**3. Results**

#### *3.1. Blood Donors (Study Groups)*

Table 2 presents basic characteristics of 39 investigated blood donors—AD patients and controls (age-matched, over 55 years old)—according to a few variables. There were 22—15 female and 7 male—among AD patients and 17—10 female and 7 male—among controls. MMSE score was evaluated. MMSE median for female was 18.75 and for male 18.5. Among the AD group, there were no differences between men and women in the level of dementia. Patients were collected randomly to estimate DGN and DSMV differences between sex. Chi-square test statistic *χ*<sup>2</sup> *d f*=<sup>2</sup> = 0.598, *p* = 0.856.

**Table 2.** Characteristics of blood donors—AD patients and controls.



Sn, measure of variability; MMSE, mini-mental state examination; DGN, the diagnostic according to the guidelines of the German Society of Neurology; DSMV, fifth edition of diagnostic and statistical manual of mental disorders classification (criteria for major neurocognitive disorder).

### *3.2. EGb Characteristic (Cytotoxicity, Antioxidant Activity)*

First, the starting solution of EGb was prepared. EGb at 20 mg/mL in DMSO was diluted to final concentrations of 25–500 μg/mL in RPMI 2% FBS. Freshly isolated PBLs were treated with several concentrations of EGb and incubated in 37 ◦C/5% CO2 for 24 h. After that, time morphological changes of the cells were observed under the inverted microscope. Total number and viability of PBLs were determined by 0.4% trypan blue staining. The viable cells—with intact cell membranes—did not take up impermeable trypan blue (stayed non-colored), whereas dead cells (with damaged cell membranes, cell shadows, shrunken cells) were permeable and took up the dye (dyed with distinctive blue). Fresh EGb dilutions were prepared before each experiment. Experiments was performed three times in two independent repetitions each. It was noticed that EGb in concentration over 200 μg/mL resulted in cytotoxicity (cell viability below 90%). EGb in the range of 25–150 μg/mL was nontoxic for PBLs (cell viability over 90%). Control were PBLs incubated only with culture medium RPMI 2% FBS. The final concentrations of DMSO < 2% were nontoxic. Estimated cytotoxic concentration of EGb, which reduced viability of PBLs by 50% (CC50), was calculated and equal to *CC*<sup>50</sup> ∼= 743.5 μg/mL. Based on cytotoxicity test for the future experiments of an influence of the extract on innate immune response of PBLs, the highest nontoxic concentration of EGb, − 150 μg/mL, was used. The cell viability for this concentration was about 95%.

An antioxidant activity (in vitro), i.e., ferric ion reducing antioxidant power, Fe2+ chelating ability, and DPPH radical scavenging activity of EGb were analyzed (Table 3). EGb preparation exerted strong DPPH scavenging activity; only 10 μg of preparation has the same effect as 0.03 μM Trolox. The antioxidant properties (inhibition concentration, IC50) for DPPH scavenging activity reached the value 41.13 μg. EGb preparation also exerted strong concentration-dependent ferric ion reducing antioxidant power and Fe2+ chelating ability (Table 3).


**Table 3.** Concentration-dependent antioxidant activity (in vitro) of EGb.

#### *3.3. EGb Improves Innate Immune Response of PBLs*

The level of innate immunity of AD patients and healthy age-matched controls was estimated based on the test with vesicular stomatitis virus (VSV) replication in freshly isolated PBLs ex vivo. To evaluate the effect of EGb (150 μg/mL) on innate immunity VSV titers were examined after EGb treatment in the collected supernatants. Results are presented in Table 4. The level of innate immunity/PBLs resistance to VSV infection was assessed with the scale: the lack of virus replication (0–1 log TCID50/mL) indicated complete immunity; VSV replication over 1 log (about 2–3 log) indicated deficiencies and partial immunity; and VSV replication over 4 log evidenced high deficiency in innate immunity. The EGb effect in every patient was calculated as a difference between the level of innate immunity after EGb treatment and before EGb treatment (EGb effect = after−before). The average change of innate immunity in AD patients was −0.75; i.e., EGb increased the level of innate immunity of AD. The results were significant (*p* = 0.0002), with confidence interval CI95(-Inf; −0.41). In the control group an increase in innate immunity (*p* = 0.0001) was also observed. There was no difference, however, in the effect of EGb treatment between AD and controls (*p* = 7539). In summary, EGb expressed a beneficial effect on innate immune response of PBLs. EGb treatment significantly increased the level of innate immunity in AD patients as well as controls (Figure 1).

**Table 4.** Influence of EGb treatment on PBLs resistance/level of innate immunity in AD patients and healthy age-matched controls (*n* = 39).


Δ, shift parameter of location (as a part of Wilcoxon rank test, Hodges-Lehman estimator of (pseudo) median); Sn, measure of variability (higher value means higher variability; robust estimator equivalent of standard deviation); 1, one side (left side) test and one side confidence interval; 2, Inf—infinity; *βwoman*, standardized difference between men and women adjusted with other variables present in ANOVA; MMSE, mini-mental state examination; DGN, the diagnostic according to the guidelines of the German Society of Neurology; DSMV, fifth edition of diagnostic and statistical manual of mental disorders classification (criteria for major neurocognitive disorder).

In addition, Table 4 presents an analysis of variance (ANOVA) where EGb effect was dependent variable in AD and control group. The analysis showed that among AD patients, the effect of EGb treatment on the level of innate immunity was not related to the severity of the disease. Interestingly, an important variable was sex. The statistic effect size here was partial eta-squared η<sup>2</sup> equals for sex effect η\_Sex2 = 0.3983, which is commonly interpreted as a huge effect size according to Cohen's interpretation. Thus, EGb increased the level of innate immunity much stronger in women with AD than in men with AD (*p* = 0.0049). Simultaneously, the sex differences in EGb effect was smaller and not statistically significant in the control group (*p* = 0.1883). The difference between AD women and AD men was more than 5.5 times higher than between women and men in the control group. Results are presented in Figure 2.

**Figure 1.** EGb effect on VSV replication (level of innate immunity) in PBLs from AD patients and healthy age-matched controls. EGb effect was measured as difference between VSV titer (log TCID50/mL) after EGb treatment and VSV titer (log TCID50/mL) before EGb treatment. Red and blue squares are individual observations.

**Figure 2.** Sex-dependent differences in EGb effect on VSV replication/level of innate immunity in AD patients and healthy age-matched controls. EGb effect was measured as difference between VSV titer (log TCID50/mL) after EGb treatment and VSV titer (log TCID50/mL) before EGb treatment. Red and blue squares are individual observations.
