*Review* **The Use of Medicinal Plants in Blood Vessel Diseases: The Influence of Gender**

**Guglielmina Froldi**

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131 Padova, Italy; g.froldi@unipd.it; Tel.: +39-049-827-5092; Fax: +39-049-827-5093

**Abstract:** Data available in the literature on the use of herbal products to treat inflammation-related vascular diseases were considered in this study, while also assessing the influence of gender. To this end, the articles published in PubMed over the past 10 years that described the use of plant extracts in randomized clinical trials studying the effectiveness in vascular pathologies were analyzed. The difference in efficacy of plant-derived preparations in female and male subjects was always considered when reporting. The safety profiles of the selected plants were described, reporting unwanted effects in humans and also by searching the WHO database (VigiBase®). The medicinal plants considered were *Allium sativum*, *Campomanesia xanthocarpa*, *Sechium edule*, *Terminalia chebula*. Additionally, an innovative type of preparation consisting of plant-derived nanovesicles was also reported.

**Keywords:** vascular dysfunction; endothelium; plant extracts; gender; safety; botanicals; garlic

#### **1. Introduction**

Inflammation is a well-known biological response of the organism to chemical and physical injuries that help the healing of tissues [1]. However, the inflammatory reaction can be harmful when it is excessively great, causing acute organ failure, or when too persistent, triggering chronic systemic inflammation [2]. In general, the inflammatory status is manifested by an increase in serum C-reactive protein (CRP) level and the release of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [3]. Arterial and venous vessels are directly involved in inflammatory progression with alterations in endothelial permeability, causing liquid and protein leakage and tissue edema formation. TNF-α increases intracellular calcium and regulates myosin light chain kinase and RhoA, which disrupts endothelial junctions, reducing barrier function and enabling leukocyte transmigration [4,5]. Subjects with high levels of homocysteine, cholesterol, and triglycerides show a greater risk of stroke by approximately 50% than those with normal values [6]. Furthermore, the correction of hyperhomocysteinemia involves a reduction in stroke risk from 34% to 70% [7]. Among the factors that affect the amount of homocysteine present in the blood, are various physiological factors, such as age, sex, and body mass index [8,9]. In fact, women generally have lower levels than men [10]; therefore, homocysteine may be a possible factor responsible for gender differences in atherosclerosis and coronary artery disease [11].

In the blood vessel, acute and chronic inflammation results in endothelial dysfunction and vascular remodeling, affecting cardiovascular function. Several endogenous modulators play a role in vascular inflammatory processes; in particular, an increase in advanced glycation end-products (AGEs) causes activation of inflammatory pathways, oxidative stress, and procoagulant activity, leading to endothelial dysfunction [12,13]. Fishman et al. suggested that AGEs and their receptors may be useful biomarkers of the presence and severity of coronary artery disease [12]. Other inflammatory modulators are proinflammatory cytokines, such as TNF-α and interleukins (ILs) [14]. IL-1α and IL-1β are responsible for the disruption effects on the endothelial barrier, while nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB) and the mitogen-activated protein kinase (MAPK)

**Citation:** Froldi, G. The Use of Medicinal Plants in Blood Vessel Diseases: The Influence of Gender. *Life* **2023**, *13*, 866. https://doi.org/ 10.3390/life13040866

Academic Editors: Azahara Rodríguez-Luna and Salvador González

Received: 21 February 2023 Revised: 17 March 2023 Accepted: 23 March 2023 Published: 23 March 2023

**Copyright:** © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

signaling cause pro-atherogenic effects [15]. Moreover, the vascular adhesion protein-1 (VAP-1), also known as amine oxidase copper-containing 3 (AOC3), is a pro-inflammatory modulator with a greater expression in endothelial cells during inflammation [16,17]. VAP-1 is an ectoenzyme that catalyzes the oxidative deamination of primary amines and produces hydrogen peroxide, ammonium, and aldehydes, regulates leukocyte extravasation, and causes vascular damage and atherosclerosis [16,17]. Furthermore, endothelial dysfunction is mediated by the activation of enzymes such as heparinase and metalloproteinases (MMPs), which cleave glycoproteins anchored to the endothelial glycocalyx, which are activated by pro-inflammatory cytokines and reactive oxygen species (ROS) [18]. Furthermore, monosodium urate and cholesterol crystals, and islet amyloid polypeptides can damage the phagolysosome membrane and promote persistent activation of the NLRP3 inflammasome, also known as NALP3 or cryopyrin, causing severe inflammatory diseases such as gout, atherosclerosis, and type 2 diabetes mellitus [19–21]. Recently, extracellular vehicles such as platelet and endothelial microparticles have been reported to be involved in vascular regulation, including inflammatory and thrombotic homeostasis [22,23]. Therefore, vascular inflammation is clearly a complex condition that has several modulators.

The literature findings suggest that the treatment of inflammation with targeted drugs may promote the regression of vascular dysfunction [4,24,25]. However, non-steroid and steroid anti-inflammatory drugs have not shown protective effects against arterial stiffening; however, some promising results have been obtained with the use of selective inhibitors of IL-1β, IL-6, and TNF-α [4,25]. Unfortunately, the use of these inhibitors can cause the appearance of significant side effects, and their risk/benefit ratio remains to be further determined. In this context, plant-derived products with potential activity in the treatment of vascular dysfunction may be of interest. For this purpose, this study focused on the literature delineating potential plant products of utility in the treatment of cardiovascular diseases related to inflammatory damage. Particular attention was paid to the identification of differences in response to herbal medicines in women versus men.

#### **2. Sex Differences in Vascular Function**

Few studies considered the sex difference related to inflammation and metabolic and cardiovascular diseases. The general opinion is that men tend to have a worse risk factor profile than women, although this relationship changes with advancing age [26]. Differences in the prevalence of cardiovascular diseases were observed in premenopausal women compared to men of the same age; clinical data suggest that women are protected against various cardiovascular diseases primarily during the fertile period of life [27–30]. In fact, estrogens increase vascular NO• signaling, improving vasodilatation and insulin responsiveness, protecting against diabetes mellitus [30]. However, the relative risk of cardiovascular disease morbidity and mortality in subjects affected by diabetes mellitus ranges from 1 to 3 in men and from 2 to 5 in women, related to those without diabetes [31]. Furthermore, clinical studies failed to demonstrate that hormone replacement therapy (HRT) in postmenopausal women could improve cardiovascular outcomes [30]. It can also be observed that men and women have different lifestyle risk factors, such as male behavior that may include more frequently cigarette smoking, alcohol abuse, higher intake of red meat, and lower fruit and vegetable consumption [32]. Effectively, in part, behavior differences could explain why women live longer than men [33]. Focus has been on the relationship between the Western lifestyle and chronic metabolic inflammatory diseases and also on finding preventive approaches [34]. A meta-analysis that considered a total of 462,194 participants demonstrated that high ingestion of fruits and vegetables (flavonoids) was inversely associated with the risk of total cardiovascular disease mortality [35]. Recently, Parmenter et al. (2022) found that in older women, a higher intake of habitual dietary flavonoids, mainly black tea, is associated with less extensive abdominal aortic calcification [36]. Previous authors have also shown that high black tea consumption is associated with lower coronary artery and abdominal aorta calcifications [37–39].

