP and Khar RK (2002). Targeted & controlled drug delivery novel carrier systems. 1st ed.: CBS Publishers and Distributors, New Delhi, pp Address correspondence to: Abhishek Chandel, Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra , Haryana, India. Tel: abhishek.k.chandel@gmail.com