DEVELOPMENT AND EVALUATION OF PHOSPHATIDYLCHOLINE COMPLEXES OF ARBUTIN AS SKIN WHITENING AGENT

DEVELOPMENT AND EVALUATION OF PHOSPHATIDYLCHOLINE COMPLEXES OF ARBUTIN AS SKIN WHITENING AGENT

R. K. Patel and M. B. Jadeja ^*

Shree S.K. Patel College of Pharmaceutical Education and Research, Ganpat University, Mahesana - 384012, Gujarat, India.

ABSTRACT: Introduction: Disorders of hyperpigmentation are difficult to treat, particularly in dark skin individuals. There is a dramatic enhancement of skin permeability of phytoconstituents due to their complexation with phospholipids. Complexation leads to better permeation through the skin, which is otherwise not possible. Research work focused on the development and evaluation of phos-phatidylcholine complexes of arbutin, having the issue of poor skin permeability as skin whitening agents. Materials and Methods: Arbutin, phosphatidylcholine, and petroleum ether have been used for the development of complexes. Authentication of arbutin has been performed by Fourier Transform Infrared study. UV–visible spectrophotometric method has been developed for estimation of arbutin followed by the development of complexes by rotary flask evaporation technique, preliminary trials for selection of dependent and independent variables, optimization of complexes by Box- Behnken reduced surface response design, evaluation of optimized batch by percentage entrapment efficiency, particle size, zeta potential, and in-vitro drug release study by Franz diffusion cell. Result and Discussion: Arbutin sample FTIR data and standard FTIR data are matched. Final polynomial equation is, % Entrapment Efficiency = + 161.924 - 25.954 * Arbutin: PC ratio - 3.8654 * Film formation temperature 0.991 * Hydration temperature +0.29737654320988 * Hydration time +17.0566 * Arbutin: PC ratio 2 + 0.03880416666667 * Film formation temperature 2 + 9.941E-003 * Hydration temperature 2 -1.499E-003 * Hydration time 2. Results show that there is ≥ 92% similarity between predictive and actual values. Developed complexes show a better in-vitro drug release profile compared to an aqueous solution of arbutin, which indicates optimized complexes improve permeability.

Phosphatidylcholine complexes, Arbutin, Box-Behnken design, Skin whitening agents, Rotary flask evaporation method

INTRODUCTION: Variability of skin tones throughout the world is well-documented, some skin tones being reported as more susceptible to pigmentation disorders than others, especially in Asia and India. Furthermore, exposure to ultraviolet radiation is known to trigger or exacerbate pigmentation disorders.

strategies for photoprotection and treatment modalities, including topical and other medical approaches, have been adopted by dermatologists to mitigate these disorders ¹². There are various issues relating to the available marketed formulation of synthetic compounds.

So, there is a clinical need for the development and optimization of the formulation, which may provide better absorption of the drug over the skin and treat the diseases related to hyperpigmentation. A phytosomes is a complex between a natural product and natural phospholipids, like soy phospholipids. Such a complex result from the reaction of stoichiometric amounts of phospholipid with the selected phytoconstituents. On the basis of their physicochemical and spectroscopic data, it has been shown that the main phospholipid-substrate interaction is due to the formation of hydrogen bonds between the polar head of phospholipids (i.e., phosphate and ammonium groups) and the polar functional groups of the substrate. They are lipophilic substances with a clear melting point, freely soluble in non-polar solvents (in which the hydrophilic moiety was not), and moderately soluble in fats.

When treated with water, phytosomes assume a micellar shape is forming liposomal-like structures. In liposomes, the active principle is dissolved in an internal pocket or floats in the layered membrane, while in phytosomes the active principle is anchored to the polar head of phospholipids, becoming an integral part of the membrane. Molecules are anchored through chemical bonds to the polar head of the phospholipids, as can be demonstrated by specific spectroscopic techniques ^3-10.

