INTRODUCTION:

Oral bioavailability of drugs is directly related with their solubility and permeability. The solubility and bioavailability of poorly soluble drugs has been improved by several techniques including particle size reduction, complex formation, nanotechnologies, solid dispersion, polymeric micelle, salt formation, prodrugs, polymorphs, and solvates^1-2. Each of these techniques has its pros and cons. Different solubilization techniques may have different influence

on the solubility and other physicochemical characteristics of the same drug molecule (Sharma, D. K. 2016). So, there is no universal technology which can be used in all cases of solubility enhancement, while preserving the safety and efficacy of drugs. Among these techniques of solubility enhancement, solid dispersion seems to be an easy and simple technique. In solid dispersion, a poorly soluble drug substance is dispersed in a quickly water soluble solid carrier matrix³. The present investigation has modified the solid dispersion technique (fusion) and compared its application on solubility enhancement of poorly soluble drugs with conventional fusion technique.

Resveratrol consists of two aromatic rings joined by an ethylene bridge and bearing three hydroxyl groups to form the 3, 4’, 5-trihydroxystilbene a molecule correlated to the synthetic estrogen diethylstilbestrol. Thanks to the presence of the hydroxyl groups. Resveratrol is able to form a radical stabilized by the electron delocalization on the two aromatic rings and the ethylene bridge, explaining the potent antioxidant properties typical of polyphenols. Resveratrol can exist in the two stereo-isomeric cis and trans forms. Although both forms have been found in natural products, only the trans-isomer is present in grapes and appears to be responsible for its numerous benefits. The cis-isomer is readily formed upon light exposure of trans- resveratrol and was found to be quite unstable⁴.

MATERIALS AND METHODS:

Resveratrol was procured from Yarrow-chem., Pvt. Ltd. Mumbai. Mannitol was procured from S.D. Fine Chem. Mumbai; Sodium Bicarbonate and Citric Acid was obtained from Rankem, Gujarat. All other chemicals and ingredients used were of analytical grade and were used as obtained.

Methods:

Drug-Excipients Interaction Study:

The drug and excipients compatibility study was carried out using FTIR and DSC resp.

Fourier Transformation Infra-red Spectroscopy:

The study by FTIR of the drug and excipient was carried out by conventional KBr plate method in order to study the interaction of the drug and polymer so as to determine the physical as well as chemical changes that can occur during the formulation. For this the mixture of powder of excipient and pure was mixed in a ratio of 1:1 with potassium bromide and the small pellet was formed by pressing the mixture in a hydraulic press and the FT-IR was carried out in the frequency range 400-4000cm-1^5-6.

Standard Calibration Curve:

The standard calibration curve of resveratrol was carried out on UV spectrophotometer by using phosphate buffer of pH 7.4 as the solvent. From the solution which is now having a concentration of 100μg/ml samples of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5ml were pipette out into 10ml volumetric flasks. The volume was made up to the mark with Phosphate buffer 7.4 to get the final concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/ml respectively. The absorbance of concentration was measured at 304nm⁷.

Preparation of Dispersion of Resveratrol:

Mannitol was melted in a glass beaker (at 175–180°C), and citric acid was added to the molten mannitol. This mixture of mannitol and citric acid was melted and uniformly mixed by continuous stirring. Powder of resveratrol was added to this molten mixture under continuous stirring. To the molten mixture of mannitol, citric acid and drug, the sodium bicarbonate (carbonic base) was added under rapid stirring. The ratio of sodium bicarbonate and citric acid was according to their molar reactivity. Since one molecule of citric acid may react with three molecules of sodium bicarbonate, the quantity of citric acid was in 1:3 molar ratio of sodium bicarbonate. After addition of sodium bicarbonate, the effervescence was generated due to acid–base reaction and the molten mixture turns into white froth. This frothy molten mixture was continuously stirred until the effervescence slowed down. The froths were cooled down and allowed to solidify. This cooled solid dispersion was crushed and ground gently using a mortar and pestle^10-11. Powdered formulation was stored in desiccators for further use^8-9.

