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Improvement of anti-inflammatory and anti-angiogenic activity of berberine by novel rapid dissolving nanoemulsifying technique.

ABSTRACT

Berberine, an isoquinoline alkaloid, has wide biological and pharmacological actions. Despite the promising pharmacological effects and safety of berberine, poor oral absorption due to its extremely low aqueous solubility results in poor oral systemic bioavailability. This limits its clinical usage. This study describes the development and characterization of self-nanoemulsifying drug delivery system (SNEDDS) of berberine in liquid as well as solid form with improved solubility, dissolution and in vivo therapeutic efficacy. The SNEDDS of berberine were prepared using Acrysol K-l 50, Capmul MCM and polyethylene glycol 400, The formulations were characterized for various in vitro physicochemical characteristics. In vivo efficacy was evaluated in acetic acid induced inflammatory bowel model in rats. Anti-angiogenic activity of the developed SNEDDS of berberine was studied using chick chorioallantoic membrane assay. SNEDDS of berberine rapidly formed nanoemulsions with globule size of 17-45nm. The in vitro rate and extent of release of berberine from SNEDDS was significantly higher than berberine alone. Chick chorioallantoic membrane assay revealed potent anti-angiogenic activity of SNEDDS of berberine. These studies demonstrate that the SNEDDS of berberine is a promising strategy for improving its therapeutic efficacy and have potential application in the treatment of chronic inflammatory conditions and cancer.

Keywords:

Berberine

SNEDDS

Dissolution

Anti-inflammatory

Anti-angiogenic

Introduction

Angiogenesis, formation of new blood vessels from a preexisting vasculature, is a complex process involving an extensive interaction between cells, the extracellular matrix, and soluble factors (Lee et al. 2010). Angiogenesis plays an important role in pathologic processes such as the growth and metastasis of tumours (Huang et al. 2006). The success of anti-angiogenic therapy for cancer treatment has led to the research for anti-angiogenic agents (He et al. 2009). The newly formed blood vessels promote cancer growth by supplying nutrients and oxygen and by removing waste products. Metastasis also depends on angiogenesis, as tumour cells are shed from primary tumour and grow at their target organs. Thus, antiangiogenic activity is a promising move towards development of novel drugs to treat cancers and other diseases related to angiogenesis (He et al. 2009; Mathur et al. 2006).

There is now enough evidence that the chronic inflammatory process provides a microenvironment that includes up-regulation of inflammatory mediators. Inflammatory mediators suppress cell mediated immune responses and promote angiogenesis, facilitate tumour promotion and progression (Kundu and Surh 2008). Chronic inflammation causes the progression of the neoplastic process and therefore, disruption of inflammatory pathway, provides a promising opportunity for the treatment of cancer (Aggarwal et al. 2006). Traditional herbal medicines have long been recognized as a rich source for discovering new therapeutic agents. Several anti-inflammatory phytoconstituents have shown chemopreventive activities and can be used for prevention as well as treatment of cancer. In addition, bioactive phytoconstituents are safe and lack toxicity.

Berberine (BR) is an isoquinoline alkaloid of the protoberberine type, with a long history of medicinal use in traditional eastern medicine. BR is usually administered in a salt form for several clinical applications like anti-bacterial, anti-fungal, and anti-inflammatory, and has been used as a gastrointestinal remedy for thousands of years (Tan et al. 2011). BR also possesses anti-HIV, anti-fungal, cardioprotective, immunoregulative, anti-malarial, anti-inflammatory, anti-oxidant, cerebro-protective, anti-mutagenic, vasorelaxing, anxiolytic and analgesic activities (Zuo et al. 2006). However, due to its hydrophobic nature, poor stability and low bioavailability, the actual therapeutic application of BR is hampered for a long time. Poor water solubility of BR reflects in limited absorption in the gastrointestinal tract and subtherapeutic plasma concentrations. These issues have hindered the development of BR as a pharmaceutical formulation. Therefore, a novel delivery system to improve the solubility and bioavailability of BR is a matter of exigency (Tan et al. 2011; Zhu et al. 2013).

The use of nanotechnology in formulation enhances solubility and permeability of drugs with low solubility and poor permeability. It also improves the therapeutic activity by either active or passive targeting, while preventing physical and chemical degradation of the drug. Self micro/nanoemulsifying drug delivery systems (SM/NEDDS) are a current formulation strategy to enhance the oral bioavailability of poorly soluble drug. SM/NEDDS are isotropic preconcentrates of drug, oil, surfactant, cosolvent which readily generate ultrafine micro/nanoemulsions after dispersion in water with mild agitation. These systems avoid the dissolution step as observed for solid crystalline compounds. Furthermore, upon dispersion these systems form micro/nanosized globules of oil in which drug remains dissolved thus facilitating the absorption process (Gershanik and Benita 2000; Gursoy and Benita 2000; Pouton 1997, 2006).

In the present study, we combined the pharmacological benefits of BR with a lipid based nanoemulsifying delivery system to obtain more effective anti-inflammatory formulation having potential application in the treatment of chronic inflammation and cancer.

Materials and methods

Materials

BR was a generous gift from Indian Institute of Integrative Medicine (Jammu, India). Following excipients from respective sources were used as received. Capryol 90, Cremophor RH 40, Labrafac CC, Labrafil M1944CS, Labrasol, and Transcutol HP were gifted by Gattefosse India Pvt. Ltd. (Mumbai, India). Ethyl oleate, isopropyl myristate, olive oil, sesame oil, ethanol, polyethylene glycol 400 (PEG), propylene glycol, Tween 20, Tween 60 and Tween 80 were purchased from Loba Chemie Ltd. (Mumbai, India). Captex 200 and Capmul MCM were received as gift samples from Abitec Corporation, Mumbai, India. Acrysol K-150 and Neusilin US2 were gifted by Corel Pharma Ltd. (Ahmedabad, India) and Gangwal Chemicals Pvt. Ltd. (Mumbai, India) respectively.

