**3. Results**

## *3.1. Evaluation of FPP* ®*-Supplemented Water E*ff*ectiveness*

The experimental design has been set up in order to evaluate *in vivo* the e ffectiveness of FPP ® supplementation on redox balance and molecular signature of aging. Although the greater e fficacy of FPP ® was proven when taken sublingually [28], we found that this administration was very stressful for mice [30], so we decided to dissolve FPP ® in the daily water.

In order to verify the real *in vivo* effectiveness of the non-orthodox FPP ® administration following dissolution in water, we have first evaluated the papaya antioxidant capacity when dissolved in water by a colorimetric test. Each day a sachet of FPP ® (3 g) was dissolved in 500 mL of water and administered to mice; each FPP ® mouse drank about 1 mL a day, with the resulting dose of 6 mg/mouse/day FPP ® taken every day. Therefore, same doses of papaya dissolved in water (500 mL for each cage) and taken by each mouse were analyzed for quantification of antioxidant power. We used a test that allowed us to estimate the total content of both hydrophilic (for example Ascorbic Acid) and hydrophobic antioxidants. As shown in Table 1, FPP ® in 500 mL of water had a Total Antioxidant Power of 6.7 ± 0.6 M, and in 1 mL taken by each FPP-treated mouse 13.48 ± 0.9 mM. Ascorbic acid is one of the hydrophilic antioxidants that can be quantified and, as for the Total Antioxidant Power, we measured ascorbic acid concentrations of a FPP ® sachet dissolved in 500 mL and of FPP ® dose taken by the mouse daily by fluorimetric assay. The papaya sachet in 500 mL of water had an ascorbic acid content equal to 192.2 ± 3.5 ng, while the mouse daily dose was 0.4 ± 0.03 ng (Table 1).


**Table 1.** Total Antioxidant Power and Ascorbic Acid quantification in FPP®-supplemented water.

> Data are expressed as mean ± SE of three experiments.

#### *3.2. Early Treatment with FPP*®*: from 6 to 51 Weeks of Age*

3.2.1. Oral Administration of FFP® Increases Plasma Levels of Antioxidants

To the purpose of evaluating comparable effect in our experimental model, we first measured the Total Antioxidant Power in plasma samples of FPP® in both treated and untreated mice; this test allows to detect both hydrophilic and hydrophobic antioxidant in blood samples. The results showed that mice daily treated with FPP® (Figure 2A) had an increased antioxidant power (ET- FPP® 11.9 ± 1.4 mM, *p* < 0.05) as compared to the control group (ET-CTR 7.6 ± 0.4 mM).

**Figure 2.** Antioxidant effect of FPP® in C57BL/6J female mice by measuring the plasma antioxidant levels (antioxidant power, GSH and SOD-1). Plasma samples collected from both untreated (ET-CTR group) and treated (ET-FPP®group) mice were analyzed. (**A**) Analysis of the quantification and detection of the total antioxidant power (mM). (**B**) Analysis of the quantification and detection of GSH activity (μM). (**C**) Analysis of the quantification and detection of SOD-1 activity (U/mL). Data are normalized on total plasma and expressed as means ± SE. \* *p* < 0.05, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

Thus, we measured the enzymatic activities of superoxide dismutase-1 (SOD-1) and plasmatic levels of reduced glutathione (GSH). SOD-1 is an enzymatic antioxidant responsible for the dissociation of superoxide anion into hydrogen peroxide and dioxygen; glutathione is a non-enzymatic antioxidant as represented by the glutathione reduced form (GSH), that plays the important role of protector against oxidative stress neutralizing reactive oxygen species.

