3.1. Method A

This is based on the Diffey-Robson's method [14]. The support used is a Transpore™ surgical perforated tape, cut to have an area of 20 cm<sup>2</sup> , in which an amount of 0.0400 0.002 g (2 mg cm<sup>2</sup> ) of the product is weighed and laid in small spots through all the area. The tape is then positioned on a scale where the spreading phase is carried out with a finger cot, performing a pattern of six movements in horizontal, vertical, and circular directions and checking the pressure applied in all the movements. As far as the spreading pressure is concern, an internal procedure must be developed by the performing laboratory in order to be repeatable (see the end of this paragraph). At least three tapes have to be prepared for each product, recording five measures each, collecting therefore 15 spectra [14].

#### 3.2. Method B

This method has been recently proposed by us [14], adapting to UVB the ISO 24443:2012 standard for the in vitro UVA protection determination. The support used is a PMMA (polymethyl methacrylate) plastic plate with an area of 25 cm2 and standardized 5 μm roughness, in which an amount of 0.0320 0.0005 g (1.3 mg cm<sup>2</sup> ) of the product is weighed and laid in small spots through all the area. The plate is then positioned on a scale where the spreading phase is carried out performing with pre-saturated finger cot a sequence of six movements in horizontal, vertical, and circular direction and checking the pressure applied throughout the spreading. Before the measurement, the sample lies for a minimum of 15 min in a dark place, allowing the evaporation of volatile components. Three plates have to be prepared for each product, recording five measures each, collecting therefore 15 spectra [14].

In both methods, the spectra were recorded with an appropriate spectrophotometer, wavelength ranging from 290 nm up to 400 nm, with increment step set at 1 nm. The tests carried out for the evaluation of new herbal compounds are usually performed including them, at a known concentration, in a stable formulation suitable for cosmetic use. The obtained SPF data are then compared to those of the same formulation without the studied compounds.

SPF in vitro is defined as follows:

• The thickness and the homogeneity of the applied sunscreen

The main concern, about this type of evaluation, is the lack of data to support correlation to

At present, the in vivo method is still the official standard for UVB protection (ISO 2444:2010), and product developers should perform the in vivo test on the final product and the in vitro one during all the phases of the development bringing attention to the ethical issue and on the

In order to provide practical indications, we suggest two methods that have proven, in our

This is based on the Diffey-Robson's method [14]. The support used is a Transpore™ surgical

This method has been recently proposed by us [14], adapting to UVB the ISO 24443:2012 standard for the in vitro UVA protection determination. The support used is a PMMA (polymethyl methacrylate) plastic plate with an area of 25 cm2 and standardized 5 μm rough-

laid in small spots through all the area. The plate is then positioned on a scale where the spreading phase is carried out performing with pre-saturated finger cot a sequence of six movements in horizontal, vertical, and circular direction and checking the pressure applied throughout the spreading. Before the measurement, the sample lies for a minimum of 15 min in a dark place, allowing the evaporation of volatile components. Three plates have to be prepared for each product, recording five measures each, collecting therefore 15 spectra [14]. In both methods, the spectra were recorded with an appropriate spectrophotometer, wavelength ranging from 290 nm up to 400 nm, with increment step set at 1 nm. The tests carried out for the evaluation of new herbal compounds are usually performed including them, at a known concentration, in a stable formulation suitable for cosmetic use. The obtained SPF data

are then compared to those of the same formulation without the studied compounds.

) of the product is weighed and laid in small spots through all the area. The tape is then positioned on a scale where the spreading phase is carried out with a finger cot, performing a pattern of six movements in horizontal, vertical, and circular directions and checking the pressure applied in all the movements. As far as the spreading pressure is concern, an internal procedure must be developed by the performing laboratory in order to be repeatable (see the end of this paragraph). At least three tapes have to be prepared for each product, recording

, in which an amount of 0.0400 0.002 g (2 mg

) of the product is weighed and

• The type of spectrophotometer

in vivo results [14].

costs.

48 Herbal Medicine

cm<sup>2</sup>

3.1. Method A

3.2. Method B

• Substrates used and their relative roughness

experience, to be among the most reliable:

perforated tape, cut to have an area of 20 cm<sup>2</sup>

five measures each, collecting therefore 15 spectra [14].

ness, in which an amount of 0.0320 0.0005 g (1.3 mg cm<sup>2</sup>

$$\text{In vitro SPF} = \frac{\int\_{\lambda=290 nm}^{\lambda=400 nm} E(\lambda)I(\lambda)d(\lambda)}{\int\_{\lambda=290 nm}^{\lambda=400 nm} E(\lambda)I(\lambda)10^{-A(\lambda)}d(\lambda)}\tag{1}$$

E(λ) is the erythema action spectrum (CIE-1987) at the wavelength λ. I(λ) is the spectral irradiance received from the UV source at the wavelength λ. A(λ) is the monochromatic absorbance of the test product layer at the wavelength λ. d(λ) is the wavelength step (1 nm).

Both methods have to be conducted in highly standardized operating conditions with regard to the operator, the environmental conditions, the substrates used, and the instruments. We have worked with spreading pressures of 100 � 15 g and 200 � 15 g, and comparing different application pressures on the same substrate, no statistically significant difference subsists in terms of repeatability.

The two fundamental parameters in the in vitro SPF measurement process are in vivo correlation and reproducibility. In our experience, Method B with a spreading pressure of 200 � 15 g is the most reliable method with respect to reproducibility and accuracy. Nevertheless, Method A can be still considered as a useful in vitro method during the early research phase, especially in laboratories with limited financial resources and limited equipment. In this case, the correlation is not influenced by the choice of operator's pressure (100 � 15 g or 200 � 15 g).

The problem of photostability of UV filters should also be considered at this stage. It is necessary, seeking potential human applications, to verify that a new compound or vegetal extract does not present any photostability problems. This evaluation can be performed using solar radiation simulators; this procedure is also indicated in the ISO 24443:2012.

It is very important to assume that, at present, it is possible for a single laboratory to optimize internal methods and protocols to achieve repeatable and predictive in vitro results, whereas it is extremely difficult to develop methods reproducible and equally reliable between different laboratories due to external variables (e.g., the environmental, operator, etc.) [14].