Table 1: Preparation of Effervescence Assisted Solid Dispersion of Resveratrol

Batch	Drug	Mannitol	Citric Acid	Sodium Bicarbonate
F1	1gm	100mg	100mg	100mg
F2	1gm	100mg	100mg	200mg
F3	1gm	100mg	200mg	100mg
F4	1gm	200mg	100mg	100mg
F5	1gm	200mg	200mg	100mg
F6	1gm	200mg	200mg	200mg

Evaluation Parameters:

Micromeretics Study^10-15:

Bulk Density:

An accurately weighed sample was carefully introduced into a 10ml graduated cylinder with the aid of funnel. Typically, the initial volume was noted. Carefully level the product without copacting, if necessary, and read the unsettled apparent volume V0, to the nearest graduated unit. Calculate the bulk density in g/cm³ by the formula.

Bulk Density = Weight of Sample/Volume of Sample

Tap Density:

The tapped density was obtained by dividing the mass of a powder by the tapped volume in cm³. The sample is carefully introduced into a 10 ml graduated cylinder. The cylinder was dropped at 2 second intervals onto a hard wood surface 100 times from a height of 1 inch. The tapped density of each formulation was then obtained by dividing the weight of sample in grams by the final tapped volume in cm3 of the sample contained in the cylinder. It was calculated by using equation given below

Tap Density = Weight of Sample/Tapped Volume

Carr’s Index:

The Carr’s index was evaluated for the flow ability of the powder by comparing the pour density and tapped density of dispersion.

Carr’s Index = Tap density-Bulk density/Tap density ×100

Hausner’s Ratio:

Hausner’s ratio (H), another index of flow ability

Hausner’s Ratio = Tap density/Bulk density

Angle of Repose:

A weighed quantity of prepared dispersion was passed through a funnel fixed on a stand at a specific height. A static heap of powder with only gravity acting upon it was tending to form a conical mound. The height of the heap (h) and radius (r) of lower part of cone were measured.

Tan θ = height of pile/radius of pile

Percent Yield:

The percent yield of the prepared dispersion was calculated on the basis of the drug and polymer used and the amount obtained after the preparation of dispersion.

Percent Yield = Practical yield/ Theoretical yield×100

Percent Drug Content:

Samples containing 100mg of resveratrol dispersion were accurately weighed and dissolved in phosphate buffer of pH 6.8 and sonicated for 10 min. The solution is then filtered using a whatman filter paper from the above solution 10ml is pipette out and made up to 100 ml using phosphate buffer of pH 6.8. The resultant solution is then analyzed spectrophotometrically at 304nm and then the percent drug content is estimated¹⁶.

In-vitro Dissolution Study:

USP dissolution apparatus Type I (basket type) was used to study the drug release behavior of pure drug and dispersion. Dissolution behavior of pure resveratrol and prepared dispersion was studied using phosphate buffer pH 6.8 as dissolution medium. Drug (1000mg) in muslin cloth which was tightly tied was placed in basket containing 900ml of solution of pH 6.8 for 2 hrs. After each regular interval of time 5ml of solution was withdrawn and diluted with buffer 6.8 solution. After each withdrawal of the samples the fresh dissolution medium was added into the apparatus in order to maintain the sink condition. The diluted samples were then analyzed at 304nm using UV-spectrophotometer. The cumulative drug release (% CDR) was then calculated for every batch of prepared dispersion^17-19.

Kinetics Study:

The drug release data were fitted to zero order (cumulative vs. %drug release versus time), first order (log of cumulative vs. %drug retained versus time), and Higuchi models (cumulative vs. %drug released versus square root of time) and Korsmeyer-Peppas model (log of %cumulative drug release vs. log of time) to calculate the kinetics of drug release and determine the release mechanism of the drug from the prepared dispersion^20-22.