TABLE 1: COMMERCIAL PRODUCTS ^{4, 11, 12}

Proposed research work focused on development and evaluation of phosphatidylcholine complexes of some phytopharmaceuticals having an issue of poor skin permeability as skin whitening agents. Selection of phytoconstituents or standardized herbal extracts having skin whitening activity and poor skin permeability for a dermatological disease like hyperpigmentation. Selection of a suitable method of preparation for the development of phosphatidylcholine-phytoconstituents complexes by preliminary screening or trials. Development of phosphate-dylcholine-phytoconstituents complexes by the selected method. Optimization of process parameters and variables for the selected method of preparation and selection of optimized batch by suitable statistical design and experimental design. Evaluation and characterization of optimized batch on selected criteria.

TABLE 2: PATENT ON PHOSPHOLIPID-PHYTOCONSTITUENTS COMPLEXES ^13-20

MATERIALS AND METHODS: Chemicals and instruments used for research work are in tabulated form as Table 3 and Table 4. Research work performed from January 2016 to January 2019.

TABLE 3: CHEMICALS

TABLE 4: EQUIPMENT AND INSTRUMENTS

Authentication of Active Pharmaceuticals: The Fourier Transform Infrared (FTIR) spectrum of Arbutin was recorded on Shimadzu FTIR spectrophotometer.

The sample was prepared by using the press pellet technique and scanned for transmission in the wavenumber. Then it was compared with the standard FTIR spectrum of Arbutin.

UV–Visible Spectrophotometric Method Development for Estimation of Arbutin: All solutions utilized for sample preparation were of analytical grade. A stock solution of Arbutin at a concentration of 1000 ppm was prepared by dissolving 100 mg Arbutin in 100 ml distilled water. Working solution of Arbutin at concen-tration of 100 ppm was prepared by 10 times dilution of stock solution. Calibration sample solutions were prepared by serial dilution from the working solution at a concentration of 10, 15, 20, 25, 30 ppm. The baseline was corrected across 400 to 200 nm, using distilled water as a sample and blank. ʎ_max of Arbutin was observed in spectrum mode, using 30 ppm calibration solution in the sample holder. Absorbance was recorded for rest of the calibration samples in photometric mode at 221 nm as ʎ_max. A calibration curve was prepared by plotting absorbance versus concentration of arbutin. From plot, slope and intercept were determined.

Method for Development of Complexes by Rotary Flak Evaporation Technique: Phosphati-dylcholine dissolved in petroleum ether in a round bottom flask of rotary flask evaporator. Applied defined temperature for formation of a film of phosphatidylcholine by rotation of flask. For the hydration of film, a previously prepared aqueous solution of Arbutin in a defined concentration was poured into the flask at a defined temperature and time.  At the end of defined time, development of complexes in the form of suspension ²¹.

TABLE 5: PRELIMINARY TRIALS

Optimization of Arbutin – PC Complexes: Based on results of Preliminary trials and literature reviews, selection of independent and dependent factors was carried out. Box–Behnken design was selected for optimization.

TABLE 6: EXPERIMENTAL DESIGN DETAIL FOR OPTIMIZATION OF ARBUTIN – PC COMPLEXES

TABLE 7: BOX-BEHNKEN DESIGN POINT

Drug: phosphatidylcholine ratio, 0.5:1 = 100 mg of arbutin drug and 200 mg of phosphatidylcholine, 1:1 = 200 mg of arbutin drug and 200 mg of phosphatidylcholine, 1.5:1 = 300 mg of arbutin drug and 200 mg of phosphatidylcholine

TABLE 8: DETAIL OF CHECK POINT BATCHES

TABLE 9: FORMULA FOR OPTIMIZED BATCH OF ARBUTIN – PC COMPLEXES

Evaluation of Optimized Batch of Arbutin – PC Complexes: ²¹

% Entrapment Efficiency: Take 10 ml of a suspension of complexes in a centrifugal tube. Centrifuge the suspension at 10000 RPM for 3 hours using an ultracentrifuge. Then collect the 1ml of supernatant, and dilute with the desired quantity of distilled water. If the supernatant not clear, use a membrane filter. Then absorbance was measured at 221 nm wavelength in UV- Visible Spectrometer.