Methods

Determination of solubility in various solvents

The solubility of BR in various oils/modified oils (Acrysol K150, Captex 200, ethyl oleate, isopropyl myristate, olive oil and sesame oil), surfactants (Capmul MCM, Capryol 90, Cremophor RH 40, Labrafac CC, Labrafil M1944CS, Labrasol, Tween 20, Tween 60 and Tween 80) and co-solvents (ethanol, polyethylene glycol 400, propylene glycol and Trancutol HP) was determined using shake flask method. An excess of BR (about 500 mg) was added to 1 ml of above mentioned vehicles and the resulting suspensions were shaken on a flask Shaker (Kytose EOS- 10M, Electrolab) at room temperature for 3 days and centrifuged at 3000 rpm for 15 min (Spinwin MC 01, Tarson, Mumbai). The clear supernatant was analyzed for the content of BR by validated RP-HPLC method. Determinations were carried out in triplicate. The HPLC system consisted of Pump (Jasco PU-2080 plus. Intelligent LC pump, Japan) with a Interface (Jasco LC-Net II/ADC, Japan) connected to Detector (Jasco UV-2075 plus, Intelligent UV-vis detector, Japan). The chromatographic separation was performed using an isocratic elution. The mobile phase consisted of a mixture of acetonitrile, 0.05 M K[H.sub.2]P[O.sub.4] and methanol (4:4:3) and delivered at a flow rate of 1 ml/min. The separation was carried out at 20 [degrees]C, on a reversed phase HiQ Sil C8 column (250 mm x 4.6 mm, 5 [micro]m particle size). An injection volume of 20 [micro]l was used. Detections were carried out at 343 nm.

Construction of pseudo-ternary phase diagram

The ternary phase diagrams were constructed by water titration method (Bachhav and Patravale 2009). From the results of solubility studies, Acrysol K-150, Capmul MCM and PEG 400 were selected as the oil, surfactant and co-solvent respectively. Distilled water was used as an aqueous phase for construction of phase diagrams. Surfactant and cosolvent were mixed in ratios 1:1,1:2 and 2:1 ([S.sub.mix], w/w). Ternary mixtures with varying compositions of [S.sub.mix], and oil were prepared. Nine different combinations of oil and [S.sub.mix], 1:9,2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, were made so that maximum ratios were covered to define the boundaries of phase formed in the phase diagrams. Homogeneous mixtures of oil and [S.sub.mix], at given volume ratio were prepared in dust-free glass vial. The preconcentrates were mixed on orbital shaker (Kytose EOS--10 M, Electrolab) for 10 min. After equilibrium was reached, the mixtures were further titrated with aliquots of distilled water. During the titration, samples were stirred to ensure homogeneity and visually monitored against a dark background by illuminating the samples with white light. Slow titration with aqueous phase was done to each weight ratio of oil and [S.sub.mix]. The formation of the nanoemulsion was visually observed as transparent or slightly bluish o/w nanoemulsion and marked on the pseudo-ternary phase diagram. Clear and isotropic samples were deemed to be within the nanoemulsion region. These titration results were then used to determine the boundaries of the emulsion regions corresponding to the selected optimum ratios of combination vehicles for developing phase diagram (Setthacheewakul et al. 2010). The tendency to spontaneously emulsify was also examined. After being equilibrated, the efficiency of self-emulsification, dispersibility, and appearance were graded based on rapidity in emulsification and the colour of the emulsion (Singh et al. 2008).

BR loaded SNEDDS

Based on solubility studies and the pseudo-ternary phase diagrams, SNEDDS composition was optimized composed and were composed of Acrysol K-150, Capmul MCM and PEG 400; 1:0.33:0.66. Calculated amount of BR was added in the oily phase: Acrysol K-150 in small increment with continuous stirring. The [S.sub.mix] was prepared by mixing separately Capmul MCM and PEG 400 in 1:2 ratio and added to BR containing Acrysol K-150 with continuous stirring. The stirring was continued till the homogenous mixture was formed. BR loaded SNEDDS were further characterized for various physicochemical parameters.

Determination of globule size by photon cross correlation spectroscopy and Zeta potential

BR loaded SNEDDS (100 mg) were diluted to 100 ml with double distilled water, 0.1 N HC1 and buffer pH 6.8. Visual observations were made immediately after dilution for assessment of self-emulsification efficiency, phase separation, and precipitation of drug. Each sample was placed in transparent polystyrene cuvette (path length = 1 cm) and placed in thermostatic sample chamber. Mean globule size and the polydispersity index of the resulting emulsions were determined by photon cross-correlation spectroscopy (Nanophox, Sympatec, Germany). Sample temperature was set at 25 [degrees]C and 3 runs of 60s were performed. Detection was carried out at a scattering angle of 90G From the resulting correlation curves, a 2nd order analysis was performed to calculate the mean globule size and standard deviation. The globule size distribution was expressed in terms of polydispersity index, which is a measure of the width of the globule size distribution. It is defined as:

Polydispersity index = {D(v, 0.9)- D(v, 0.1)} / D(v, 0.5)

where, D(v, 0.5), D(v, 0.1) and D(v, 0.9) are standard percentile readings from the globule size estimation. D(v, 0.5) is the mean globule size. D(v, 0.1) and D(v, 0.9) are the size of the globules below 10% and 90% respectively of the sample lies (Matos et al. 2013).

Zeta potential was measured on Zetasizer (ZS 90, Malvern Zetasizer, Malvern, UK) after diluting the BR loaded SNEDDS (100 mg) with 100 ml double distilled water.

Stability studies

Robustness to dilution. BR loaded SNEDDS (100 mg) were diluted to 100 ml with double distilled water, 0.1 N HC1 and phosphate buffer pH 6.8. The globule size of the resulting nanoemulsions was measured immediately after dilution and after 24 h storage at room temperature (Kale and Patravale 2008; Kallakunta et al. 2012).