Solubility Studies:

Solubility of resveratrol dispersion was studied in different solvents such as distilled water and phosphate buffer pH (6.8). Pure drug and an excess amount of dispersion formulation were added in 10ml of the chosen solvents. The mixtures are mixed in a mechanical stirrer for 24 hrs. Visual inspection is carefully made to ensure there are excess resveratrol solids in the mixture, indicating saturation have been reached. The mixtures are then filtered and the resultant solution was diluted suitably to determine the solubility of resveratrol in each solvent by using UV spectrophotometer at 304nm²³.

X-ray Diffraction Study (XRD):

X-ray diffraction spectra of resveratrol and prepared dispersions were recorded with x-ray diffractometer employing a voltage of 45 Kv and a current of 40 mA. The instrument was operated in continuous scan mode over 2θ range at 20°- 80°. The relative intensity I/I0 and interplanar distance (d) like the 2θ values were reported and compared²⁴.

Scanning Electron Microscopy (SEM):

Scanning electron microscopy of resveratrol and prepared dispersions were taken using scanning electron microscope. The form and surface morphology were observed using SEM. The dispersions were observed at various magnifications so as to research the effect of additives on surface morphology and crystallization efficiency²⁵.

RESULTS AND DISCUSSION:

1). Drug –Excipient Interaction Study:

The results from FTIR of pure resveratrol represented the following band characteristics at 3309cm^-1 of Free O-H stretching vibration, 1458, 1504, 1597cm^-1 exhibited benzene skeleton vibrations, and 995cm^-1 represented bending vibration of C=C-H, the typical transolefinic band. The results of FTIR were compared with the standard and it was found that the pure drug was having the same peaks as that of the standard which confirmed that the drug was pure and the optimized formulation was then matched with the peaks of pure drug and it was seen that there was no new formation, disappearance, mismatching of peaks.

Figure 1: FTIR of Pure Drug

Figure 2: FTIR of Optimized Formulation

Standard Calibration Curve:

The results of standard calibration curve revealed that it follows the beers lamberts law as the equation obtained was linear with the values of y =0.014x + 0.005 and the regression value of R² = 0.999.

Figure 3: Standard Calibration Curve of Resveratrol

Micromeretics Study:

The micromeretics study of the prepared batches showed that the flow property of the prepared batches was good. The tap density was found to be in the range of 0.713±0.36-0.929±0.07 gm/cm³, the bulk density was found to be in the range of 0.798±0.29-0.878±0.17 gm/cm³, the Carr’s index and Hausner’s ratio was found to be in the range of 4.8-7.2 and 1.03-1.08. The angle of repose was found to be in the range of 13.37^o+0.32-21.25^o±0.71. The evaluation of percent drug content revealed that the percent entrapment was found to be in the range of 46.18 ± 1.55%- 58.07 ± 1.02% from which the batch F5 shows the maximum drug entrapment efficiency.

Table 3: Evaluation of Percent Yield and Drug Entrapment Efficiency

Batch	Percent Yield (%)	Drug Entrapped (%)
F1	79.36±0.67	46.18 ± 1.55
F2	85.64±0.03	55.15 ± 2.17
F3	86.12±0.72	55.72 ± 3.01
F4	82.24±0.08	51.60 ± 3.61
F5	89.38±0.25	58.07 ± 1.02
F6	79.20±0.28	57.01 ± 1.26

In-Vitro Drug Release:

The percent cumulative drug release of the prepared microspheres showed that the drug release was found to be in the range of 1.203% - 99.08% in the time of 12hrs. It can be seen that the batch F5 was having the maximum drug content entrapment and it also released the maximum amount of drug in the predicted time period. From the results it can be concluded that the batch F5 was the perfect combination for the microspheres and therefore from the results it was concluded that the batch F5 was optimum. This batch was further studied for the kinetics study.

