Calibration curve

5 6

Clay W4 Clay SGY Clay SP Clay SW

Cumulative frequency (%)

Particle size (µm)

Figure 9. Particle size distribution curves of the Clay minerals.

4.2 Centrifugation and pH test

The centrifugation test is a useful tool for assessing and predicting the shelf life of emulsions based on the principle of using centrifugal force to separate two or more substances of different densities. No phase separation was observed after centrifugation in any of the formulation samples tested.

The pH is a unique parameter with high significance once a healthy skin normally presents pH range from 4.5 to 6.0, and in order for a formulation to possibly gain acceptance for industrial application, it should have a pH that is close to this range. The different emulsions prepared for this study had a mean pH value of: 6.1 for F-Base; 5.51 for F-SP; 5.82 for F-SW; 5.74 for F-SGY and 5.74 for F-W4, which is close to the ideal pH.

4.3 Calibration curve

Table 4. Calibration curves and detection (LD)/quantification (LQ) limits

Compound Concentration (µg/mL) Regression equation* r²* LD* LQ*

Ascorbyl

glucoside 180, 140, 100, 60, 20, 5 y = 23.136x + 1.2441 0.9991 0.95 3.17

*(n=3)

The results showed satisfactory and consistent behavior of the HPLC stability-indicating method. Least-squares regression analysis was used to evaluate the concentration range data that showed excellent linearity, with r² ≥0.99(Jenke, 1996).

Linear regression was y = 23.136X + 1.2441, where y is the ratio of ascorbyl glucoside peak area and x is the concentration of ascorbyl glucoside (µg/mL⁻¹).

4.4 Specificity

Figure 10. a) Chromatograms of active-free o/w emulsion , b) o/w emulsion containing AA2G (ascorbyl glucoside) and c) active AA2G at 40µg/mL.

Specificity for ascorbyl glucoside quantification on all the active-free formulations and o/w emulsion containing the active was investigated in order to obtain an indication of possible interferents from the formulations excipients. As shown in Fig. 10, the presence of the excipients in O/W emulsions did not cause any interference with the active peak, and the same chromatographic profile was observed for all formulations(Maia et al., 2007).

Formulation

AA2G

AA2G 3.680 min.

Formulation

4.5 Precision

Table 5. Relative standard deviations of intraday and interday analysis

Mean ± S.D. (R.S.D.%)

Compound Concentration (μg/mL) Intraday* Interday**

Ascoryl glucoside 80

**n=6, triplicate injection each day for 2 consecutive days.

Precision was expressed as relative standard deviation (R.S.D., %) in terms of area ratio of ascorbyl glucoside. Low percentage values of R.S.D. < 5.0 % were an evidence of the appropriate precision of this stability-indicating method which provided an irrelevant variability of the data (Huang et al., 2004).

4.6 Accuracy/Recovery

Table 6. Recoveries of ascorbyl glucoside spiked in 5 formulation samples Formulation containing the samples ranged from 92.74% to 104.67% (Table 4). The R.S.D. of recovery rate in these 5 formulations was 0.10~2.64%.

4.7 Robustness

Table 7. Changes in the method for robustness evaluation

Mean ± S.D. (R.S.D.%) Compound Concentration

(μg/mL) Mobile Phase* Temperature* Flow*

Ascorbyl glucoside

100 2267.51 ± 12.35 (0.54)

2328 ± 39 (1.67) 2328 ± 71 (3.03)

Mobile Phase: Ortho-Phosphoric acid 0.1% : MeOH (99:1) Temperature: 23 ⁰C

Flow: 0.980 mL/min

*n=3

Robustness was expressed as relative standard deviation (R.S.D., %) in terms of area ratio of ascorbyl glucoside. Low percentage values of R.S.D. < 5.0 % were an evidence of robustness, once, the changes in the method provided an irrelevant variability of the data(Huang et al., 2004).