Thermodynamic stability. The BR loaded SNEDDS were subjected to heating and cooling cycle. The product was exposed to temperature 4 [degrees]C and 45 [degrees]C. At each temperature, the product was held for 45 h. These cycles were repeated two more times. After total 3 cycles of heating and cooling, the product was centrifuged at 3000 rpm for 30 min. Product was observed visually for precipitation, if any, at every stage. Freeze thaw stability was also analyzed by storing the product at 4 [degrees]C and -4 [degrees]C with storage at each temperature for 12 h (Kallakunta et al. 2012).

Cloud point. The BR loaded SNEDDS preconcentrate and diluted nanoemulsion were heated in a water bath from 25 [degrees]C to a temperature at which formulation shows first sign of turbidity. Temperature was increased gradually at a rate of 5[degrees]C/min. Appearance of the product was closely monitored with increase in temperature. Cloud point was considered as the temperature at which turbidity appears (Kallakunta et al. 2012).

Preparation and characterization of solid SNEDDS of BR (S-SNEDDS)

Preparation of solid SNEDDS of BR (S-SNEDDS). S-SNEDDS of BR were prepared by adsorbing BR loaded SNEDDS preconcentrate on highly porous magnesium aluminometasilicate (Neusilin US2, Fuji Chemical Ind. Ltd., Gangwal Chemicals Pvt. Ltd., Mumbai). The BR loaded SNEDDS were added drop wise over the solid adsorbent; Neusilin US2 (1:1 by weight). After each addition, the mixture was mixed thoroughly to ensure uniform distribution. The granular mass obtained was passed through sieve with aperture of 400 [micro]m, to get free flowing and lump free powder. The powder sample was stored in a desiccator until further evaluation.

Characterization of S-SNEDDS. S-SNEDDS of BR were evaluated for angle of repose, true density, porosity, specific surface area, surface topography by scanning electron microscope (SEM) and in vitro dissolution. Angle of repose ([theta]) was determined by pouring the SSNEDDS through a funnel with its tip positioned at a fixed height (H) on a horizontal surface until the apex of the powder pile just touches the tip of the funnel. The angle of repose was calculated using the formula tan [theta] = H/r where r is radius of the pile of powder (Kallakunta et al. 2012). True density of Neusilin US2 and S-SNEDDS was measured using Helium Pycnometry (Smart Pycno 30, Smart Instruments, Mumbai). Before measurement samples were regenerated by heating at 50 [degrees]C for 30 min. Measurements were done in triplicate. Specific surface area was measured using Single Point Dynamic [N.sub.2] BET Method (Smart Instruments, Mumbai). The surface of Neusilin US2 and S-SNEDDS of BR was examined using SEM (JEOL JSM-6100, Tokyo, Japan). The particles were vacuum dried, coated with thin gold-palladium layer by sputter coater unit (JEOL JFM-1100, Tokyo, Japan) and observed microscopically at an accelerating voltage of 5.0 kv.

In vitro dissolution study

SNEDDS loaded with BR (2%, w/v) composed of Acrysol K-150, Capmul MCM and PEG 400; 1:0.33:0.66 were filled in Flofit capsules (ACG Associated capsules, Mumbai). Pure BR powder and S-SNEDDS were filled in hard gelatin capsules. The dissolution, for these three capsule formulations, was carried out in 900 ml, pH 6.8 phosphate buffer using the USP dissolution apparatus I (Basket) at a rotational speed of 50 rpm at 37 [degrees]C (Dissolution tester TDT06, Electrolab, Mumbai). The samples (5 ml) were withdrawn at predetermined time intervals, filtered through syringe filter (0.45 [micro]m, 25 mm, Qualisil, LCGC, Mumbai) and analyzed immediately after the completion of dissolution test by UV-vis spectrophotometer (V-630, Jasco UV-vis spectrophotometer, Japan). Equal volume of pH 6.8 phosphate buffer was replaced into the vessel after each sampling. BR dissolved in the dissolution media was measured at [[lambda].sub.max] 343 nm by validated spectrophotometric method. The anlaytical method was specific, linear in the concentration range of 2-10 [micro]g/ml, precise (% CV < 2) and accurate (99.5-102%). For each dissolution run, a mean of 6 determinations was recorded. Dissolution profiles were compared using Similarity factor; [f.sub.2] and dissolution efficiency at 30 min (DE30, %). DE30 was calculated from the area under the dissolution curve at time t (measured using the trapezoidal rule) and expressed as a percentage of the area of the rectangle described by 100% dissolution in the same time (30 min) (Costa and Lobo 2001).

In vivo chick embryo chorioallantoic membrane (CAM) assay

CAM assay was performed as per the reported method with modifications (Jung et al. 2007; Seow et al. 2011; Song et al. 2004). Fertilized chicken eggs were procured from hatchery and were cleaned and decontaminated using alcohol and were incubated in an incubator for 3 days at 37 [degrees]C under a constant relative humidity of 80%. Eighteen eggs per sample were prepared to allow for the 30-40% mortality inherent to the procedure and to yield a minimum of 8 available eggs per sample. A window was then cut in the shell using a fine-cutting tool, and the shell was then removed with sterile forceps. This window served as a portal of access for the CAM. Albumin (1-1.5 ml) was removed using a syringe with a 21gauge needle through the pointer end of the egg in order to allow detachment of the developing CAM from the eggshell and incubated for 8 days. Gelatin sponge was cut in approximately 2 [mm.sup.3] pieces and loaded with 2 [micro]l of test samples i.e. blank SNEDDS without BR and BR SNEDDS. Grafts were placed on developing CAMs on day 8 and further incubated to day 12. On day 12 CAMs were fixed with formaldehyde and dissected. Photograph of the embryos were captured with digital camera (Sony Cyber-shot DSC) and examined for anti-angiogenic effect at site of sample application. Images of the surface of the CAM within the same test sample were compared. The anti-angiogenic effect was quantified solely in the area of the CAM using a modified semi quantitative score system with a scale of 0-2 was used for grading (Burgermeister et al. 2002; Krenn and Paper 2009). The degree of anti-angiogenic effect was recorded blindly by two independent observers. The average score was calculated and interpreted based on grade scale.