The results showed that ET-FPP® -treated mice presented a significant increase of GSH plasmatic levels (*p* < 0.0001) of about 7.5-fold higher as compared to control plasma samples (ET-FPP® 21558 ± 1100 μM, ET-CTR 2896 ± 574 μM) (Figure 2B). Comparable results were obtained with SOD-1 analysis, where SOD-1 plasmatic levels in FPP® treated mice were significantly higher (ET-FPP® 361 ± 9 U/mL, *p* < 0.001) as compared to control mice (ET-CTR 282 ± 13 U/mL) (Figure 2C). These results supported a potentially powerful in vivo antioxidant action exerted by FPP® administered to mice from 6 weeks of age (early treatment), even when administered as dissolved in the water.

#### 3.2.2. Oral Administration of FFP® Reduces Plasma Levels of ROS

Although Reactive Oxygen Species (ROS) and ROS-induced oxidative damage are not considered as the sole cause of aging, it is believed that ROS play a key role in the molecular mechanisms regulating longevity. For this reason, we evaluated and determined the effect of FPP® supplementation on plasmatic levels of ROS in our experimental model. The results in Figure 3 showed a significant decrease (*p* < 0.005) of plasmatic ROS levels in ET-FPP® mice (7737 ± 331 a.u.) as compared to ET-CTR group (10962 ± 692 a.u.).

**Figure 3.** Effect of FPP® on total ROS blood levels in C57BL/6J female mice. Analysis of the total ROS levels (arbitrary units, a.u.) on the plasma samples collected from both ET-CTR and ET-FPP®. Data are normalized on total plasma and expressed as means ± SE. \*\* *p* < 0.005.

#### 3.2.3. Oral Administration of FFP® Increases Plasmatic Telomerase Activity

Telomerase (TE) is an enzyme that adds repetitive sequences of DNA to the chromosomal ends (telomeres); at each DNA replication, the telomeres undergo shortening and the task of telomerase is to maintain their integrity. In fact, in the absence of telomerase, the telomeres progressively shorten until they reach a threshold value where cell division stops, thus inducing cell senescence. Telomerase activity and telomeres length are currently considered the molecular signature of aging. To this purpose, we first determined and quantified the telomerase activity in the plasmas of FPP®-treated mice (ET-FPP®) as compared to the control group (ET-CTR).

As shown in Figure 4, we observed an increase in telomerase concentration of mice daily treated with FPP® as compared to mice drinking tap water. More in details, ET-FPP® mice had a concentration of TE 1.6-fold higher (*p* < 0.005) as compared to ET-CTR (ET-FPP® mice: 88.5 ± 4.5 ng/mL, ET-CTR mice: 55.9 ± 6.6 ng/mL).

**Figure 4.** Effect of FPP® on telomerase (TE) activity in plasma samples from C57BL/6J female mice. Quantitative determination of mouse telomerase (TE) activity (ng/mL) was performed on plasma samples obtained from both ET-CTR and ET-FPP® groups immediately before the sacrifice. Data are normalized on total plasma and expressed as means ± SE. \*\* *p* < 0.005.

3.2.4. Oral Administration of FFP® Increases Telomeres Length in Bone Marrow Cells and Ovarian Germ Cells

In order to evaluate the effect of FPP® treatment on telomeres length, we analyzed single cell suspensions obtained from both bone marrow and ovaries of either FPP® treated or untreated mice. To this purpose bone marrow and ovaries were obtained from each mouse and the single cell suspensions were isolated as described in the Materials and Methods section; subsequently bone marrow and ovarian germ cells were counted by trypan blue exclusion under optical microscope. In ET-FPP® mice bone marrow and ovarian germ cells were respectively almost 4-fold and 2-fold more than the ET-CTR cells (data not shown).

Comparable numbers of cells obtained from both organs were analyzed by hybridization of a fluorescein-conjugated probe (PNA) recognizing the sequence of six nucleotides (TTAGGG) repeated in the telomeres. The results, expressed as mean intensity of fluorescence (M.I.F.), are summarized in Figure 5. TTAGGG sequence in telomeres correlated with the value of the M.I.F.

**Figure 5.** Effect of FPP® on telomeres length in bone marrow cells and in ovarian germ cells from C57BL/6J female mice. The analysis of telomeres length was performed on nucleated haematopoietic cells from (**A**) bone marrow and (**B**) on ovarian germ cells. Cells were retrieved from both ET-CTR and ET-FPP® groups immediately after the sacrifice. Data are expressed as mean ± SE of M.I.F. (Mean Intensity Fluorescence) normalized on total cells. \*\*\*\* *p* < 0.0001.

The results showed that ET-FPP® mice had an impressive increase of telomeres length than ET-CTR in both organs. In details the telomeres lenght in bone marrow cells was 4-fold higher than in control group (ET-FPP®: 5020 ± 542 M.I.F, ET-CTR: 1228 ± 88 M.I.F., *p* < 0.0001) (Figure 5A), while in ovarian germ cells the telomeres length was 2.7-fold higher as compared to controls (ET-FPP®: 91 ± 5 M.I.F., ET-CTR 33 ± 3 M.I.F., *p* < 0.0001) (Figure 5B).

#### *3.3. Late Treatment with FPP*®*: from 51 to 96 Weeks of Age*

This set of experiments was aimed at evaluating the FPP® anti-aging effect in a group of mice that started the treatment later in their life (10 months) (LT-FPP®). As for ET-FPP® mice, we measured antioxidants and ROS levels and telomerase in the blood and the telomeres length in single cell suspensions obtained from the bone marrow and the ovaries of the mice.

#### 3.3.1. Oral Administration of FFP® Increases Plasma Levels of Antioxidants

The results showed that Total Antioxidant Power levels in LT-FPP® (8 ± 0.17 mM) were comparable (*p* > 0.05, not significant) to LT-CTR (7.5 ± 0.13 mM) (Figure 6A); similarly to previous results in ET-FPP® mice, GSH plasmatic levels resulted to be 279.5 ± 24.9 μM in LT-FPP® mice (*p* < 0.05) and 208.9 ± 11.2 μM in LT-CTR (Figure 6B), and SOD-1 levels 89.9 ± 1.5 U/mL in LT-FPP® mice (*p* < 0.05) and 78.3 ± 4.3 U/mL in LT-CTR group (Figure 6C). It is clear from the figures that while the significant increase in the SOD-1 and GSH plasma levels in the FPP® -treated mice, the absolute values are lower than in the group of mice that started the FPP® treatment earlier in their life (Figure 2).

**Figure 6.** Antioxidant effect of FPP® in C57BL/6J female mice by measuring the plasma antioxidant levels (antioxidant power, GSH and SOD-1). Plasma samples collected from both untreated (LT-CTR group) and treated (LT-FPP® group) mice were analyzed. (**A**) Analysis of the quantification and detection of the total antioxidant power (mM). (**B**) Analysis of the quantification and detection of GSH activity (μM). (**C**) Analysis of the quantification and detection of SOD-1 activity (U/mL). Data are normalized on total plasma and expressed as means ± SE. \* *p* < 0.05.

3.3.2. Oral Administration of FFP® Reduces Plasma Levels of ROS

This set of results did not show significant difference in the plasmatic ROS levels between the FPP®-treated and untreated mice (*p* > 0.05, not significant). In fact, LT-FPP® mice had ROS levels of 10727 ± 157 a.u. and LT-CTR mice 11266 ± 198 a.u. (Figure 7).

**Figure 7.** Effect of FPP® on total ROS blood levels in C57BL/6J female mice. Analysis of the total ROS levels (arbitrary units, a. u.) on the plasma samples collected from both LT-CTR and LT-FPP®. Data are normalized on total plasma and expressed as means ± SE. *p* = NS (>0.05).

#### 3.3.3. Oral Administration of FFP® Increases Plasmatic Telomerase Activity

Analysis of telomerase activity was performed also for LT-FPP® mice LT-CTR controls. As for the early treatment, in this case the mice that received water supplemented with FPP® showed a higher telomerase concentration (LT-FPP®: 124.0 ± 9.0 ng/mL, *p* < 0.05) than the mice that drank only tap water (LT-CTR: 92.5 ± 6.5 ng/mL) (Figure 8).