#### **3. Materials and Methods**

This study considered articles identified using a PubMed search strategy up to January 2023. The terms included in the search query were 'blood vessel', 'inflammation', and 'plantderived compound'. The inclusion criteria for the search were as follows: 1. Published in the last 10 years, 2. Full texts, 3. English language. Of a total of 16,395 articles included, some were excluded according to other inclusion criteria, such as 'endothelium' and 'female', excluding the term 'review' (Figure S1). A total of 20 articles was reviewed according to their relevance to the selected topic. Additionally, other references were examined also through Google Scholar to find additional relevant articles, including in vitro and in vivo studies to facilitate the explanation of the pharmacological mechanisms. The safety profiles of the selected plants were described, along with the unwanted effects reported in humans, also by searching VigiBase®, the WHO global database of possible side effects of medicinal products [40].

#### **4. Results**

The selected medicinal plants evaluated in this review were *Allium sativum*, *Campomanesia xanthocarpa*, *Sechium edule*, and *Terminalia chebula*. Additionally, an innovative type of preparation consisting of plant-derived nanovesicles was considered. Table 1 reports a synthesis of the clinical trials that detected the efficacy of the medicinal plants considered in this review.

#### *4.1. Allium sativum L.*

#### 4.1.1. Botanical Characteristics

Garlic (Amaryllidaceae) is a herbaceous plant whose bulbs are often used to flavor different types of food, widely known for its typical smell and taste [41]. Its alimentary use is widespread throughout the world, and its medicinal use is well-known in popular medicine as crude drug, standardized extracts, and also as a food supplement [42]. The bulbs are harvested in late spring and early summer, then dried in the shade at 40 ◦C, to enable storage [41]. Several traditional uses are known, such as antimicrobial, diuretic, vermifuge, adjuvant in the prevention of atherosclerosis and the relief of the common cold [43,44].

#### 4.1.2. Phytoconstituents and Preclinical Activity

Among the most peculiar constituents, there are several sulfur compounds, including alliin, an odorless compound, which in turn is transformed by the alliinase enzyme into allicin that has the typical garlic smell [45–48]. Allicin is a diallyl thiosulfinate considered one of the most active components, although other sulfur compounds provide garlic properties, such as ajoene, allyl propyl disulfide, diallyl trisulfide, and S-allylcysteine [49]. Additionally, garlic contains saponins, flavonoids, vitamins, and minerals [41,50]. Numerous in vitro and in vivo studies suggested its efficacy in several human diseases [51,52]. Garlic powder, aged garlic, and garlic oil have shown antiplatelet and anticoagulant effects by interfering with cyclooxygenase-mediated thromboxane synthesis [49,53]. Garlic extracts showed antioxidant property, decreased expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1 alpha (HIF-1α), inducible nitric oxide synthase (iNOS), and metalloproteinase (MMP)-9 [54–56]. Furthermore, garlic prevents the expression of inflammatory cytokines such as IL-6 and monocyte chemoattractant protein-1 (MCP-1) in lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes [57].


*Life* **2023** , *13*, 866

#### 4.1.3. Therapeutic Efficacy: Clinical Trials

Garlic is a very well-known plant that is used around the world. Studies on garlic preparations have mainly tested hypocholesterolemic, antihypertensive, antimicrobial, and, also, antitumor activities [54–56]. A randomized placebo-controlled trial (RCT) enrolled a total of 100 pregnant women at high risk of pre-eclampsia who were treated during the third trimester of pregnancy for 8 weeks with 800 mg garlic tablets (dried garlic powder containing 1 mg allicin, and ajoene) per day or placebo [60]. The treatment prevented the increase of total cholesterol and reduced hypertension [60]. Another RCT conducted in 44 pregnant women treated with 400 mg garlic tablet (equal to 400 mg garlic and 1 mg allicin) for 9 weeks showed reduced serum CRP levels and increased plasma glutathione in the treated women compared to the untreated group [61]. Another study showed that garlic supplementation (400 mg/day, garlic extract 2% allicin) positively modifies endothelial biomarkers of cardiovascular risk, suggesting that treatment can reduce chronic inflammation in obese individuals of both sexes [58]. Furthermore, one trial also suggested that 500 mg granulated garlic powder (2–3 mg allicin) can be considered as an adjuvant treatment in patients with diabetic macular edema [62]. Recently, the acute efficacy of 180 mg fermented garlic extract enriched with 7 mg inorganic nitrite (NO2 −) in healthy women and men showed a significant decrease in both systolic and diastolic pressure 30 min after ingestion of the product [70].

Systematic reviews and meta-analysis evaluating the effects of garlic supplementation have generally reported an improvement of lipid profile and insulin-resistance; however, the low quality of the trials does not permit at the moment the real effectiveness of garlic preparations to be defined [71,72]. Overall, the data available in the literature support the use of garlic extracts in the treatment of vascular diseases mainly with an atherosclerotic basis, without apparent differences between the two sexes. However, additional RCTs with standardized extracts are required to confirm the therapeutic use of garlic in cardiovascular diseases.

#### 4.1.4. Safety

According to toxicity data from experimental studies and medicinal use, garlic preparations are generally considered safe in the usual dosage regimen. Female and male rats treated with 300 and 600 mg/kg day of an aqueous garlic bulb extract for 21 days showed changes in weight growth, biological parameters, and histological structures [73]. In humans, common side effects included odor and skin rash, and gastrointestinal upset [60,74,75]. In VigiBase® *Allium sativum* has 232 reports of potential side effects from all countries, mainly Europe (31%), in patients of both sexes (female 49%, male 47%, unknown 5%), in adults and older people [40]. Figure 1 shows the types and percentages of reported side effects. Among these, there are mainly gastrointestinal disorders (18%, such as vomiting, gastrointestinal pain, diarrhea, etc.), general disorders (13%, such as drug interaction, asthenis, etc.), nervous system disorders (11%, such as dizziness, hemorrhages, skin disorders, etc.) and various others (Figure 1). The large number of side effects related to garlic use may depend on the very high consumption of this plant worldwide. In general, the use of garlic even for curative purposes is considered safe in humans, avoiding use in atopic subjects [51,76,77]. The medicinal use of garlic is not recommended during pregnancy and breastfeeding due to the absence of clinical evidence showing both efficacy and non-toxicity [44]. Furthermore, it is also not recommended in patients being treated with antiplatelet and anticoagulant drugs due to the increased risk of bleeding [44,75].

#### 4.1.5. Future Needs

The use of garlic preparations is widespread both in the diet and for healing purposes. To optimize its medical use, it is essential to define the type of formulation and the titer of sulfur derivatives, the dose, and the mode of administration, to standardize treatments and compare data from different clinical trials. Certainly, double-blind randomized clinical trials, with a sufficient number of subjects of both sexes are needed, to validate the use of

garlic in therapy in the treatment of vascular diseases. Otherwise, unfortunately, it would remain only a traditional use, without valid evidence of effectiveness, the use on the basis of scientific evidence being renounced.

**Figure 1.** *Allium sativum*: potential side effects reported in VigiBasis®.

#### *4.2. Campomanesia xanthocarpa Berg.*

#### 4.2.1. Botanical Characteristics

*Campomanesia xanthocarpa* (Myrtaceae) is a semi-deciduous tree, commonly known as "guavirova", which grows in Brazil, Argentina, Paraguay, and Uruguay. It has edible fruits with a succulent pulp and a sweet flavor [78]. Leaves are traditionally used in herbal teas to treat inflammatory, urinary, rheumatic diseases, high blood pressure, and high cholesterol [78,79].

#### 4.2.2. Phytoconstituents and Preclinical Activity

Leaves contain phenolic compounds, such as chlorogenic, gallic, ellagic and rosmarinic acids, glycosylated flavanols mainly of quercetin and myricetin, and pro-anthocyanidins [80–84]. Alkaloid theobromine (3,7-dimethyl-xanthine) was also identified in an aqueous infusion of leaves [83]. Acute administration of an aqueous extract showed a dose-dependent hypotensive effect in rats, by inhibiting the renin–angiotensin system through the block of the angiotensin II type 1 receptor (AT1R) and of calcium currents, as well as by KATP channel activation [83,84]. Furthermore, several studies reported the antioxidant activity of the fruits and leaves [80,82,85].

#### 4.2.3. Therapeutic Efficacy: Clinical Trials

The authors studied *Campomanesia xanthocarpa* on inflammatory processes, oxidative stress, and lipid biomarkers of hypercholesterolemia, showing a decrease in total cholesterol and LDL levels in treated hypercholesterolemic subjects [63,64]. A small trial in 33 hypercholesterolemic subjects treated with 250 and 500 mg capsules that contained dried *Campomanesia xanthocarpa* leaves for 90 days revealed a significant reduction in total cholesterol and LDL levels in hypercholesterolemic subjects with total cholesterol >240 mg/dL (*n* = 22) [63]. In addition, another trial involving a larger number of subjects treated for 90 days with 500, 750, and 1000 mg of dried encapsulated leaves also demonstrated anticholesterolemic activity [64]. These authors also suggested that this treatment attenuates oxidative stress and pro-inflammatory reactions, improving blood flow and endothelial function [64]. Furthermore, healthy subjects were treated with 1000 mg of powdered *Campomanesia xanthocarpa* leaves (*n* = 8) and compared to those treated with 100 mg of acetylsalicylic acid (ASA, *n* = 7), or 500 mg of *Campomanesia xanthocarpa* plus 50 mg of ASA

(*n* = 7). The authors showed that *Campomanesia xanthocarpa* leaves have antiplatelet activity when administered at 1000 mg for 5 days alone, or at 500 mg with low doses of ASA [65].

#### 4.2.4. Safety

The extract of *Campomanesia xanthocarpa* leaf administered to male rats at 300 mg/kg iv caused cardiac depression with a dramatic drop in blood pressure and animal death [84]. In contrast, a 5000 mg/kg ethanol leaf extract administered orally to five male and five female mice did not show toxicity [81]. Genotoxic effects were observed after treatment with an aqueous leaf extract administered to male rats at 1000 mg/kg [82]; this observation should be taken into account in future studies. No adverse reactions were reported for the use of *Campomanesia xanthocarpa* (guavirova) in VigiBase® [40]. No information is available in the literature on its safety during pregnancy or breastfeeding in humans.

#### 4.2.5. Future Needs

Preparations with dry leaves of *Campomanesia xanthocarpa* seem to have interesting hypocholesterolemic and antiplatelet activities, potentially useful in vascular diseases. However, no specific active compounds were identified in the formulations administered to the subjects, and therefore there is no reference compound for titration. Data from clinical trials are very limited, and there is certainly a need for double-blind randomized clinical trials with a sufficient number of subjects of both sexes to define the clinical usefulness of this medicinal plant.

#### *4.3. Sechium edule (Jacq.) Sw.*

#### 4.3.1. Botanical Characteristics

*Sechium edule* (Cucurbitaceae) is a perennial herbaceous climbing plant cultivated mainly by Asian and Latino-American populations for food use, in particular for Chayote fruits [86–88]. Likewise, fruits, roots, and leaves are known in traditional medicine against kidney stones, as a diuretic and antihypertensive [89–93]. Furthermore, alcoholic extracts showed a very good antimicrobial efficacy against all strains of multi-resistant Staphylococci and Enterococci [94]. Moreover, various leaf and seed preparations have shown remarkable antioxidant activity [95]. Several investigations in different animal models, such as rats, mice, and dogs, defined the capacity of this plant to reduce blood pressure [86,89,91,96]. The hydroalcoholic extract and the acetone fraction obtained from the roots of *Sechium edule* showed antihypertensive activity by a relaxant effect on blood vessels [91,97].

#### 4.3.2. Phytoconstituents and Preclinical Activity

Leaf, seed, stem, and also the fruit *Sechium edule* are rich in various bioactive components, as well as flavonoids, phenolics, vitamin C, and carotenoids [94,95,98]. The leaves contained the highest concentration of luteolin glycosides, while the most significant concentration of apigenin derivatives (C-glycosidic and O-glycosidic bonds) was found in the root extract [92]. Trans-cinnamic acid, phenylacetic acid, and α-linolenic acid were identified in the leaf extract [99]. Fruits can contain bitter principles called cucurbitacins [86,100,101].

In isolated aorta rings without endothelium, a hydro-alcoholic root extract caused a concentration-dependent vasorelaxation of angiotensin II-induced vasocontraction [91]. Furthermore, the authors reported in vivo antihypertensive effects in mice treated with angiotensin II [91]. The distinctive components of the highest active fraction were identified as cinnamic compounds, such as cinnamic acid methyl ester [91,97]. An aqueous leaf extract administered at 200 mg/kg showed nephroprotective activity against various types of chemically induced renal damage in rats [102]. The extract used at 100–200 mg/kg showed anti-inflammatory activity reducing levels of TGF-β, TNF-α, and ICAM-1 [103,104]. In rats fed with a high-fat diet, *Sechium edule* shoots can prevent hepatic steatosis and attenuate fatty tissue by inhibiting lipogenic enzymes and stimulating lipolysis by upregulating AMP-activating protein kinase (AMPK) [105]. The same authors also showed that the

shoot extracts inhibited the expressions of fatty acid synthase and HMG-CoA reductase in rats, while also isolated caffeic acid and hesperetin, the main characteristic components of *Sechium edule* shoots, prevented hepatic lipid accumulation [105]. The acetone fraction of the hydro-alcoholic extract of *Sechium edule* roots administered to female mice at 10 mg/kg per day, orally, for 10 weeks was able to control hypertension, as well as the oxidative and inflammatory status in the kidneys, as efficiently as losartan, returning mice to normotensive levels [97,103]. Furthermore, the acetonic fraction was more effective than losartan in preventing liver and kidney damage. Therefore, the fraction was able to control endothelial dysfunction and related diseases [97,103].

#### 4.3.3. Therapeutic Efficacy: Clinical Trials

As far as was found in the literature, no clinical studies have been conducted in patients using this plant as a single treatment. A clinical trial studied a commercial antioxidant supplement containing three components, including *Sechium edule*, showing an improvement of the hemorheology in alcoholics [106]. Based on available data, clinical studies are required on the use of *Sechium edule* in hypertension, diabetes mellitus, obesity, and, in general, in vascural-related diseases.

#### 4.3.4. Safety

Acute toxicity was tested in rats and mice that received a single oral dose of 2000 mg/kg of an aqueous leaf extract. Treated animals showed no change in the normal behavior pattern and no evidence of toxicity and mortality [102]. Negative effects on humans were not documented. In VigiBase® there are no reports of potential side effects related to *Sechium edule* [40], and the literature does not provide data on the safety of medicinal use during pregnancy or breastfeeding.

#### 4.3.5. Future Needs

Although traditional use and preclinical data suggest great interest in the use of *Sechium edule* in vascular diseases, the total absence of clinical studies strongly limits its use. Therefore, proper clinical trials are desirable.

#### *4.4. Terminalia chebula Retz.*

#### 4.4.1. Botanical Characteristics

*Terminalia chebula* (Combretaceae), also known as black myrobalan, is a deciduous tree that grows up to 30 m, widely known in India and Southern Asia for its use in Ayurvedic medicine [107,108]. Fruits are used in traditional medicine to treat various diseases, such as used as a laxative, stomachic, tonic, and antispasmodic [107,108].

#### 4.4.2. Phytoconsituents and Preclinical Activity

The main components of the fruit are phenolic compounds, such as hydrolysable tannins and flavonoids, saccharides, such as D-glucose, D-fructose, and saccharose [67]. The aqueous extract of *Terminalia chebula* fruits contains chebulagic acid, chebulinic acid, and other low molecular weight hydrolysable tannins [68]. The leaves contain polyphenols such as punicalin, punicalagin, terflavins B, C, and D [108].

The antioxidant activity of *Terminalia chebula* has been reported in vivo and in vitro assays [108–111]. A study described a significant decrease in glucose level in normal and alloxan-induced diabetic rats four hours after oral administration of a methanolic fruit extract (100 mg/kg) [110]. Similarly, *Terminalia chebula* fruit extract at a concentration of 200 mg/kg administered for 30 days significantly reduced blood glucose, glycosylated hemoglobin, urea, and creatinine levels in diabetic rats [112]. Furthermore, a cardioprotective effect of an ethanolic extract of fruits (500 mg/kg) was described in rats [113].

#### 4.4.3. Therapeutic Efficacy: Clinical Trials

Few trials have been reported in the literature on the potential therapeutic use of *Terminalia chebula*. A 12-week prospective trial showed that an aqueous extract of *Terminalia chebula* fruits administered at 250 mg and 500 mg, twice daily, significantly improved endothelial function, systemic inflammation, and lipid profile in 60 subjects with type 2 diabetes mellitus of either gender, compared to placebo treatment [66]. *Terminalia chebula* extract significantly increased NO• and GSH levels, reducing oxidative stress, malondialdehyde, and CRP levels [66]. Previously, the same authors reported similar beneficial effects in 56 patients of either gender with metabolic syndrome [68]. In this study, *Terminalia chebula* reduced malondialdehyde levels and increased glutathione levels, improving antioxidant status. Furthermore, the treatment significantly decreased total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and increased high-density lipoprotein cholesterol, while the placebo did not have a significant effect on endothelial function or any of the other clinical parameters [68].

#### 4.4.4. Safety

Treatment with *Terminalia chebula* extracts was well tolerated with very few side effects; in fact, very few patients experienced dyspepsia [66]. Other authors reported increased libido, dry mouth, colic, and confusion [114]. In general, the fruit preparations are welltolerated and do not adversely affect health [67,108]. In VigiBase®, *Terminalia chebula* has five reports of potential side effects, such as gastrointestinal disorders (i.e., diarrhea and gastrointestinal pain), and other general disorders [40].

#### 4.4.5. Future Needs

The few clinical studies that have evaluated the efficacy of *Terminalia chebula* fruit extracts suggest a potential use in the treatment of vascular dysfunction in diabetes mellitus and/or metabolic syndrome. Unfortunately, data are limited, and other studies are certainly necessary to determine the efficacy and safety of this medicinal plant.

#### *4.5. Plant-Derived Nanovesicles*

#### 4.5.1. General Characteristics

In recent years, various investigations have suggested that plant cells, through an exosome-like process, may release nanosized particles, which are involved in plant cellcell communication [115,116]. Furthermore, various studies suggest that plant-derived nanovesicles may also play a role in the properties of medicinal plants in human diseases, mainly based on their biological cargo [117–120]. It is assumed that the interaction between vegetal extracellular vesicles and mammalian cells may have beneficial effects through antioxidant and anti-inflammatory activities [121,122]. Plants produce nanovesicles in response to numerous biotic and abiotic environmental stresses, including pathogen infection and attack. Plant nanovesicles carry a wide variety of molecules, including proteins, lipids, miRNAs, vitamins, and various plant metabolites [123].

#### 4.5.2. In Vitro and In Vivo Studies

Lemon and strawberry-derived nanovesicles showed antioxidant activity in mesenchymal stem cells [124,125]. The authors verified the potential anti-osteoporotic effects of apple-derived nanovesicles using MC3T3-E1 cells, inhibiting osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells, by regulating the BMP2/Smad1 pathway [126]. Lemon-derived nanovesicles have been found to be rich in citric acid and vitamin C, which have a significant protective effect on oxidative stress in mesenchymal stromal cells [124].

Few studies have been carried out in animal models and humans. Oral administration of grape exosome-like nanoparticles showed beneficial effects in dextran sulfate sodium (DSS)-induced experimental colitis in mice, via induction of intestinal stem cells [120]. Similarly, broccoli-derived nanoparticles administered orally protected against various types

of mice colitis, through activation of adenosine monophosphate-activated protein kinase (AMPK) in dendritic cells [127]. Additionally, ginger-derived nanoparticles protected mice from alcohol-induced liver injury, activating nuclear factor erythroid 2-related factor 2 (Nrf2) and inhibiting ROS production [128].

#### 4.5.3. Therapeutic Efficacy: Clinical Trials

Very few trials considered the administration of plant-derived nanovesicles in the treatment of human diseases. In a prospective open-label study, 20 healthy volunteers (9 women and 11 men) were treated with a commercial preparation of extracellular vesicles from *Citrus limon* L., administered at 1000 mg daily for 3 months [69]. A decrease in waist circumference was found in women after 4 and 12 weeks of treatment, while no significant reduction was detected in men [69]. In the same study, the authors also observed a significant reduction in low-density lipoproteins (LDL) [69]. Significant correlations were also found in the stratified analysis between alkaline phosphatase enzymes (ALP) and glucose for women and between ALP and LDL for men [69]. A phase 1 clinical trial is currently underway studying the ability of a grape exosome preparation, administered orally for 35 days, to act as an anti-inflammatory agent against oral mucositis during radiation and chemotherapy treatment for head and neck tumors (NCT01668849) [129]. Another clinical study with ginger and aloe-derived exosomes studying the ability to mitigate insulin resistance and chronic inflammation in patients diagnosed with polycystic ovary syndrome was withdrawn because the investigator left the university before study approval (NCT03493984).

#### 4.5.4. Safety

As reported in the literature, there are no toxicity studies or reports of undesirable effects related to human administration of these types of preparations. The use of plantderived extracellular vesicles represents a new and very interesting approach in the treatment of diseases; however, other studies are needed to explore the advantages and, also, the disadvantages of plant-derived nanovesicles in therapy [129].

#### 4.5.5. Future Needs

Plant-derived nanovesicles are certainly an innovative type of plant preparation, which also has considerable industrial implications. However, there are many aspects to be validated, starting from the techniques for obtaining and the definition of the constituents, up to the possible uses in the prevention or treatment of human pathologies.

#### **5. Conclusions**

Based on the data collected, it can be observed that clinical studies concerning the use of products of plant origin in the treatment of human pathologies and, in particular, in cardiovascular diseases are few and consider only small groups of subjects. Furthermore, the studies generally do not examine the differences in treatment response comparing the female or male gender. In studies in which the efficacy of the products used were reported separately, women versus men, it was not possible to obtain evidence of the difference in efficacy because the number of subjects enrolled in the trials was too small to perform any statistical estimation.

Among the plants considered, garlic has been the most studied and there are several data on its effectiveness in the treatment of vascular-related disorders. However, the available data are insufficient to validate the pharmacological use of garlic preparations for any of the conditions under consideration. Additional research that recruits more patients is desirable. Some plants, such as *Campomanesia xanthocarpa*, *Sechium edule*, and *Terminalia chebula*, have been proposed for their potential use in vascular problems in diabetic or hypertensive subjects. Finally, a new type of innovative preparation based on plant-derived extracellular vesicles has been suggested, but this is only an idea that still requires long investigation. Importantly, greater attention must be paid in carrying out clinical trials with the aim of obtaining a personalized use of plant products, noting the differences in the effectiveness between women and men. Improved consideration of gender-based medicine is required to improve the efficacy of therapeutic interventions and reduce adverse reactions.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/life13040866/s1, Figure S1: Flowchart of criteria used in PubMed research.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


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### *Review* **Neuroprotective Potential of Biflavone Ginkgetin: A Review**

**˙ I. ˙ Irem Tatlı Çankaya 1, Hari Prasad Devkota 2, Gokhan Zengin <sup>3</sup> and Dunja Šamec 4,\***


**Abstract:** Neurological disorders are becoming more common, and there is an intense search for molecules that can help treat them. Several natural components, especially those from the flavonoid group, have shown promising results. Ginkgetin is the first known biflavonoid, a flavonoid dimer isolated from ginkgo (*Ginkgo biloba* L.). Later, its occurrence was discovered in more than 20 different plant species, most of which are known for their use in traditional medicine. Herein we have summarized the data on the neuroprotective potential of ginkgetin. There is evidence of protection against neuronal damage caused by ischemic strokes, neurotumors, Alzheimer's disease (AD), and Parkinson's disease (PD). Beneficial effects in ischemic strokes have been demonstrated in animal studies in which injection of ginkgetin before or after onset of the stoke showed protection from neuronal damage. AD protection has been the most studied to date. Possible mechanisms include inhibition of reactive oxygen species, inhibition of β-secretase, inhibition of Aβ fibril formation, amelioration of inflammation, and antimicrobial activity. Ginkgetin has also shown positive effects on the relief of PD symptoms in animal studies. Most of the available data are from in vitro or in vivo animal studies, where ginkgetin showed promising results, and further clinical studies should be conducted.

**Keywords:** Alzheimer's disease; biflavonoids; ginkgetin; neuroprotection; ginkgo

#### **1. Introduction**

Since 1840, human life expectancy has increased at a rate of nearly 2.5 years per decade, and this trend has continued to this day [1]. According to the World Health Organization [2], by 2030, 1 in 6 people in the world will be 60 or older. At that time, the proportion of the population aged 60 and over is estimated to increase from 1 billion in 2020 to 1.4 billion. By 2050, the global population aged 60 and older will double (2.1 billion). Similarly, the number of people aged 80 or older is estimated to triple between 2020 and 2050, reaching 426 million. On a biological level, aging is a complex process in which a variety of molecules and cellular damage accumulate, leading over time to a gradual decline in physical performance and cognitive functions, as well as an increased risk of disease. Older people are more susceptible to various chronic diseases, especially diseases of the central nervous system, such as strokes, epilepsy, Parkinson's disease (PD), Alzheimer's disease (AD), neuropathy, and other dementias [3]. Neurological disorders are disorders of the nervous system and can affect the activity and physiology of the brain, spinal cord, and nerves. They occur in 5% to 55% of people who are aged 55 and older and are associated with a high risk of adverse health effects, including mortality, disability, and hospitalization [3]. Therefore, scientists have made considerable efforts to understand the pathophysiology of these disorders and to develop effective prevention methods and therapies. However, the reported compounds/active ingredients, which are mostly synthetic, are not considered to be very reliable and therapeutically effective due to their complexity and off-target problems [4]. On the other hand, several natural products

**Citation:** Tatlı Çankaya, ˙ I.˙ I.; Devkota, H.P.; Zengin, G.; Šamec, D. Neuroprotective Potential of Biflavone Ginkgetin: A Review. *Life* **2023**, *13*, 562. https://doi.org/ 10.3390/life13020562

Academic Editors: Salvador González and Azahara Rodríguez-Luna

Received: 19 December 2022 Revised: 9 February 2023 Accepted: 11 February 2023 Published: 16 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

may prove to be viable preventive therapeutics to fill the large gap in the treatment of neurological disorders [4].

One of the groups of natural products associated with neuroprotective properties is the flavonoids [5–7], a large and diverse group of specialized plant metabolites characterized by a 15-carbon flavone backbone (C6-C3-C6) with two benzene rings (A and B) linked by a trinuclear pyran ring (C) [8]. Flavonoids can be mainly divided into six groups: flavones, flavonols, flavan-3-ols, flavanonols, flavanones, isoflavones, and anthocyanins. They can be present in free form in plants, but are more often glycosylated, methylated, acetylated, prenylated, or polymerized [9]. The pattern of conjugation, glycosylation, or methylation is responsible for the different chemical and biological properties of these compounds [10]. Flavonoid dimers known as biflavonoids are formed by two linked flavonoid monomers and consist of flavone-flavone, flavane-flavane, flavane-flavone subunits, and in rare cases, dimers of chalcones and isoflavones. Today, nearly 600 different biflavonoids are known to occur in ferns, bryophytes, angiosperms and gymnosperms [11,12]. They are often found in plants used in traditional medicine and are considered responsible factors in the health benefits of these plants [13]. Biflavonoids possess diverse biological activities including therapeutic potential against neurodegenerative diseases [14].

The first biflavonoid isolated was ginkgetin from the yellow leaves of ginkgo (*Ginkgo biloba* L.) (Figure 1a). Chemically, it is a 7,4 -dimethyl ether derivative of the 3 ,8"-dimer of the apigenin, known as amentoflavone. Thus, ginkgetin consists of apigenin and apigenin 7,4 -dimethyl ether.

**Figure 1.** Yellow leaves of *Ginkgo biloba* L. (**a**) and the chemical structure of ginkgetin (**b**).

Ginkgetin is a compound found in ginkgo whose standardized extract (EGb 761) has been used for many years as a supportive therapy and to prevent cognitive impairment [15]. Ginkgo extract can slow the progression of memory loss in AD, usually at a high dose of 240 mg or more per day [16,17], and may have supportive and/or protective effects in the treatment of PD [18]. It is not entirely clear which molecules from the extracts contribute to this activity. Recently, natural products, especially polyphenols, have been intensively studied as potential neuroprotective molecules. One of these molecules is ginkgetin, but as far as we know, there is no review paper summarizing the research to date. Therefore, the review aimed to summarize the data on the potential of ginkgetin in the treatment of neurodegenerative diseases in order to highlight the neuroprotective properties of ginkgetin.

#### **2. Ginkgetin**

Ginkgetin (Figure 1b) is a flavonoid dimer, a 7,4 -dimethyl ether derivative of the apigenin dimer amentoflavone. It is the first isolated biflavonoid obtained in the form of a yellow powder from the leaves of ginkgo (Figure 1a) and the first biflavonoid whose structure was described. To date, its occurrence has been confirmed in other ginkgo plant parts [13,19], as well as in more than 20 other plant species [20]. The list of plant species in which the presence of ginkgetin was detected is given in Table 1. It should be noted that the presence of ginkgetin in mosses and liverworts has not been yet reported, and in ferns and fern allies it has been reported only in *Sellaginela* sp. It is commonly found in the conifers, cycads and allies group, and in flowering plants. So far, it has not been found in plants commonly used as food, but rather in plants used in traditional medicine.

**Table 1.** List of plant species with associated division in which the presence of ginkgetin has been reported.


Most of the plants listed in Table 1 have been used in the traditional medicine systems of various cultures, suggesting that ginkgetin also may have biological activity. As the first known biflavonoid, its biological activity has been studied over the last 30 years. Research shows its potential in treating various inflammation-related diseases such as cancer, cardiovascular disease, inflammation caused by viruses and bacteria, and neurodegenerative disorders [20] (Figure 2).

Most commonly, its anticancer activity has been studied. Recently, Adnan et al. [20] summarized that ginkgetin combats cancer progression by various mechanisms such as arresting the cell cycle, inducing apoptosis, stimulating autophagy, and targeting many deregulated signaling pathways such as JAK / STAT and MAPKs in the colon, lung, prostate, osteosarcoma, breast, leukemia, cervical, medulloblastoma, ovarian, neck, and kidney cell lines. In animal studies, ginkgetin inhibited tumor growth in xenotransplanted nude mice, down-regulated p-STAT3Tyr705 and survivin in tumor tissues [46] and decreased tumor size and weight without apparent toxicity [47]. Ginkgetin may also enhance the therapeutic effects of cisplatin [48] and 5-fluorouracil [49].