In vivo anti-inflammatory activity in inflammatory bowel disease/experimental acute colitis induced rats: preliminary screening

The experimental protocol was approved by the Institutional Animals Ethics Committee and the experiment was carried out according to the guidelines of Committee for The Purpose of Control and Supervision on Experiments on Animals (CPCSEA) for experimental animal care. Male Wistar rats weighing 200-220 g were obtained from National Toxicology Centre, Pune. Rats were housed in standard wire mesh plastic cages in a room maintained at 22 [+ or -] 0.5 [degrees]C and 12 h light and 12 h dark cycle. Animals were given standard pellet food and water ad libitum. Experiments were carried out between 09:00 and 15:00h. During experimentation, the rats were divided in four groups; (I) normal, (II) control, (III) BR powder treatment group and (IV) BR loaded SNEDDS treatment group (n = 3 each). Rats from the same group were housed in one cage and fasted overnight with free access to water. Acute colitis was induced with acetic acid in three groups of rats namely; II, III and IV as per the method described by Paiva et al. (2004). Animals were anesthetized with ether and 2 ml of 4% (v/v) acetic acid was slowly administered via a polyethylene tubing (6 cm long, 0.7 mm thick) fitted to 1 ml syringe. The tubing was inserted through anus to reach the colon (Paiva et al. 2004). Before taking the catheter out, 1 ml air was applied in order to spread the acid completely within the colon. Three groups namely; II, III and IV, received a single intra-rectal administration of acetic acid on day 0. Thereafter, for the following 7 days, group III received BR powder suspended in 0.5% sodium CMC equivalent to 7.5 mg/kg, and group IV received BR loaded SNEDDS equivalent to BR 5 mg/kg, administered per orally. To analyze improvement in efficacy, dose of BR in SNEDDS was 1.5 times less than BR powder. On day 8, animals were sacrificed by cervical dislocation. Colon tissue specimens of control, BR treated animals were isolated and immersed in 10% neutral buffered formalin till further processing. Sections from these tissues were stained with haematoxylin and eosin and investigated by light microscopy for the presence of inflammatory changes. At least three specimens per treatment were observed and studied for histopathological changes.

Results and discussion

Treatment of chronic diseases requires long term therapy, and chronic use of synthetic drugs may cause unpredictable adverse effects. World Health Organization in support of natural remedies encourages the use of traditional medicines of natural origin. Many research based pharmaceutical companies are investing relatively more time and money on the development of herbal rather than synthetic drug formulations. Plant extracts are the most widely used natural medicine due to their ease of availability, comparatively low production cost and biocompatibility. However, the delivery of phytoconstituents is problematic due to poor solubility and low bioavailability and in vivo instability. These limitations of bioactive phytoconstituents can be overcome by nanotechnology.

In order to improve poor water solubility and limited dissolution rate, novel formulations are needed. One popular approach is the use of lipid and surfactant based drug delivery systems, in particular SM/SNEDDS. These formulations are clear monophasic liquids composed of drug, oil, surfactants, cosurfactants/cosolvents. When introduced into aqueous medium, present the drug in solution form ready for absorption. Therefore, in the formulation of SM/NEDDS the selection of suitable oil, surfactant and co-surfactant plays a key role to potentiate the solubility and drug loading.

Solubility studies and pseudo-ternary phase diagram

The components in the formulation of SNEDDS were selected to have maximum solubility of BR along with good miscibility with each other to produce an isotropic and stable system. The results of solubility of BR in various vehicles/excipients screened are shown in Table 1. The components used for developing a SM/SNEDDS should have high solubilization capacity for BR, ensuring maximum solubilization of BR after dispersion in aqueous media resulting in isotropic system. In the present study, amongst the various vehicles tested, highest solubility of BR was observed in Acrysol K-l 50, Capmul MCM and PEG 400; as oil, surfactant and co-solvent respectively. Therefore, these ingredients were selected for formulation SNEDDS of BR and evaluated further. The construction of pseudo-ternary phase diagram makes it easy to find out the concentration range of components that results in self-emulsification. Although self-emulsification is a dynamic process involving interfacial phenomena, information can be obtained about self-emulsification using equilibrium phase behaviour. There is a correlation between emulsification efficiency, formation of lamellar liquid crystalline dispersion phase, region of enhanced water solubilization and phase inversion region, after addition of water. To develop an optimum self-emulsifying formulation, self-emulsifying region in the phase diagram should be located (Singh et al. 2008). Pseudo-ternary phase diagrams were constructed by using Acrysol K-150, Capmul MCM and PEG 400 are presented in the Fig. l(a-c). It can be deduced from Fig. 1 that [S.sub.mix] ratio 1:2 and 2:1 have more emulsification area as compared to [S.sub.mix] ratio 1:1. Acrysol K-l 50 approximately 10-30% for all [S.sub.mix] ratios showed extensive and easy nanoemulsification, at room temperature. After analyzing the saturation solubility in all the 9 combinations of oil and [S.sub.mix] (1:9-9:1) for all the three [S.sub.mix] ratios (1:1,1:2 and 2:1), Acrysol K-l 50, Capmul MCM and PEG 400; 1:0.33:0.66, was selected for further characterization. This premix showed maximum solubility of BR; 27.53 [+ or -] 0.6 mg/ml (mean [+ or -] SD, n = 3). However, to avoid precipitation of BR, we incorporated 2% (w/v) BR to make BR loaded SNEDDS and used for further evaluation.

Characterization of BR loaded SNEDDS

Globule size is an important parameter in the development of self-emulsifying systems as it influences the rate of drug release as well as their in vivo stability. The drug can diffuse faster from smaller globules into the aqueous phase, thereby increasing the drug dissolution. Smaller globule size presents large surface area for drug absorption. Globule size of the emulsion BR loaded SNEDDS formed after dilution with water, 0.1 N HC1 and phosphate buffer pH 6.8 was determined by photon cross-correlation spectroscopy and was found to be 18.78 [+ or -] 1.01,16.12 [+ or -] 0.98,19.53 [+ or -] 1.58 nm. The polydispersity index values of the SNEDDS formulations were less than 0.5 for all the three above mentioned dilutions. The system may be considered as self-nanoemulsifying as the droplet size of emulsion was less than 100 nm. In addition, the mean globule size remained fairly constant even after varying the ratio of oil to surfactant:cosolvent. The pH of the dilution medium showed negligible effect on the mean globule size of emulsions indicating robustness of the formulation to dilution. Visual observations were made immediately after dilution for assessment of self-emulsification efficiency, phase separation, and precipitation of BR. Formulation required less than a 60s to form a slightly bluish-green coloured emulsion. As the BR is bright yellow coloured, nanoemulsion was bluish green coloured as compared to corresponding placebo mixture which had slight blue tinge.

Zeta potential was measured after dilution with double distilled water was found to be -9.14 mV. The presence of little negative charge on the dispersed phase of emulsion could be due to the anionic groups of the fatty acids and glycols present in the oil and surfactant. It is reported that positively charged emulsions get electrostatically attracted to the mucosal cell surface resulting in an improved bioavailability of the drug. However, such observations were found true in case of the coarse emulsions (Gershanik et al. 1998). In case of nanoemulsions, bioavailability is irrespective of its surface charge. The emulsion stability is directly related to the magnitude of the surface charge. It has been suggested that zeta potential may serve as a partial indicator for the physical stability of the emulsion being formed. High absolute zeta potential values (above 30 mV) should preferably be achieved in order to make sure about the repulsion of globules due to charge which will stabilize the system against coalescence. These recommended zeta potential values are predicted based on experiments. However, a wide range of zeta potential values have been reported for stable SNEDDS in previous studies (Zhao et al. 2010).

Stability was also analyzed by storing the diluted samples for 24 h at room temperature. There was no significant change in the droplet size as well as zeta potential. The developed system is a classical example of type Ill-A systems as described by Pouton and Porter (2008).

Thermodynamic stability study was designed to identify and avoid the metastable SNEDDS formulation. In thermodynamic stability studies, formulation was subjected to different stress tests like centrifugation and freeze-thaw test. The BR loaded SNEDDS was found to be stable to three freeze thaw cycles and showed no signs of precipitation and phase separation. Cloud point of the BR loaded SNEDDS was found to be >80 [degrees]C. Cloud point is the temperature above which an aqueous solution of emulsifying system becomes turbid. Turbidity at elevated temperatures is attributed to the phase inversion resulting from the increased mobility of the surfactant. When the temperature is higher than the cloud point, an irreversible phase separation will occur and the cloudiness of the preparation will affect absorption of the drug adversely. BR loaded SNEDDS exhibited cloud point >80 [degrees]C, indicating very few chances of flocculation or coagulation of the system in the biological environment and also during its shelf life.

Solid SNEDDS of BR (S-SNEDDS)

Converting liquid SM/NEDDS of a poorly water-soluble drug into solid enables the development of capsules or tablets. In addition to providing the obvious in vivo benefits of a SM/NEDDS, a high content of liquid SM/NEDDS can be loaded onto variety of carriers which maintains good micromeritic properties and helps in production of tablets with good cohesive properties and good content uniformity in both capsules and tablets. The transformation of SM/NEDDS into a solid dosage form can also be carried out by liquid capsule filling, spray drying, adsorption on to solid carriers, and melt granulation as well as other techniques. This clearly expands the formulation choices available. In terms of functionality and performance, the solubilizing properties of the final solid dosage form remain unaffected by both the adsorption of the liquid SM/NEDDS onto a carrier and the state of the drug in the lipid formulation. The formulation and the process are remarkably simple and straightforward and can be easily scaled up to industrial production level. Capsule filling and adsorption onto solid carrier techniques are more popular (Kohli et al. 2010).

Neusilin US2, is a highly porous magnesium aluminometasilicate capable of adsorbing up to three times its weight of oil (Sander and Holm 2009). Adsorption of BR loaded liquid SNEDDS on adsorbent Neusilin US2 (1:2) resulted in free flowing powder. The S-SNEDDS showed angle of repose ([theta]) 31.2[degrees] when determined using fixed funnel standing cone method indicatinggood flow. True density of Neusilin US2 and S-SNEDDS was measured using Heluim Pycnometry and was found to be 2.2 and 1.282 g/[cm.sup.3] respectively. Porosity was calculated using this true density and was found to be 93.18% and 72.69% respectively. Significant decrease in porosity of the material is due to the adsorption of SNEDDS of BR in the voids and interior spaces of Neusilin US2. Specific surface areas of Neusilin US2 and S-SNEDDS were measured using Single Point Dynamic [N.sub.2] BET Method and were found to be 300 [m.sup.2]/gm and 1.274 [m.sup.2]/gm, respectively. Tremendous decrease in surface area as well as porosity can be attributed to the adsorption of SNEDDS on the surface of Neusilin US2. BR crystals are acicular (Fig. 2A and B). Morphological examination of the surface of Neusilin US2 and S-SNEDDS of BR was carried out using a SEM. Microphotographs of powder samples were obtained using SEM are shown in Fig. 2C and D. From the Fig. 2C, Neusilin US2 appears to be spherical porous particles of size of approximately 100p,m. Scanning electron microphotographs of S-SNEDDS shows liquid SNEDDS adsorbed onto the surface of Neusilin US2 particles (Fig. 2 D). Since the formulation process involved facilitating adsorption through physical mixing, partially covered particles are also visible in the field of vision. Crystalline structure of solid BR is not seen in S-SNEDDS microphotographs suggesting that the BR is present in a completely dissolved form.

Dissolution studies of BR powder, liquid SNEDDS of BR and S-SNEDDS were carried out in 900 ml, pH 6.8 phosphate buffer using the USP dissolution apparatus I (Basket) at a rotational speed of 50 rpm at 37 [degrees]C. The dissolution profiles are plotted in Fig. 3. BR release from SNEDDS was significantly rapid as well as complete than the BR powder. More than 85% BR dissolved in 45 min. from S-SNEDDS and liquid SNEDDS. The dissolution profiles were compared using the model independent approach. Similarity factor; [f.sub.2]. All the three profiles were different with similarity factor<50. The dissolution of BR from S-SNEDDS was comparatively faster than liquid SNEDDS in initial stage of dissolution. However, the extent of dissolution was same at 45 min. This can be attributed to extensive surface area of adsorbent; Neusilin US2 over which the SNEDDS were adsorbed. [DE.sub.30] was calculated for BR powder, liquid SNEDDS of BR and S-SNEDDS and was found to be 19.5%, 25.9% and 37.5%, respectively. This indicates significant improvement in dissolution efficiency of BR when formulated as S-SNEDDS.

In vivo chick embryo chorioallantoic membrane (CAM) assay

Nowadays, increasing interest in anti-angiogenic therapy for cancer requires the development of a angiogenesis assay to investigate novel antitumor drugs. CAM has become a widely used tool for the determination of both angiogenesis and anti-angiogenic activities of many drugs including herbal extracts (Song et al. 2004). In the present study, the anti-angiogenic activity of SNEDDS of BR was evaluated in vivo using the CAM assay. CAM offers the advantage of being simple to use and low cost. In addition, the vascular system of CAM is directly accessible to observation and experimentation and there are no metabolic or hormonal influences (Mathur et al. 2006). Quantification of vascular changes in the CAM can be easily compared. In the present assay, the different qualitative changes in the capillaries for SNEDDS of BR treated CAM were observed and used to quantify the anti-angiogenic effect. It was observed that the area of the CAM below the disc containing blank SNEDDS without BR (blank control) showed normal vasculature and no change in vascular density (Fig. 4A). The appearance of normal branching pattern of blood vessels indicated that the weight of the gelatin graft and blank SNEDDS had no influence on growth of blood vessels. SNEDDS of BR demonstrated significant inhibition on the small capillaries below the graft, however, very few larger vessels remain unaffected (Fig. 4B). Modified semi quantitative score system with a scale of 0-2 as described below was used for grading the anti-angiogenic effect (Burgermeister et al. 2002; Krenn and Paper 2009).

Average score < 0.5 = no anti-angiogenic effect (inactive). 0.5 [less than or equal to] average score [less than or equal to] 1 = weak anti-angiogenic effect. 1 < average score < 1.5 = good anti-angiogenic effect. Average score [greater than or equal to] 1.5 = strong anti-angiogenic effect.

The degree of anti-angiogenic effect was recorded blindly by three independent observers and the average score for BR loaded SNEDDS was found to be >1.5 which indicates strong anti-angiogenic effect. Though formulation showed strong antiangiogenic effect, no lethal toxicity was observed in the chick embryos, indicating safety of the product.

In vivo anti-inflammatory activity in inflammatory bowel disease/experimental acute colitis induced rats: preliminary screening

The association of chronic inflammation with a variety of epithelial malignancies has been recognized for centuries. Well established examples include, among many others, oesophageal adenocarcinoma associated with chronic oesophagitis and bowel cancer associated with chronic inflammatory bowel diseases. There is now enough evidence that the chronic inflammatory process provides a microenvironment that includes upregulation of inflammatory mediators (inflammatory cytokines and prostaglandins). These inflammatory mediators suppress cell mediated immune responses and promote angiogenesis (Coussens and Werb 2002; Mignogna et al. 2004). It has been reported that the structural changes in the microenvironment of diseased tissues enable both passive and active targeting of therapeutic agents to these tissues. Due to increase in inflammation specific intestinal vascular permeability, inflammatory bowel disease may be targeted systemically using nanotechnology based formulations (Crielaard et al. 2012). A lower size of ~200nm enhances targeting of the drug by means of the Enhanced Permeability and Retention (EPR) effect while preventing the glomerular filtration. The long circulation time increases the statistical probability for sufficient accumulation of the drug at the target. Significantly higher drug concentrations may be obtained in the target tissue by employing such passively targeted drug delivery systems (Crielaard et al. 2012). Therefore, we opted pharmacodynamic studies, to verify the improvement in efficacy, at the site of action, at a lower dose.

Induction of inflammatory bowel disease/experimental colitis by acetic acid in rats is one of standardized methods to produce an experimental model of inflammatory bowel disease. Acetate ions cause massive intracellular acidification resulting in injury of epithelial cells and inflammatory response. The causative factors in the initiation of colitis are enhanced vasopermeability, prolonged neutrophils infiltration and increased production of inflammatory mediators (Elson et al. 1995). The present study has shown that acetic acid induced ulcerative colitis was associated with macroscopic as well as microscopic changes. The daily body weight changes in rats were analyzed. Normal rats (group I) alone gained body weight and showed normal colonic mucosal epithelium and absence of inflammation (Fig. 5A). In group II i.e. rats with acetic acid induced colitis, body weight gradually decreased and did not recover at the end of the experiment. On the other hand, the body weight of rats in groups III and IV significantly recovered compared with the acetic acid induced colitis rats (group II). Rats treated with acetic acid alone showed hypomotility, prostration and piloerection. Blood adhesion to the anus was also noted in group II rats. Histological study in the colons of rats of group II showed extensive destruction of mucosal epithelium, inflammatory cell infiltration with necrotic foci, loss of goblet cells and oedema (Fig. 5 B). The architecture of the crypts was distorted. The histologic sections of the colonic mucosa of rat treated with BR suspension (group III) and BR loaded SNEDDS (group IV) (Fig. 5C and D, respectively) showed progressive restoration, improvement of the crypt architecture. Furthermore, there was significant reduction in the congestion and oedema in comparison to those of the acetic acid control group. It should be noted that the dose of BR given to the group IV animals was 1.5 times (~33%) less than that of the group III. This indicates potentiation of anti-inflammatory effect of BR by nanoemulsification. Nanotechnology can offer reduction in dose because of targeting the drug and accumulation at the site of inflammation. The ability of the nanosized formulations to target the bioactives, precisely and differentially, at the site of action, due to EPR effect can establish a new paradigm in the management of chronic inflammation and cancer.

