2.2.1. Pre-Selection of an Optimal Physical Filter/Antioxidant Ratio

The method comprises three parts: (1) using DPPH as probe to measure radicals generated by four physical filters, (2) determining the capacity of six antioxidants using DPPH, (3) determining each antioxidant’s concentration required to eradicate radicals generated by a chosen physical filter under the condition described in (1), and comparing the concentration of each antioxidant with its “theoretical” value from (2).

Six percent (% w/w) of physical filter of Table 1 was dispersed in propylene glycol by homogenizer (Primix Robomix, Tsukishima Kikai Co, Japan). The dispersion was irradiated by a UV source (350–400 nm, 882.59 mW) (Zhongshan Tiandou UV LED, Xiaolan Town, China) for 15 min. Therefore, the lamp was placed into a dark box, the sample was transferred into a glass bottle and was irradiated with UV light for the specified time.

The dispersion was then mixed 1:1 with a DPPH solution (water: ethanol = 2.5:7.5). The final concentration is 2% for physical filter and 0.008% for DPPH. The mixture was stirred for 1 min (1000 mot) and then centrifuged for 5 min at the rate of 4000 r/min (CH80-2, Shanghai Medical Instruments CO., LTD, Shanghai, China). The supernatant was collected and subjected to spectrometric analysis (PerkinElmer, Lambda 750S, Connecticut, USA) for DPPH (517 nm), the value is denoted as A1.

Calculation of the scavenged DPPH:

Ra=1−A1−A2A0 100%

(1)

Ra = radical generation by physical filters

A2 = A1 without DPPH,

A0 = 0.008% DPPH [propylene glycol/water/ethanol solution].

DPPH reduction without irradiation was considered by subtraction.

A propylene glycol/water/ethanol solution was used for antioxidant and DPPH studies (the solvent ratio is the same as shown in (1)). The DPPH concentration was fixed at 0.008% and the antioxidants were added at various concentrations. The calibration curve of each antioxidant against DPPH was plotted at different concentrations by ELISACalc (Shanghai Bluegene Biotech CO.,LTD, Shanghai, China). The IC50 where 50% of the ECGC is consumed was calculated.

Preparation and irradiation of pigment dispersion were performed exactly as described in step (1). The dispersed physical filters (without antioxidants) were irradiated to generate radicals. The mixture was then mixed with different concentrations of selected antioxidants solubilized in water/ethanol solution to neutralize the radicals. It was stirred for 10 min at 1000 Mot, then adding DPPH solution (water/ethanol) and stirred for another minute, the final mixture has a solvent that is the same as described in steps (1) and (2). The DPPH concentration was 0.008%. The DPPH solution was introduced to measure the radicals, which had not been neutralized, or excessive antioxidants. The final mixtures were centrifuged for 5 min at the rate of 4000 r/min (CH80-2, Shanghai Medical Instruments CO., LTD, Shanghai, China). The supernatant was collected and subjected to spectrometric analysis (PerkinElmer, Lambda 750S, USA) for DPPH (517 nm), the value is denoted as B1.

Calculation of the scavenged DPPH:

Rb=1−B1−B2 B0 100%

(2)

Rb= radical scavenging

B2 = B1 without DPPH,

B0 = 0.008% DPPH [propylene glycol/water/ethanol solution].

We searched for an Rb inferior to 5% implying the antioxidant concentration is sufficient to almost completely neutralize the radicals generated by a pigment so that less than 5% of DPPH was sacrificed. This value was then compared with the corresponding theoretical value calculated from step (2).

X is defined as the theoretical concentration of an antioxidant that is supposed to completely neutralize radicals generated by a pigment divided by the experimentally obtained concentration when Rb < 5%.

If X = 1, the capacity of an antioxidant to scavenge the radicals generated by a physical filter is in line with expectations.

If X > 1, the capacity of an antioxidant to scavenge the radicals generated by a physical filter outperforms expectations (good for production).

If X < 1 the capacity of an antioxidant to scavenge the radicals generated by a physical filter underperforms expectations.

2.2.2. Measurements of Antioxidant Capacity of the Creams by Radical Protection Factor (RPF)The radical scavenging activity of the cream formulations including physical filters and antioxidants was analyzed by EPR spectroscopy using again the test radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, Steinheim, Germany). The number of reduced DPPH represents the radical scavenging activity of the investigated extracts/cream formulation, which is normalized to 1 mg input. The measuring unit of the RPF is 1014 radicals/mg [16,19].The RPF analysis was performed with an X-band MiniScope MS5000 EPR spectrometer (Magnettech, Freiberg Instruments GmbH, Freiberg, Germany) at a microwave frequency of 9.4 GHz. The method was in principle applied as previously described [16]. The calculation of the spin concentration of the 1 mM ethanolic DPPH solution and the final calculation of the RPF was performed by the MS5000 device control software “ESR Studio” (Freiberg Instruments GmbH, Freiberg, Germany).

For the RPF analysis of the samples, 20 to 500 mg of the formulations were solubilized in 10 mL ethanol, followed by a dilution (1:1) with a 1 mM DPPH ethanol solution. The EPR measurements were performed directly after sample preparation (0 h) and 23 to 28 h after sample incubation, until a stabilization of the DPPH signal was achieved. During the incubation time, the samples were kept dark at room temperature by constant panning. The samples were measured in glass capillaries (Hirschmann Laborgeräte GmbH & Co. KG, Eberstadt, Germany) with the following parameter settings: frequency 9.4 GHz, central magnetic field 338.43 mT, magnetic field sweep width 9.5 mT, modulation frequency 100 kHz, modulation amplitude 0.2 mT attenuation 15 dB, sweep time 20 s.

2.2.3. Evaluation of the Optical Properties of the Creams by UV-VIS SpectroscopyThe double integrating sphere technique combined with inverse Monte Carlo simulation (iMCS) was used to determine the optical parameters absorption µa and effective scattering coefficient µs’ of the different cream formulations [23]. The method was performed as previously described in detail [24]. An integrating sphere spectrometer (Lambda 1050, PerkinElmer, Rodgau-Jügesheim, Germany) was used for the measurements. The cuvette can be fixed in a defined position at a constant distance from the sphere opening in front of or behind the integrating sphere to measure the transmission (TtM) or reflection (RtM) spectra. For the measurement of Tt, the reflectance port was closed with a diffuse reflecting Spectralon® standard. Rt was measured relative to the reflectance standard by replacing the special Spectralon® standard by the sample, which is inclined at an angle of 8° to the incident light.In order to obtain reproducible reflectance and transmittance measurements, a homogeneous sample distribution was ensured. The measurement inaccuracy of repeated measurements was less than 2%. The optical parameters µa and µs’ were calculated by inverse Monte Carlo simulation (iMCS) as previously described [23,24]. Thereby, the iMCS uses forward Monte Carlo simulations iteratively to calculate the optical parameters µa and µs’ on the basis of a given phase function and the experimentally measured values for reflection and transmission (RtM and TtM). By systematic variation of μa and μs’ the deviation of the simulated RtSand TtSvalues from the ones measured is minimized until a set of optical parameters is found, where the deviations are within an error threshold of 0.20%.