Figure 4: Percent Cumulative Drug Release of the Prepared Formulations

Kinetics Study:

From the results of kinetics study as shown in Table 4 it can be seen that the prepared optimized formulation possesses a drug release by korsmeyers-peppas model of diffusion with the R²value of 0.9711.

Table 4: Kinetics Study of the Optimized Formulation

Batch	Zero Order (R²)	First Order (R²)	Higuchi (R²)	Korsmeyers-Peppas (R²)	Best Fit Model
F5	0.6969	0.8726	0.9146	0.9711	Korsmeyers-Peppas

Solubility Studies:

The solubility study of the prepared dispersion and the pure drug was studied in distilled water and in phosphate buffer of pH 6.8. The formulations showing highest amount of drug release was chosen for the study with the optimized formulation. From the results it was clear that the optimized formulation was having the highest solubility of drug in the solvents selected for the test. The pure drug resveratrol showed the solubility of 4.174±0.02mg in distilled water and 20.547±0.05mg in phosphate buffer while the optimized formulation F5 showed the solubility of 40.068±0.09mg in distilled water and 160.182±0.14mg in phosphate buffer.

Figure 5: Solubility Study of the Prepared Dispersion.

X-ray diffraction study (XRD):

The XRD pattern of resveratrol exhibited intense, sharp well resolved peaks whereas XRD pattern of prepared dispersion exhibited less intense and denser peaks compared to resveratrol. The XRD pattern of resveratrol showed its characteristics peaks at 2θ of 5.77, 10.88, 12.09, 13.03, 16.79, 18.02, 18.60, 19.47, 20.26, 21.00, 22.90 and 23.36 which are the characteristic of a crystalline compound.

Figure 6: XRD of Pure Drug

Figure 7: XRD of Optimized Formulation

Scanning Electron Microscopy (SEM):

The SEM study of the pure drug and the prepared dispersion was studied and it was seen that the drug is having a tile like crystal appearance which might be due to the compression during the packaging while the prepared dispersion revealed the flakes surface.

Figure 8: SEM of Pure Drug

Figure 9: SEM of Optimized Formulation

CONCLUSION:

As resveratrol is water insoluble medication the straightforward justification for this low impact on the solvency of resveratrol might be the immaterial effect of combination measure on the dynamic surface space of scattered medication particles. Bubbling helped strong scattering of resveratrol showed a striking improvement in the fluid dissolvability of these medications. Adequacy of solvency improvement through bubbling helped strong enhancement of solubility of resveratrol was up to 10 times. In fizz helped strong scattering, the consistently circulated drug particles with diminished molecule size showed improvement insolvency in light of the fact that these little medication particles were covered/restricted to the water solvent transporter framework, and their dynamic surface region was expanded.

ACKNOWLEDGEMENT:

The authors are thankful to the management of P.R. Pote Patil College of Pharmacy for providing us with the facilities to carry out this research work without which this work could not have been performed.

CONFLICT OF INTEREST:

None

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Received on 28.03.2022 Modified on 14.06.2022

Res. J. Pharma. Dosage Forms and Tech.2022; 14(4):293-298.

DOI: 10.52711/0975-4377.2022.00048

Batch No.	Tap Density (gm/cm³)	Bulk Density (gm/cm³)	Carr’s Index	Hauser’s Ratio	Angle of Repose
Pure Drug	0.803±0.36	0.741±0.04	7.7	1.08	24.56 +0.22
F1	0.713±0.36	0.752±0.04	6.7	1.07	19.60⁰±0.25
F2	0.797±0.02	0.786±0.36	4.8	1.03	21.25⁰±0.71
F3	0.833±0.06	0.878±0.17	7.2	1.04	16.87⁰±0.06
F4	0.883±0.58	0.784±0.29	6.3	1.08	19.58⁰±0.02
F5	0.922±0.12	0.862±0.07	4.3	1.03	18.84⁰±0.08
F6	0.929±0.07	0.902±0.03	6.1	1.05	15.88⁰±0.47