Conclusion

Our studies demonstrated that the new SNEDDS of BR is a promising strategy for improving solubility and in turn, therapeutic efficacy of BR. The developed novel formulation has a potential application in treatment of chronic inflammatory conditions and cancer. SNEDDS is commercially viable formulation of choice to give a new lease of life to old but potential phytochemical; BR. Conversion of liquid SNEDDS into solid dosage form using adsorbents like Neusilin US2 offers an additional alternative in the pursuit to improve product design, manufacturability, product performance and efficacy.

http://dx.doi.org/10.1016/j.phymed.2013.09.013

ARTICLE INFO

Article history:

Received 30 April 2013

Received in revised form 31 July 2013

Accepted 20 September 2013

Acknowledgements

Authors are thankful to Indian Institute of Integrative Medicine, Jammu, for the gift sample of berberine. The generosity of Gattefosse India Pvt. Ltd. (Mumbai, India), Abitec Corporation (Mumbai, India), ACG Associated Capsules Pvt. Ltd. (Mumbai, India), Corel Pharma Ltd. (Ahmedabad, India) and Gangwal Chemicals Pvt. Ltd. (Mumbai, India) is gratefully acknowledged for providing the gift samples of variety of excipients. The authors wish to thank Dr. U. Aswar, Head, Department of Pharmacology for her assistance in conducting animal studies.

References

Aggarwal, B.B., Shishodia, S., Sandur, S.K., Pandey, M.K., Sethi, G., 2006. Inflammation and cancer: how hot is the link? Biochem. Pharmacol. 72, 1605-1621.

Bachhav. Y.G., Patravale, V.B., 2009. SMEDDS of Glyburide, formulation, in vitro evaluation, and stability studies. AAPS PharmSciTech 10, 482-487.

Burgermeister, J., Paper, D.H., Vogl, H., Linhardt, R.J., Franza, G., 2002. LaPSvS1, a(1 [right arrow] 3)-[beta]-galactan sulfate and its effect on angiogenesis in vivo and in vitro. Carbohydr. Res. 337, 1459-1466.

Costa., P. Sousa, Lobo, J.M.S., 2001. Modelling and comparison of dissolution profiles. Eur.J. Pharm. Sci. 1, 123-133.

Coussens, LM., Werb, Z., 2002. Inflammation and cancer. Nature 420, 860-867.

Crielaard, B.J., Lammers, T., Schiffelers, R.M., Storm, G., 2012. Drug targeting systems for inflammatory disease: one for all, all for one. J. Control. Release 161, 225-234.

Elson, C.O., Sartor, R.B., Tennyson, G.S., Riddell, R.H., 1995. Experimental models of inflammatory bowel disease. Gastroenterology 109, 1344-1367.

Gershanik, T., Benita, S., 2000. Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs. Eur. J. Pharm. Biopharm. 50, 179-188.

Gershanik, T., Benzeno, S., Benita, S., 1998. Interaction of a self-emulsifying lipid drug delivery system with the everated rat intestinal mucosa as a function of droplet size and surface charge. Pharm. Res. 15, 863-869.

Gursoy, R.N., Benita, S., 2000. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs. Biomed. Pharmacother. 58, 173-182.

He, Z.H., He, M.F., Ma, S.C., Paul, B.P.H., 2009. Anti-angiogenic effects of rhubarb and its anthraquinone derivatives. J. Ethnopharmacol. 121, 313-317.

Huang, S.T., Yang, R.C., Lee, P.N., Yang, S.H., Liao, S.K., Chen, T.Y., Pang, J.H., 2006. Anti-tumor and anti-angiogenic effects of Phyllanthus urinaria in mice bearing lewis lung carcinoma. Int. J. Immunopharmacol. 6, 870-879.

Jung, H.J., Jeon, H.J., Lim, E.J., Ahn, E.K., Song, Y.S., Lee, S., Shin, K.H., Lim, C.J., Park, E.H., 2007. Anti-angiogenic activity of the methanol extract and its fractions of Ulmus davidiana var japonica. J. Ethnopharmacol. 112, 406-409.

Kale, A., Patravale, V., 2008. Design and evaluation of self-emulsifying drug delivery systems (SEDDS) of nimodipine. AAPS PharmSciTech 9, 191-196.

Kallakunta, V.R., Bhandari, S., Jukanti, R., Veerareddy, P.R., 2012. Oral self emulsifying powder of lercanidipine hydrochloride: formulation and evaluation. Powder Technol. 221, 375-382.

Kohli, K., Chopra, S., Dhar. D., Arora, S., Khar, R.K., 2010. Self-emulsifying drug delivery systems: an approach to enhance oral bioavailability. Drug Discovery Today 15, 958-965.

Krenn, L, Paper, D.H., 2009. Inhibition of angiogenesis and inflammation by an extract of red clover (Trifolim pratense L). Phytomedicine 16, 1083-1088.

Kundu, J.K., Surh, Y.J., 2008. Inflammation: gearing the journey to cancer. Mutat. Res. 659, 15-30.

Lee, Y.S., Kim, Y.H., Shin, E.K., Kim, D.H., Lim, S.S., Lee, J.Y., Kim, J.K., 2010. Antiangiogenic activity of methanol extract of Phellinus linteus and its fractions. J. Ethnopharmacol. 131, 56-62.

Mathur, R., Gupta, S.K., Singh, N., Mathur, S., Kochupillai, V., Velpandian, T., 2006. Evaluation of the effect of Withania somnifera root extracts on cell cycle and angiogenesis. J. Ethnopharmacol. 105, 336-341.

