*2.2. In Vivo Studies*

For our analysis, we have chosen an aging female mouse model (C57BL/6J), in order to have available cells from organs with either gender-independent (i.e., the bone marrow) or gender-dependent (i.e., ovaries) functions, and divided mice into two groups: FPP® was daily administered to the first group for 10 months from 6 weeks old (6 to 51 weeks of age) (ET-FPP®: early treatment with FPP®) and to the second group for 10 months from 51 weeks old (51 to 96 weeks of age) (LT-FPP: late treatment with FPP®); in both conditions a control group was included receiving tap water only (ET-CTR and LT-CTR). Each group consisted of 10 animals for statistical significance. To compare the mice treatment groups to the human age, ET treatment corresponded to women starting FPP®at 13 years and ending FPP® at 41 years of age; while LT treatment starting at 41 years and ending at 63 years of age. (Figure 1).

**Figure 1.** Equivalence between mice age and human age. Early treatment of mice from 6 weeks to 51 weeks of age corresponds to treatment in humans from 13- to 41-years old. Late treatment of mice from 51 weeks to 96 weeks of age corresponds to treatment in humans from 41- to 63-years old.

Each treated mice drank 1 mL of FPP®-supplemented water every day, corresponding to 6 mg/mouse/day of FPP®. Just before mice sacrifice, blood was withdrawn from mice eyes. Immediately after the sacrifice, bone marrow was isolated from both tibias and femurs of the mice hind legs, while ovaries were retrieval from reproductive system. Blood, bone marrow cells and ovarian germ cells were used for subsequent experimental analysis of aging parameters.

All the studies were approved by the ethical committee of the Italian National Institute of Health (Rome, Italy) and were conducted in accordance with the current Italian Law (Law 26/2014), authorization n◦792/2017-PR (prot. D9997.49 27/06/2017), that regulates experiments in laboratory animals. 40 C57BL/6J female mice between 16 and 20 g (4 weeks of age) were purchased from Charles River Laboratories Italia s.r.l., (Calco, Lecco, Italy), and housed in the animal facility of the Italian National Institute of Health. Mice had 10 and 14 h periods of light and darkness respectively, were housed in a different number of animal cages, depending on the experiment, with ad libitum mice chow (Mucedola, Settimo Milanese (MI), Italy) and water intake. Accordingly to the guidelines for a correct laboratory practice and signs of poor quality of life, a veterinarian responsible for animal welfare checked mice twice a week, to monitor signs of sufferance such as weight loss, decreased water and food consumption, poor hair coat, decreased activity levels and tumor ulcerations

#### *2.3. Total Antioxidant Power Assay (PAO Test Kit)*

Detection and quantification of Total Antioxidant Power was performed in FPP®-supplemented water using a colorimetric assay: PAO Test kit for Total Antioxidant Capacity (JaICA, Japan). The assay can detect not only hydrophilic antioxidants such as Vitamin C, glutathione, but also can detect hydrophobic antioxidants such as Vitamin E. The determination of the antioxidant power is carried out using the reduction of the cupric ion (Cu++ to Cu+). Briefly, samples were incubated for 3 min at room temperature with Cu++ solution, Cu++ are reduced by antioxidants to form Cu+ that reacts with chromatic solution (bathocuproine), and can be detected by absorbance at wavelength 480 to 490 nm. Antioxidant capacity can be calculated from the Cu+ formed. Absorbance was recorded at 490 nm.