**Figure 8.** Effect of FPP® on telomerase (TE) activity in plasma samples from C57BL/6J female mice. Quantitative determination of mouse telomerase (TE) activity (ng/mL) was performed on plasma samples obtained from both LT-CTR and LT-FPP® groups immediately before the sacrifice. Data are normalized on total plasma and expressed as means ± SE. \* *p* < 0.05.

3.3.4. Oral Administration of FFP® Increases Telomeres Length in Bone Marrow Cells and Ovarian Germ Cells

As previously described, bone marrow and ovarian germ cells were obtained from each mouse and hybridizated of with a fluorescein-conjugated probe (PNA) for telomeres length analysis. As for the previous group of experiments cells were counted by trypan blue exclusion under optical microscope and the results showed that the number of bone marrow and ovarian germ cells in LT-FPP® mice was increased 1.8-fold and 2-fold, respectively, as compared to cells obtained from LT-CTR mice. The results on telomeres length showed that cells from the bone marrow of LT-FPP® treated mice had 2-fold longer telomeres (121 ± 6 M.I.F., *p* < 0.0005) as compared to LT-CTR 59 ± 9 M.I.F. (Figure 9A). Similarly, telomeres analyzed from ovarian germ cells of LT-FPP® mice were significantly longer (8.69 ± 0.25 M.I.F., *p* < 0.05) than telomeres from untreated mice (7.29 ± 0.44 M.I.F.) (Figure 9B). Consistent with the anti-oxidant reaction the M.I.F. signals in both bone marrow and ovarian germ cells were significantly decreased in LT-FPP® as compared to ET-FPP®.

**Figure 9.** Effect of FPP® on telomeres length in bone marrow cells and in ovarian germ cells from C57BL/6J female mice. The analysis of telomeres length was performed on nucleated haematopoietic cells from (**A**) bone marrow and (**B**) on ovarian germ cells. Cells were retrieved from both LT-CTR and LT-FPP® groups immediately after the sacrifice. Data are expressed as mean ± SE of M.I.F. (Mean Intensity Fluorescence) normalized on total cells. \* *p* < 0.05, \*\*\* *p* < 0.0005.

#### *3.4. Comparison of FPP*® *E*ff*ectiveness between Early Treatment and Late Treatment Supplementation*

We thus wanted to compare the early to the late FPP® treatment in terms of percentage of ratio between FPP® -treated mice and untreated controls. As shown in Table 2, the most beneficial effects are observed with the early treatment.

Comparing ET-FPP® and LT-FPP® values with their respective control, we could observe that the early treatment with FPP® was impressively more effective in increasing the plasmatic levels of the antioxidant power (increase of 56%) as compared to the late treatment (1%) that was in fact comparable to CTR.

In the case of GSH the differences between early and late treatment were straightforward: in the case of ET mice GSH plasmatic level was increased of 640% compared to control, while we observed only 34% of increase in the LT group. Also SOD-1 levels were higher in the early treated mice (30%), as compared to the late treatment (15%). Moreover, FPP® showed a greater effect in decreasing total ROS levels when administered early (30% of decrease) than in LT-FPP® group (5% of decrease).

Comparable results were obtained with telomerase levels (58% increase in ET-FPP® mice and 34% in LT-FPP®) as compared to controls, and telomeres length in bone marrow and ovarian germ cells (length increase of 300% and 174% in ET-FPP® and 101% and 19% in LT-FPP®, respectively). All in all this analysis allows to conclude that the early treatment with FPP® starting from 6 weeks of life was the most effective.


**Table 2.** Comparison between ET- and LT-treatment.

Results are expressed as percentage ratio between value of FPP® mice and respective control.
