3. Guidelines for the determination of SPF in vitro

generation of reactive oxygen species suppressing immune reactions. The benefits of natural antioxidants in topical products are nowadays generally accepted in light of the information available [9]. There are several effective in vitro tests for antioxidant activity. Each of them is based on different mechanisms and, thus, evaluates different kinds of oxidative protection. In order to obtain a sufficient evaluation on in vitro antioxidant, it is thus necessary to perform different types of tests to assess the studied compounds on different kinds of oxidative species. Listed below are some examples of tests that should be carried out to define the whole


UV radiation is capable of damaging DNA and therefore participating in cancer pathogenesis through multiple mechanisms such as immunosuppression, oxidative stress, direct DNA damage, inflammatory response, and p53 tumor suppressor gene mutations. On the other hand, it should be taken into account that immunosuppression might be the desired effect in subjects

Several methods are available for a predictive in vitro antimutagenic or anticancer activity evaluation, and it is quite difficult to identify a list of preferred tests [11]. However, a good practice should include at least one of the validated methods in the screening of a new

UV radiation induces the inflammatory response. UVB-induced cyclooxygenase-2 (COX-2) expression leads to an increase in the production of prostaglandin (PG) metabolites. COX-2 expression in the skin has been linked to the pathophysiology of inflammation and cancer. Exposure to UV radiation is also known to increase the expression of pro-inflammatory cytokines like tumor necrosis factor, interleukin (IL)-1, and interleukin IL-6. These antiinflammatory properties including various herbal substances and medicines can be evaluated

This topic is yet quite complex because it is relatively new and not fully explored. There are already known compounds that can boost the SPF of UV filters [10, 14], but the mechanisms that are responsible for booster effects are heterogeneous and often unpredictable; some are linked to the nature of the UV filter(s) that the formulator wants to enhance. It is, in fact, difficult to uniquely define the general characteristics of an ingredient with booster effect, but it is possible to describe the two main aspects of this topic. The three main strategies available

spectrum of protection. The tests include:

affected by autoimmune diseases [10].

substance or mixture [11].

2.3. Anti-inflammatory activity

by a number of methods [12, 13].

2.4. Booster effect

• 2,2<sup>0</sup>

46 Herbal Medicine

• 1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay • Luminol photochemiluminescence (PCL) assay

• Oxygen radical absorption capacity (ORAC)

2.2. Antimutagenic activity and anticancer properties

Currently, several in vitro tests for determination of SPF exist. They are all used for screenings performed in the research and developing phase. The first method proposed is the one by Diffey (1989); it is still the most accredited reference [14]. The fundamental characteristic of all the in vitro methods is that they are based on spectrophotometric measurement of the absorbance (calculated from transmittance) of a thin film of product applied on UV transparent substrates. Substrates should be as close as possible to the physical characteristics of the skin. The amount of product applied varies from 0.7 to 2.0 mg cm2 . There are different types of suitable substrates; they can range from plastic perforated surgical tape such as Transpore™ to standardized plastic plates such as polymethyl methacrylate (PMMA) plates [14]:


Our experience in the research of useful compounds in the solar protection field and an accurate bibliographic research indicate that it is possible to point out several factors and variables which are able to affect the accuracy and the repeatability of the in vitro SPF tests. The most important ones are:


The main concern, about this type of evaluation, is the lack of data to support correlation to in vivo results [14].

SPF in vitro is defined as follows:

terms of repeatability.

In vitro SPF ¼

Ð <sup>λ</sup>¼400nm

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

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 corre-

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

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

lation is not influenced by the choice of operator's pressure (100 � 15 g or 200 � 15 g).

solar radiation simulators; this procedure is also indicated in the ISO 24443:2012.

laboratories due to external variables (e.g., the environmental, operator, etc.) [14].

tigated in biological activities useful for sunscreen products.

4. Natural compounds in solar radiation protection: current knowledge

In recent years, many plant species have been investigated for their potential uses in the field of solar radiation protection, but much remains to be accomplished. As stated above, this depends on both a large number of under-investigated species and the lack of an official standard in vitro SPF evaluation method, to speed up the screening procedure. Depending on this, and on the many different and incomplete approaches led by different research groups, it is also complex to have a general picture of the existing knowledge. In the aim to achieve a "state of the art," we conducted a detailed bibliographic research on the plants already inves-

In our previous investigation [2], we identified 54 plants, 5 lichens, and 14 pure molecules which have been studied in order to obtain herbal sunscreen products. It is remarkable how

Ð <sup>λ</sup>¼400nm

<sup>λ</sup>¼290nm <sup>E</sup>ð Þ <sup>λ</sup> <sup>I</sup>ð Þ <sup>λ</sup> <sup>d</sup>ð Þ <sup>λ</sup>

<sup>λ</sup>¼290nm <sup>E</sup>ð Þ <sup>λ</sup> <sup>I</sup>ð Þ <sup>λ</sup> <sup>10</sup>�Að Þ <sup>λ</sup> <sup>d</sup>ð Þ <sup>λ</sup> (1)

Guidelines for the Development of Herbal-Based Sunscreen

http://dx.doi.org/10.5772/intechopen.72712

49

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 costs.

In order to provide practical indications, we suggest two methods that have proven, in our experience, to be among the most reliable:
