**4. Discussion**

With numbers of sufferers with cognitive impairments still increasing, dementia such as Alzheimer's disease (AD) is observed. AD is an age-related disorder; however, neurodegenerative changes may begin many years before clinical manifestation of the disease. Therefore, brain health is becoming very important for the adults and the elderly. The lack of effective AD treatment means that those affected by this disease are still looking for any alternative approaches. Nutritional science and dietary supplements, such as extracts of *Ginkgo biloba* (EGb), offer an approach (preventative and restorative) to adding phytonutrients to daily consumption of food and drink [20]. EGb is the most widely studied natural compound with proven beneficial effect on cognitive functions (improving memory and concentration) in both healthy adults and patients with mild cognitive impairment (MCI) or dementia. However, its effect on the immune system is less explored, with little literature data available. We have previously reported that EGb increased an innate immune response and regulated cytokine production in healthy young people [21,22]. We suggest the possibility of using EGb for the treatment of immune deficiencies. As AD is considered as a systemic disease with evidence for changes in immune system functioning, we examined the effect of EGb on innate immune mechanisms in AD patients. In the present study, we showed that EGb is a good immunoregulator increasing an innate immune response of AD patients. We also examined the effect of EGb treatment on several inflammatory-associated genes expression.

Currently, many EGb preparation are available, with different compositions resulting in various therapeutic features [23]. We used a standardized extract, prepared according to the European Pharmacopoeia (Ph. Eur. 8.0); it is easily accessible, which is a great benefit. In addition, an advantage of herbal drugs is that they have very low side-effects compared to chemical drugs [6]. Indeed, we observed immuno-enhancing activity of EGb in an absolutely nontoxic concentration for human peripheral blood leukocytes (PBLs). The leaves of maidenhair tree (*G. biloba*) are also excellent sources of antioxidants. Ethanol extracts of that plant are well-characterized and contain about 22–27% flavonol glycosides, including polyphenols such as tannins and terpene lactones (5–7%). The high phenolics content determines strong ferric ion activity, reducing antioxidant power, copper chelating ability, peroxyl radical scavenging activity, and radical scavenging activity for *G. biloba*

extracts [24–26]. Our results correspond to the results of a study on antioxidant properties of various plant extracts carried out by Szerlauth and others (2019) [24]. It was found that the plant extracts exhibit remarkable antioxidant properties (IC50) from 0.44 (*Alliumsativum*) to 44.18 (*Juglansregia*) AAEQ values [24].

The observed age-related progressive decline in immune system functioning contributes to a development of chronic states of inflammation resulting in systemic diseases, including AD. This phenomenon—dysregulation of the immune system—is not limited to the one mechanism, but it concerns aging of the innate and adaptive immune cells, alterations in circulating inflammatory mediators, and changes in lymphoid and nonlymphoid tissues [4]. In AD patients, dysregulation of innate immune mechanisms were also observed. PBLs resistance to viral infection is a good indicator of the innate immune system condition. We previously established that the level of innate immunity, measured with the test based on vesicular stomatitis virus (VSV) replication in human PBLs, was remarkably correlated with clinical advancement of AD. The higher VSV titer means a lower level of innate immunity. More severe patients were characterized with a lower level of innate immunity [27]. We also showed as a potential therapeutic the oral administration of proline-rich polypeptide complex (PRP) isolated from bovine colostrum, a nutraceutical intended to boost an immune system by increasing an innate immune response in AD patients [27]. Here, we present for a first time the strong immune-enhancement activity of natural, herbal preparation, EGb, in an ex vivo model of PBLs from AD patients. As we suspected, EGb was capable of significantly improving innate immune response by decreasing VSV replication in PBLs of AD patients but also controls. The most interesting, however, was that EGb notably increased an innate immune response in AD women. This sex discrepancy in EGb effect was not observed in the control group. Similarly, in the above-mentioned study of EGb effect on the immune system functioning in healthy young people, sex differences were not observed. Thus, EGb may be promising for immune improving in AD patients, especially in AD women. It is important due to the fact that women are more afflicted with the frequency, prevalence, and clinical manifestation of the disease [28].