% EE = (Total amount of arbutin taken – Free arbutin measured) / Total amount of arbutin taken × 100

Particle Size: The average diameter (Z-AVE), poly dispersity index (PDI), and zeta potential of suspension was determined by photoncorrelation spectroscopy (PCS) (Zetasizer Nano-ZS, Malvern Instruments, UK) at room temperature. PCS follows the principle of LASER light diffraction, and it is based on the measurement of the Brownian motion of particles. The Brownian motion is the random movement of particles in suspension. The smaller the particle, the faster the Brownian motion. When the incident laser beam reaches the sample, the light was scattered in such a way, depending on the Brownian motion, and then detected by a photomultiplier positioned at a determined angle. Fluctuations in the intensity of scattered light were converted into output current, which is passed to an autocorrelator.

In this way, a correlation function is generated and analyzed by software. Prepared suspension was added (after suitable dilution) to the sample cell and put into the sample holder unit and measurement was carried out with the help of software of same instrument. The computer can provide the mean size and the distribution width of the nanosomes in the batch (Malvern Zeta Sizer Nano series manual).

Zeta Potential: Diluted suspension was added to the sample cell (quartz cuvette) and put into the sample holder unit, and measurement was carried out with the help of software. The Zeta potential of the optimized formulation was measured using the same instrument used in particle size measurement. The sample was added in specialized zeta cell, and the same procedure was carried out.

In-vitro Drug Release Study Using Franz Diffusion Cell: Franz diffusion cells with a receiver compartment volume of 10 mL and effective diffusion area of 2.84 cm² were used to evaluate drug release characteristics of selected compositions.

A dialysis membrane was used for the permeation study. Phosphate buffer solution of pH 7.4 used as receptor medium. The receptor phase was continuously stirred and kept at a temperature of 32 ± 0.5 °C during the experiments. Suspension of complexes and an aqueous solution of arbutin were placed in the donor compartment. At an appropriate time, 1 ml of the sample was withdrawn from the receiver compartment, and the same amount of fresh solution was added to keep the volume constant. Each experiment was run in three independent cells.

The samples were analyzed spectrophotometrically at a wavelength of 221.00 nm, and the concentration of arbutin in each sample was determined from a standard curve.

RESULTS AND DISCUSSION:

Authentication of Active Pharmaceuticals:

Sample FTIR Data:

FIG. 1:  FTIR SPECTRA OF ARBUTIN SAMPLE

Reported FTIR Data: ²²

FIG. 2: FT - IR SPECTRUM OF ARBUTIN REFERENCE

Discussion: Arbutin sample FTIR data and standard FTIR reported data were matched, so the arbutin sample was pure.

UV–Visible Spectrophotometric Method Development for Estimation of Arbutin:

Calibration Curve of Arbutin:

TABLE 10: TABULATED RESULT OF CALIBRATION SAMPLES AT 221 nm

FIG. 3: UV SPECTRA FOR ʎ_MAX DETECTION OF ARBUTIN

Regression Statistics:

FIG. 4: CALIBRATION CURVE OF ARBUTIN (ʎ_max = 221 nm)

TABLE 11: LINEAR REGRESSION RESULT (ABSORBANCE → CONCENTRATION)

Absorbance = (0.023286667 * Concentration) - 0.0804

Equation 1: Linearity Equation for Calibration Curve of UV Method of Arbutin

TABLE 12: ANOVA OF REGRESSION ANALYSIS

Development and Evaluation of Arbutin – PC Complexes:

Preliminary Trials:

TABLE 13: % ENTRAPMENT EFFICIENCY OF PRELIMINARY BATCHES

Discussion: Preliminary trials indicate that there is the effect of selected independent factors on the % entrapment efficiency of Arbutin. Literature reviews and preliminary batches results were helpful for a selection of the level of independent factors.

