**1. Introduction**

Immunity and chronic inflammation play a key role in the survival of the older adults, and according to the latest knowledge, they also represent one of the main features of Alzheimer's disease (AD) pathology [1]. AD is one of the most important age-related health problems worldwide, and it is believed that the onset and progression of the disease may depend at least in part on optimal immune system functioning. Experimental studies highlight the pathological changes in the central and peripheral immune response in AD [2,3]. An increased levels of peripheral inflammatory markers, such as IL-6, TNF-α,

**Citation:** Sochocka, M.; Ochnik, M.; Sobczy ´nski, M.; G ˛ebura, K.; Zambrowicz, A.; Naporowski, P.; Leszek, J. Ginkgo Biloba Leaf Extract Improves an Innate Immune Response of Peripheral Blood Leukocytes of Alzheimer's Disease Patients. *Nutrients* **2022**, *14*, 2022. https://doi.org/10.3390/nu14102022

Academic Editors: Omorogieva Ojo and Amanda R Amorim Adegboye

Received: 1 April 2022 Accepted: 9 May 2022 Published: 11 May 2022

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or C-reactive protein (CRP), were found to be associated with future cognitive decline and dementia [2,4]. Currently, no effective drugs are available for the treatment of AD symptoms. An accessible pharmacotherapy aims to only slow disease progression and reduce cognitive symptoms. Some hope is associated with GV-971 (sodium oligomannate capsules), which improved cognitive functions in AD patients in China in a phase 3 trial and was approved for the treatment of AD in China [5]. Thus, more attention is paid to alternative therapy, such as using drugs of natural origin. Moreover, searching for natural compounds with immunoregulatory activity seems to be a good direction for future adjunct AD therapy.

Currently, phytomedicine is gaining its popularity, and many plant-derived phytotherapeutics with medicinal properties are used in the treatment of various diseases, including age-related diseases [6]. The phytomedicine of aging provide a wide range of bioactive compounds, such as flavonoids, terpenoids, or polyphenols with therapeutic effects. Health benefits consist mainly of acting as an immunity booster and exhibiting antioxidant, cardioprotective, and neuro-protective effects [7]. One of the most popular medicinal plants is *Ginkgo biloba*. Standardized extract of *G. biloba* (EGb) contains 24% ginkgo flavonoid glycosides, 6% terpene lactones, and up to 5 ppm ginkgolic acids [8]. The therapeutic potential of EGb is manifested in beneficial effect on the circulatory system (blood flow improvement, prevention of clot formation, reinforcing the walls of the capillaries) and nervous system with protection of nerve cells from injury [9]. Phytochemical constituents from *G. biloba,* such as flavonoids and terpenoids, showed beneficial effect in the treatment of concentration difficulties, memory impairment, and AD. Thus, EGb is considered as memory enhancer [10].

The use of phytotherapeutics/nutraceuticals as an adjunct therapy to classic drug therapies is highly recommended in many diseases. However, many more studies are still needed to evaluate the therapeutic potential and clarifying the mechanism of action of natural compounds, including EGb. It is believed that this could help to choose better phytotherapeutics as the accompanied treatment of neurodegenerative pathologies such as AD. EGb is already used in the treatment of AD and cognitive deficits acting as anti-aggregating and pro-cognitive preparation. It is implemented to improve memory impairment and cognitive decline [11]. However, less attention and research are concentrated on its effect on immune system functioning in AD patients. The purpose of this study was to investigate the effect of EGb on the mechanisms of innate immune response of peripheral blood leukocytes (PBLs) of AD sufferers.

#### **2. Materials and Methods**

#### *2.1. Blood Samples*

Peripheral venous blood was obtained from 39 Subjects: 22 AD patients (15 females, 7 males) and 17 healthy adult volunteers (10 females, 7 males) of an appropriate age (43–90 years) and collected in tubes containing anticoagulant EDTA or heparin. Patients were under the care of Department of Psychiatry of the Medical University in Wroclaw, Poland. Patients did not receive any anti-dementia and other drugs before blood venipuncture as well as any other immunomodulators. Among the patients, no infectious diseases occurred in the 3-month period before the inclusion to the study.

#### *2.2. Ethics Approval and Consent to Participate*

This study has been reviewed, approved, and conducted in accordance with the guidelines of the Ethics Committee of the Wroclaw Medical University (No. KB-349/2016). Signed consent was obtained from all participants of the study or their legal representative.

#### *2.3. Clinical Examination*

Patients were under psychiatric and neurological examinations as well as laboratory tests, electroencephalographic examinations (EEG), and computer tomography (CT) or magnetic resonance imaging (MRI) structural studies. Mini-Mental State Examination (MMSE) was used for the screening of dementia. All patients met DSM-V and NINCDA-ADRDA criteria for probable AD dementia. A diagnosis of AD was made when specific symptoms were present and by making sure other causes of dementia were absent, including anemia, brain tumor, chronic infection, intoxication from medication, severe depression, stroke, thyroid disease, and vitamin deficiencies. CT and MRI of the brain were performed as well to look for other causes of dementia, such as brain tumor or stroke. Semi-structured interview with the patient and informant, physical exam, evaluation of neurological status, and psychiatric exam were obtained. Vital signs and blood screening labs (hematology, chemistry panel, urinalysis, vitamin B12 (B12), thyrotropin (TSH)) were collected. *Exclusion criteria:* patients older than 90 years, any significant neurological disease such as Parkinson's disease, multi-infarct dementia, Huntington disease, normal pressure hydrocephalus, brain tumor, progressive supranuclear palsy, seizure disorder, subdural hematoma, multiple sclerosis, or history of significant head trauma followed by persistent neurologic defaults or known structural brain abnormalities. MRI scan with evidence of infection, infarction, or other focal lesions; subjects with multiple lacunes or lacunes in a critical memory structure; psychiatric disorder/psychotic features: major depression, bipolar disorder, agitation, or behavioral problems within the last 3 months; history of schizophrenia, alcohol abuse, history of alcohol or substance abuse or dependence within the past 2 years; any significant systemic illness or unstable medical condition; clinically significant abnormalities in B12, rapid plasma regain test (RPR), or TSH; and current use of specific psychoactive medications (e.g., certain antidepressants, neuroleptics, chronic anxiolytics, or sedative hypnotics, etc.). Patients were excluded if they did not agree to respond to the test questions and/or if they had life-threatening diseases other than AD.