**Figure 2.** Biological activity of ginkgetin.

It may also be useful for the treatment of cardiovascular disease. Cell-based studies showed its potential as an inhibitor of TRPV4-mediated proatherogenic processes in macrophages [50]. In addition, ginkgetin showed inhibitory effects on human thrombin, an important serine protease that regulates the blood coagulation cascade and processes of thrombosis [51], and pancreatic lipase, an important target that regulates lipid uptake [52]. Animal studies showed its beneficial effects in preventing adipogenesis [53], local vascular damage associated with atherosclerosis [54], and ischemic reperfusion injury [55].

Due to its anti-inflammatory effects, in vitro studies have shown that ginkgetin can be used in the treatment of inflammation-related diseases such as airway inflammation [56] and diabetic nephropathy [57]. It may also be useful as an antiviral [32], antibacterial [30] and antiparasitic [58] agent and has gained attention in recent years as a target for the treatment of SARS-CoV-2 infection [59–61].

In this review, we address in more detail the potential role of gingketin in the treatment of neurodegenerative diseases.

#### **3. Neurodegenerative Diseases**

Neurodegenerative disease is a general term for several diseases mainly characterized by a progressive loss of structure or function of neurons that worsens over time [62]. They can be genetic or caused by a tumor, stroke, toxins, viruses, etc., and affect millions of people around the world. Neurodegenerative disorders are caused by various conditions such as abnormal protein dynamics with defective protein aggregation and degradation and aggregation, impaired bioenergetics and mitochondrial dysfunction, excessive free radical formation leading tooxidative stress, and exposure to environntal toxicants such as heavy metals and pesticides [63] (Figure 3).

Neuroprotection is defined as the ability of a specific molecule to prevent neuronal cell death by interfering with and inhibiting the pathogenetic cascade that leads to cell dysfunction and eventual death [64]. AD and PD are the most common neurodegenerative diseases, but others can cause serious problems for individuals and society, and lead to significant healthcare costs. These diseases are known that these diseases cause irreversible cognitive dysfunctions in individuals. It is therefore extremely important for an effective treatment strategy to slow down the prognosis of neurodegenerative diseases by diagnosing them at the earliest possible stage. AD, PD, Huntington's disease (HD), and other neurodegenerative disorders share common features at the cellular and subcellular levels, and utilize similar molecular signaling pathways that can lead to inflammation, apoptosisnecroptosis,

etc. These diseases are the consequence of misfolding and dysfunctional trafficking of proteins (Figure 3), mitochondrial dysfunction, oxidative stress, and/or environmental factors [63].

**Figure 3.** Multifactorial conditions causing neurodegenerative diseases and examples of diseases with the main site of pathology and proteins whose degradation and aggregation cause the pathology.

AD is a highly complex disorder characterized by severe synaptic losses and neuronal death, especially in regions with cognitive functions such as the cerebral cortex, hippocampus, entorhinal cortex, and ventral striatum [65]. Generally, in an average of 10 years, the stage of mild cognitive impairment passes to the advanced stage of AD, and the patient is lost in a completely helpless state at the end of this period. Due to the long duration of the disease and the fact that it affects the vital structures that determine who we are, it creates a great emotional and financial burden on patients' relatives and society [66]. Since the pioneering work of Alois Alzheimer in 1907, neuropathologists have identified amyloid plaques and NFTs in the brains of patients in autopsy examinations and stated that these pathologies cause the disease [67]. Amyloid plaques have been found to be extracellular deposits of amyloid-beta (Aβ) found in the brain parenchyma and cerebral blood vessels. The NFTs observed in the cell were found to consist of hyperphosphorylated tau protein associated with microtubules and clustered in helical filaments [68]. Additional pathological data for amyloid plaques and NFTs can be listed as intracellular granulovacuolar degeneration, decrease in the number of synapses, cholinergic cell losses in Meynert's basal nucleus, and astroglial activation [69]. Studies conducted to understand AD indicate that the disease arises as a result of complex interactions of many genetic, epigenetic, and environmental factors [69,70]. The main histopathological findings observed in the brain parenchyma of the patients have extracellularly located amyloid plaques, neurofibrillary structures consisting of intracellular tau protein clusters, glial activation, and traces of inflammation [71]. Based on these symptoms, many mechanisms have been proposed for the pathogenesis of the disease. The main ones can be listed as the amyloid cascade hypothesis, cholinergic damage hypothesis, neuronal cytoskeleton hypothesis, and oxidative stress hypothesis [72,73]. Other more debatable AD hypotheses are: inflammatory hypothesis, vascular hypothesis, cholesterol hypothesis, metal hypothesis, and cell cycle hypothesis [74].

PD is a progressive neurodegenerative disease that causes involuntary or uncontrollable movements such as tremors, stiffness, balance and coordination problems [75]. Symptoms usually begin insidiously and worsen over time. Degeneration of neurons in the compacta part of substantia nigra and the presence of Lewy bodies in its cytoplasm are the classic pathological findings of the disease [76]. Over the years of PD progression, a picture of dementia can develop that can be severe and debilitating, overshadowing the movement disorder of the disease. Dementia is defined as the presence of impairments in more than one cognitive domain, such as attention, memory, language, executive functioning, practice, and visuo-spatial functioning [77]. These losses reflect a marked decline from previous levels, and this decline is severe enough to interfere with daily, occupational, and social life. PD dementia is a mild or moderate dementia that begins insidiously, progresses slowly, affects some areas of cognitive function, especially executive function, and often develops psychosis during its course. The mechanisms underlying the pathogenesis of PD are still unclear, but there are several proposed mechanisms, such as those related to mitochondrial dysfunction, oxidative stress, ubiquitin-proteosome system, neuroinflammation, excitotoxicity, iron ion accumulation, and genetic issues [78].

HD is an autosomal dominant genetic disorder mainly characterized by progressive motor dysfunction, cognitive decline, and behavioral symptoms [79]. Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disorder that is characterized by a progressive loss of motor neurons of the CNS leading to muscle weakness, wasting, and spasticity. Patients develop progressive muscle weakness along with fasciculation and hyperreflexia. Mild cognitive deficits and frontotemporal dementia (FTD) are common. FTD is characterized by progressive deficits in executive function, behavior, and language.