Optimization of Arbutin – PC Complexes:

TABLE 14: RESULTS OF EXPERIMENTAL DESIGN POINTS (AVERAGE ± SD; N = 3)

TABLE 15: ANOVA FOR RESPONSE SURFACE REDUCED QUADRATIC MODEL

TABLE 16: FIT STATISTICS OF Y: 

FIG. 5: PREDICTED VS. ACTUAL PLOT FOR Y

FIG. 6: CONTOUR PLOT OF Y (FIXED LEVEL: X3= 60, X4 = 75)

EE = + 46.92 + 4.079167 × X1 + 0.15 × X2 + 4.02583 × X3 + 3.2583 × X4 + 4.264166 × X12 + 3.8804167 × X22 + 3.9767 × X32-3.037083 × X42

Equation 2: Polynomial equation for Y (Predictive model – code level)

% EE  = + 161.924 - 25.954  × Arbutin: PC ratio - 3.8654 × Film formation temperature 0.991 × Hydration temperature + 0.29737654320988 × Hydration time + 17.0566 × Arbutin: PC ratio 2 + 0.03880416666667 × Film formation temperature 2 + 9.941E-003 × Hydration temperature 2 - 1.499E-003 × Hydration time 2

Equation 3: Final Equation for actual factors

FIG. 7: CONTOUR PLOT OF Y (FIXED LEVEL: X1 = 1, X2 = 50)

FIG. 8: 3D SURFACE PLOT OF Y (FIXED LEVEL: X2 = 50, X3 = 60)

TABLE 17: CHECKPOINT BATCHES

Discussion: Checkpoint batches are helpful to validate polynomial equations. Results show that there is ≥ 92% similarity between predictive and actual values.

FIG. 9: CONTOUR FOR OPTIMIZATION

TABLE 18: RESULT OF ACTUAL AND PREDICTED FOR OPTIMIZATION USING DESIRABILITY FUNCTION:

FIG. 10: PARTICLE SIZE OF OPTIMIZED BATCH

Evaluation of Optimized Batch of Arbutin – PC Complexes:

% Entrapment Efficiency: As Shown in Table 18, Particle Size: Here, particle sizes were measured in terms of average particle size diameter, and the uniformity was described in the polydispersity index (PDI).

A PDI value of 0.1–0.3 indicates a fairly narrow size distribution, whereas a PDI value greater than 0.5 indicates a very broad distribution. The particle size of the optimized batch is shown in Fig. 10. The average particle size of the optimized batch is 285.6 nm.

Zeta Potential: Zeta potential was found to be –15.8 mV. The Zeta potential of the optimized batch is shown in Fig. 11.

In general, a zeta potential value of ± 20 mV is sufficient for stability suspension. Our formulation is -15.8 mV, which means it complies with the requirement of zeta potential for stability.

FIG. 11: ZETA POTENTIAL OF OPTIMIZED BATCH

In-vitro Drug Release Study Using Franz Diffusion Cell:

TABLE 19: COMPARISON OF IN – VITRO DRUG RELEASE OF ARBUTIN SOLUTION AND ARBUTIN –PC COMPLEX

FIG. 12: COMPARISON OF % DRUG RELEASE OF ARBUTIN – PC COMPLEXES AND ARBUTIN SOLUTION

DISCUSSION: As shown in graph percentage, drug release and release rate of complexes are much higher than arbutin aqueous solution. This indicates that complexes of arbutin may improve in-vitro performance of Arbutin.

CONCLUSION: Developed complexes show a better in-vitro drug release profile compared to an aqueous solution of Arbutin, which indicates optimized complexes improve permeability or absorption of the drug through the skin.

ACKNOWLEDGEMENT: We would like to express my very great appreciation to Dr. Manan Raval for his valuable and constructive suggestions during the planning and development of this research work. His willingness to give his time so generously has been very much appreciated.

CONFLICTS OF INTEREST: We have no conflict of interest to declare.

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    How to cite this article:

    Patel RK and Jadeja MB: Development and evaluation of phosphatidylcholine complexes of arbutin as skin whitening agent. Int J Pharm Sci & Res 2021; 12(2): 917-27. doi: 10.13040/IJPSR.0975-8232.12(2).917-27.

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