According to recent data in animal models, EGb showed wide-ranging anti-inflammatory and antioxidant properties [29]. The immune effect of EGb was showed by Wan at al. [30] in the model of mouse microglia cells. Microglia, brain resident macrophages, play an important role in the development of many central nervous system (CNS) diseases. Reactive microglia that produce large amounts of inflammatory mediators influence the development of AD [31]. Significantly reduced production of pro-inflammatory cytokines, such as IL-6 and TNF-α, and increased anti-inflammatory IL-4, IL-13, or TGF-β were found in the brains of animals supplemented with EGb [30]. Similar results were presented by Tao et al. [32] in allergic mice. Administration of EGb showed significant decrease in release of pro-inflammatory IL-4, IL-5, IL-6, IL-8, and TNF-α. In our study with human leukocytes model ex vivo from AD patients and healthy age-matched controls, EGb presented immunoregulatory activity. EGb significantly decreased production of pro-inflammatory TNF-α and IFN-γ as well as anti-inflammatory IL-10 by uninfected and VSV-infected PBLs of AD patients and controls. At the same time, EGb significantly increased production of IL-15 and slightly increased IL-1β in both groups of participants. Interestingly, this effect was more pronounced among AD patients.

It was established that VSV infection of PBLs induced secretion of IFN-α [33]. In the present study, IFN-α was also investigated. IFN-α was not assessed for all study participants, and this investigation needs to be continued. However, interesting observations were obtained. Although, as suspected, there was no spontaneous secretion of IFN-α, after VSV infection, leukocytes of both groups responded with production of this cytokine. After incubation with EGb, the production of IFN-α decreased to no detectable level in all AD patients as well as in controls. IFN-α was the only cytokine strongly inhibited by the extract.

EGb also downregulated expression of several genes that regulate innate immune response to viral infection and cytokine production, such as interferon regulatory factors (IRFs) *IRF-3* and *-7*, which are primary transcriptional factors regulate the type I IFN response after RNA virus infections [34]; IFN-stimulated antiviral genes *MxA* and *tetherin*, critical for controlling VSV infection [33]; but also *NFkB* transcription factors that mediate induction of various pro-inflammatory genes in innate immune cells; and *SOCS*, the main regulators of antimicrobial innate immune response [35,36]. It was presented earlier that *G. biloba* extract has anti-oxidative as well as anti-inflammatory properties. A marked suppression of transcription factor NFκB and pro-inflammatory cytokines (TNF-α, IL-1α, 1L-6) was shown [37].

#### **5. Conclusions**

Therefore, EGb may have an advantageous properties for health management in elderly and AD sufferers but especially in women afflicted with AD. Female sex is a major risk factor for developing late-onset AD, which is suggested to be implicated in the menopause transition [38]. The observed beneficial effect of EGb on innate immune response/increase PBLs resistance to VSV infection may be at least partially explained by its antioxidant activity and differential influence on cytokine production. Even though the most important antiviral response is mediated with IFN I, the role of other cytokines should not be diminished. In our study, EGb decreased IFNs production but increased IL-15 and IL-1β. IL-15 is known as playing an important role in promoting the development and homeostasis of NK cells and CD8 T; however, IL-15 also mediates the anti-viral responses of these cell populations during an active immune response [39]. IL-1β, next to type I IFNs, is also a central mediator driving innate antiviral immunity and inflammation [40]. It was suggested that activation status of peripheral innate immune cells may be a good biomarker of AD pathology [3]. Thus, improving their activity by adding EGb as accompanying treatment may be a good long-term course to modify the disease progression.

**Author Contributions:** J.L., conception and study design, clinical examination of the patients, and drafted and revised the manuscript; M.S. (Marta Sochocka), conception and study design, laboratory experiments, acquired and analyzed and interpreted all data, drafted and revised the manuscript, and edited language; M.O., laboratory experiments and drafted the manuscript; P.N. and K.G., laboratory experiments; A.Z., laboratory experiments and drafted the manuscript; M.S. (Maciej Sobczy ´nski), acquired and analyzed and interpreted all data. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by internal research funds of the Wroclaw Medical University and Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the Wroclaw Medical University (No. KB 349/2016).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Acknowledgments:** The authors wish to acknowledge the nursing staff of the Wroclaw Medical University for the support in recruiting participants.

**Conflicts of Interest:** The authors report no conflict of interest, financial or otherwise.

#### **References**