4.8 Ascorbyl glucoside solubility determination

Table 8. ascorbyl glucoside solubility profile

Sample *Concentrations of ascorbyl

glucoside (µg/mL) Ascorbyl glucoside in de-ionized water 142.76; 97.56; 21.45

Ascorbyl glucoside in PBS_pH = 7.4 140.62; 101.32; 20.78

Ascorbyl glucoside in PBS_pH = 7.4: MeOH (75:25) 144.01; 93.98; 22.01 PBS: Phosphate-buffered saline

MeOH: Methanol

*n=3

The ascorbyl glucoside is a water-soluble derivative with a solubility value of 714 g/L at 19 ± 1 ^oC (Moribe et al., 2011). The active showed good solubility in de-ionized water, PBS pH = 7.4 as well as in PBS pH = 7.4: MeOH (75:25).

4.9 In vitro release and permeation study of Ascorbyl glucoside

The past years have seen an increasing interest in utilizing clay minerals as carriers for drugs that can be adsorbed and released at a later time in target locations of the human body, thus permitting the controlled delivery of active ingredients (Moraes et al., 2017).

Equally as important as the ability to formulate a O/W emulsions containing clay minerals and vitamin C derivative, is the ability to release the active from the delivery system.

Typically, clay minerals-based delivery systems exhibit active release kinetics by the mechanistic processes of (1) diffusion in of the exchanging cation, (2) cation exchange and (3) diffusion out of the active molecules. The determination of the extent of active intercalation and also the rate of active release is directly related to the cation exchange capacity of the substrate clay mineral. The adsorption and desorption processes are essentially dependent on the nature of the ionic interactions between the active molecule and the host clay mineral structure(Zhang, and Cresswell, 2016).

To evaluate the impact of kaolinite and smectite clay minerals on release of ascorbyl glucoside content, five O/W emulsions containing or not clay minerals were evaluated in Franz cell diffusion studies. All release experiments showed a good reproducibility; the release profiles are reported in Figure 13. The results are expressed as the amount (µg/mL) of the released active in the acceptor solution referred to the total active content in the donor cells.

All the samples showed high active release during the whole analyzed time range and no significant differences can be observed among the five experiments, however when the base formulation (without clay mineral) or SGY, SP and F-SW formulations (with kaolinite clay mineral) are compared to the F-W4 formulation (with smectite clay mineral) it shows that the clay mineral smectite increased the active release at each time of the whole analyze. At the end of 10 h, approximately 77.88%, 77.59%, 81.92%, 72.98%, and 92.51% of the active was released from F-Base, F-SGY, F-SP, F-SW and F-W4 formulations.

Investigations by Bastianini et al., 2018 showed likely release profile for a formulation containing caffeic acid and layered double hydroxides (LDHs) clay as an active carrier, explaining the observed relationship between release rate and the presence or not of double layered clay mineral that has the same chemical structure as smectite clay. This result clearly indicated that the smectite clay mineral referred

to their high cation exchange capacity (CEC) had a greater bioavailability than the base formulation without clay mineral and even greater than the formulations containing kaolinite clay minerals that has little or no ionic substitution and thus low cation exchange capacity. In fact, thanks to the double layers structure of the release from the clay mineral layers occurs by exchange between ascorbyl glucoside ions and phosphates ions contained in the PBS buffer. (Ambrogi et al., 2001; Moraes et al., 2017; Perioli and Pagano, 2016; Yang et al., 2016).

During the in vitro permeation study using cadaver human skin, aliquots of 300 µL of receptor medium was collected at appropriate intervals at 2, 4, 6 and 8 h and immediately analyzed by HPLC. All the formulations were analyzed following the same protocol.

To evaluate the impact of kaolinite and smectite clay minerals on the permeability study of ascorbyl glucoside content, five O/W emulsions containing or not clay minerals were evaluated in Franz cell diffusion studies. All permeation experiments showed a good reproducibility; the permeability profiles are reported in Figure 19. The results are expressed as the amount (µg/cm²) of the permeated active in the acceptor solution referred to the total active content in the donor cells.