Matos, M., Gutierrez, G., Coca, J., Pazos, C., 2013. Preparation of water-in-oil-in-water (Wl /0/W2) double emulsionscontaining trans-resveratrol. Colloids Surf. A: Physicochem. Eng. Aspects, http://dx.doi.Org/10.1016/j.colsurfa.2013.05.065.

Mignogna, M.D., Fedele, S., Lo Russo, L., Lo Muzio, L, Bucci, E., 2004. Immune activation and chronic inflammation as the cause of malignancy in oral lichen planus: is there any evidence? Oral Oncol. 40, 120-130.

Paiva, L.A.F., Gurgel, LA., De Sousa, E.T., Silveira, E.R., Silva, R.M., Santos, F.A., Rao, V.S.N., 2004. Protective effect of Copaifera langsdorffii oleo-resin against acetic acid-induced colitis in rats. J. Ethnopharmacol. 93, 51-56.

Pouton, C.W., 1997. Formulation of self-emulsifying drug delivery systems. Adv. Drug Deliv. Rev 25, 47-58.

Pouton, C.W.. 2006. Formulation of poorly water-soluble drugs for oral administration: physicochemical and physiological issues and the lipid formulation classification system. Eur. J. Pharm. Sci. 29, 278-287.

Pouton, C.W., Porter, C.J.H., 2008. Formulation of lipid-based delivery systems for oral administration: materials, methods and strategies. Adv. Drug Deliv. Rev. 60, 625-637.

Sander, C., Holm, P., 2009. Porous magnesium aluminometasilicate tablets as carrier of a cyclosporine self-emulsifying formulation. AAPS PharmSciTech 10, 1388-1395.

Seow, L.J., Beh, H.K., Majid, A.M.S.A., Murugaiyah, V., Ismail, N., Asmawi, M.Z., 2011. Anti-angiogenic activity of Gynura segetum leaf extracts and its fractions. J. Ethnopharmacol. 134, 221-227.

Setthacheewakul, S., Mahattanadui, S., Phadoongsombut, N., Pichayakorn, W., Wiwattanapatapee, R., 2010. Development and evaluation of self-microemulsifying liquid and pellet formulations of curcumin, and absorption studies in rats. Eur. J. Pharm. Biopharm. 76, 475-485.

Singh, A.K., Chaurasiya, A., Singh, M., Upadhyay, S.C., Mukherjee, R., Khar, R.K., 2008. Exemestane loaded self-microemulsifying drug delivery system (SMEDDS): development and optimization. AAPS PharmSciTech 9, 628-634.

Song, Y.S., Kim, S.H., Sa, J.H., Jin, C., Lim, C.J., Park, E.H., 2004. Antiangiogenic and inhibitory activity on inducible nitric oxide production of the mushroom Ganoderma lucidum. J. Ethnopharmacol. 90, 17-20.

Tan, W., Li, Y., Chen, M., Wang, Y., 2011. Berberine hydrochloride: anticancer activity and nanoparticulate delivery system. Int. J. Nanomed. 6, 1773-1777.

Zhao, Y., Wang, C., Chow, A.H.L., Ren, K., Gong, T., Zhang, Z., Zheng, Y., 2010. Self nanoemulsifying drug delivery system (SNEDDS) for oral delivery of Zedoary essential oil: formulation and bioavailability studies. Int. J. Pharmacol. 383, 170-177.

Zhu, J.-X., Tang, D., Feng, L., Zheng, Z.-G., Wang, R.-S., Wu, A.-G., Duan, T.-T., He, B., Zhu, Q., 2013. Development of self-microemulsifying drug delivery system for oral bioavailability enhancement of berberine hydrochloride. Drug Dev. Ind. Pharm. 39, 499-506.

Zuo, F., Nakamura. N., Akao. T., Hattori, M., 2006. Pharmacokinetics of berberine and its main metabolites in conventional and pseudo germ-free rats determined by liquid chromatography/ion trap mass spectrometry. Drug Metab. Dispos. 34, 2064-2072.

Swati Pund *, (1), Ganesh Borade (1), Ganesh Rasve

Department of Pharmaceutics, STES's Sinhgad Institute of Pharmacy, Pune, India

* Corresponding author at: Department of Pharmaceutics, STES's Sinhgad Institute of Pharmacy, Narhe (Ambegaon), Pune 411041, Maharashtra, India.

Tel.: +91 20 66831801; fax: +91 20 66831816.

E-mail address: swatipund@gmail.com (S. Pund).

(1) These authors contributed equally to this work.

Table 1
Solubility of BR in various oils, surfactants and co-solvents
estimated at room temperature.

Vehicle               Solubility (a)

Acrysol K 150          20.8 [+ or -] 1.5
Ethyl oleate          0.071 [+ or -] 0.01
Isopropyl myristate    2.20 [+ or -] 0.05
Olive oil               0.6 [+ or -] 0.03
Sesame oil             2.58 [+ or -] 0.07
Captex 200             0.93 [+ or -] 0.11
Ethanol                  10 [+ or -] 1.2
PEG 400               46.24 [+ or -] 2.6
Transcutol HP         29.94 [+ or -] 2.8
Propylene glycol      33.03 [+ or -] 2.8
Labrafac CC            0.34 [+ or -] 0.05
Labrafil M1944CS       0.02 [+ or -] 0.01
Cremophore RH 40       0.03 [+ or -] 0.01
Labrasol               10.3 [+ or -] 1.25
Capryol 90             3.93 [+ or -] 0.37
Capmul MCM             30.2 [+ or -] 2.6
Tween 20               0.85 [+ or -] 0.03
Tween 60                1.0 [+ or -] 0.2
Tween 80                1.2 [+ or -] 0.6
Cremophore EL          0.49 [+ or -] 0.08

(a) Data expressed as mg/ml, mean [+ or -] SD (n = 3).
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Author:Pund, Swati; Borade, Ganesh; Rasve, Ganesh
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Feb 15, 2014
Words:7584
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