The samples F-SP and F-SW showed that the active AA2G permeated the human skin during the whole analyzed time range and no significant differences can be observed among these formulations, however when the F-base formulation (without clay mineral) was tested, the permeation of the active AA2G occurred only at T= 8 hours, also the formulation F-W4 showed that the active permeated the skin only at T= 4, 6 and 8 hours. The formulation F-SGY was tested following the same protocol and there was no permeation of the active among the whole analyzed time.

Although F-SGY, F-SP and F-SW formulations has kaolinite clay mineral in their compositions, each clay mineral is unique in their chemical structures and plays a role differently even when classified in the same group (Kaolinite), once in this study, the kaolinite SP and SW showed opposite results when compared with the kaolinite SGY. Moreover, both SP and SW contributed to improve the permeation of AA2G

active during the whole time analyzed, the same fact did not occur with the formulation F-Base (without clay mineral) which showed permeation of the active only at the end T = 8 hours. Furthermore, the formulation F-W4 has smectite clay which is structurally different from kaolinite clays due to the double layer showed that the active began to be permeated after 2 hours of analyze, suggesting that possibly the delay in the process of permeation was due to the double layer of this mineral when compared to both F-SP and F-SW containing kaolinite clays which are a single layer mineral.

Figure 11. The active ascorbyl glucoside entrapped in kaolinite clay mineral and smectite clay mineral.

2 4 6 8 10

0 1000 2000 3000 4000 5000 6000 7000 8000

F- Base F- SGY F- SP F- SW F- W4

Cumulative Release (µg/cm2 )

Time (h)

Figure 12. The effect of different clay minerals on release of ascorbyl glucoside. Cumulative ascorbyl glucoside release measured using Franz cell method from O/W emulsions. The results are presented as mean±SE (n=3). The p-value > 0.05 was considered to be not statistically significant between the amounts of ascorbyl glucoside released from each formulation.

2 4 6 8 10

The results of the kinetic study showed that the release of ascorbyl glucoside from the five formulations through the dialysis membrane follow zero order kinetics, because the highest values of the correlation coefficient were found when this model was applied. Thus, the release of ascorbyl glucoside occurred by diffusion, at constant speed, regardless of the concentration of the active(Monteiro et al., 2007).

The zero order release kinetics is a desirable release pattern and considered a

controlled-release delivery, which describes a system where the drug release rate is constant for a specified period of time(Maderuelo et al., 2011).

2 3 4 5 6 7 8

Figure 18. Cumulative ascorbyl glucoside permeated and measured using Franz cell method from O/W emulsions. The results are presented as mean±SE (n=4). The p-value > 0.05 was considered to be not statistically significant between the amounts of ascorbyl glucoside permeated from each formulation.

Among the results in this study, SP, SW and W4 clays played a role as penetration enhancers for the formulations analyzed. Usually, penetration enhancers act by lipid disruption and at acceptable concentrations they interact and affect the stratum corneum intercellular lipid domain or organization and make the stratum corneum more permeable (Vavrova et al., 2005). Understanding the physiochemical relationship of active/vehicle interactions through a membrane barrier is critical for selection of optimal formulation penetration enhancement efficacy (Flaten et al., 2015).

Table 10. Release and Permeation parameters of ascorbyl glucoside through dialysis membrane and human skin after 10 h and 8 h

RELEASE PERMEATION determined from slope of the cumulative amounts of ascorbyl glucoside released or permeated versus time profiles; F, flux of ascorbyl glucoside from the formulations containing clay minerals divided by the flux of ascorbyl glucoside from the base formulation without clay mineral. The rank order for ascorbyl glucoside flux for each formulation for dialysis membrane are: W4 > SP > SGY > Base> F-SW. The rank order for ascorbyl glucoside flux for each formulation for human skin are: F-W4 > F-SW

> F-SP.