Neurodegenerative diseases also share some common pathological features such as the accumulation of characteristic proteins in insoluble aggregates within and/or between neurons and the loss of synapses and death of neurons [80]. These proteins include β-amyloid (Aβ) of senile plaques and tau of neurofibrillary tangles (NFTs) in AD, α-synuclein (α-syn) of Lewy bodies (LBs) and Lewy neurites in PD, polyglutamine (PolyQ) rich huntingtin inclusions in HD, TDP-43 aggregates in ALS, and TDP-43 aggregates and tau in FTD (Figure 3). In line with the above-mentioned explanations, neurodegenerative diseases occur with the folding and proteasomal disorders of certain proteins due to environmental or genetic reasons, followed by active glial cells secreting various mediators, including proinflammatory cytokines [80]. This whole process repeats each other in a vicious circle, causing apoptosis and developing neuroinflammation. This mechanism is the underlying cause of all diseases. Therefore, it is necessary to develop treatment strategies against neuroinflammation. These may include the use of natural sources or their secondary metabolites with anti-inflammatory effects.

#### **4. Ginkgetin for the Treatment of Neurodegenerative Diseases**

#### *4.1. Oxidative Stress Mediation*

Oxidative stress is a common trigger that can be associated with the development of neurodegenerative diseases, so compounds with antioxidant activity can be considered beneficial for the development of these diseases. Although the physiological cause of aging is not fully known, the free radical theory states that increasing oxidative stress of aging and aging-related diseases plays a fundamental role in this process by causing cellular degeneration. The increase in the number of free radicals observed in age-related neurodegenerative diseases and the fact that neurons are more sensitive to this damage have both been determined to be important characteristics. Therefore, it is thought that free radical production has an important role in the development and progression of neurological diseases [81]. Neurons are more susceptible to free radical damage [82] for certain reasons shown in Figure 4.

**Figure 4.** Neurons are more susceptible to free radical damage because of differences in some parameters and biological functions. Higher parameters are marked with *↑*, lower parameters with *↓*.

Flavonoids are widely recognized as a molecule with good antioxidant activity and beneficial effects in the treatment of neurological disorders [5–83]. Li et al. [84] measured the antioxidant activity of ginkgo leaves from plants grown in different locations and reported that ginkgetin content in the leaves resulted in stronger antioxidant activity and concluded that ginkgetin together with isoginkgetin were the two most active constituents with a strong relationship with antioxidant activity. Several cell-based studies have shown that ginkgetin plays a role in oxidative stress and that ginkgetin can protect fibroblasts from UVBinduced cytotoxicity [85], alleviate oxidative stress induced by H/R injury [86], inhibited NO production from lipopolysaccharide (LPS)-induced RAW 264.7 cells [87] and reduced oxidative stress caused by hyperglycemia [57]. However, reports about the antioxidant activity of ginkgetin are contradictory. Bedir et al. [40] who compared the antioxidant activity of 29 compounds isolated from *G. biloba* reported that ginkgetin was the least potent antioxidant after amentoflavone, showing only 19% inhibition at a concentration of 62.5 μg/mL. In the same study, monomeric biflavonoids had an IC50 of less than 10 μg/mL, clearly indicating that ginkgetin itself is not a molecule with antioxidant potential. Kang et al. [14] tested protective effect of biflavonoids on H2O2-induced cell death in SH-SY5Y (triple subcloned cell line derived from SK-N-SH neuroblastoma) and showed that all biflavonoids tested, including ginkgetin, significantly reduced H2O2-induced cell death. Furthermore, they tested the antioxidant activity of using the well- known 1,1-diphenyl-2 picrylhydrazyl (DPPH) assay, and none of the nine biflavones showed radical scavenging activity at concentrations up to 100 μM. These results suggest that the neuroprotection of biflavonoids may be mediated by direct blockade of cell death cascades rather than by their antioxidant activity. Therefore, they investigated the neuroprotective effects of biflavonoids against the cytotoxic insult induced by staurosporine. Staurosporine is known to mediate apoptosis via the caspase-dependent mitochondrial pathway. Ginkgetin significantly reduced cell death induced by staurosporine at a concentration of 10 μM. They therefore suggest that neuroprotection by biflavonoids is mediated in part, if not completely, by direct blockade of the signaling events that lead to apoptosis during cellular stress. Jeong et al. [31] investigated the neuroprotective effects of four biflavonoids, including ginkgetin, using mouse HT22 hippocampal cells, a model system for studying glutamate-induced oxidative stress. They reported that ginkgetin can protect HT22 neuronal cells from glutamateinduced oxidative damage by preserving the activities of antioxidant enzymes and/or inhibiting ERK1/2 activation.

This example shows that commonly used methods for determining antioxidant activity, such as DPPH, are not ideal for predicting the ability of compounds to reduce oxidative stress at the cellular level and even more so at the tissue or organism level.

#### *4.2. Protection against Neuronal Injury Caused by Ischemic Stroke*

Ample evidence has supported the role of neuroinflammation in the development of neurological disorders. Inflammatory components such as astrocytes, microglia, the complement system, and cytokines have been associated with neuroinflammation in the CNS. In particular, inflammatory cytokines have been found to play a central role in neuroinflammation pathway as a large numberof studies have reported abnormally elevated levels of interleukin-1β (IL-1β) and tumor necrosis factor (TNF) in AD and PD patients (reviewd by [88]).

A common method to study the antineuroinflammatory potential of certain molecules is to use animal models exposed to neuronal injury caused by ischemic stroke. Most ischemic strokes occur in the middle cerebral artery territory, so many of the animal models of stroke that have been developed have focused on this artery. In the intraluminal monofilament model of middle cerebral artery occlusion (MCAO), a surgical suture is inserted into the external carotid artery and advanced into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in interruption of blood flow and subsequent cerebral infarction in the MCA area [89]. This model has been used also for the study of the effects of ginkgetin (Figure 5.).

**Figure 5.** Ginkgetin protection against neuronal injury caused by ischemic stroke.

In a study by Xu et al. [90] animals received ginkgetin at concentrations of 100 and 200 mg/kg i.p. five days prior to induction of MCAO, and they investigated the effect of ginkgetin against stroke. Treatment with ginkgetin attenuated the increased neurological score and decreased the water content in the brain. Ginkgetin-treated rats showed that the levels of pro-inflammatory cytokines NF-κB, IL-1β, and TNF-α were significantly decreased in brain tissue. The authors concluded that ginkgetin aglycone improved the PI3K/NF-κB/ TLR-4 inflammatory pathway. Tian et al. [91] used a transient MCAO procedure to establish the cerebral ischemia/reperfusion model (IR) in rats. Ginkgetin was injected at doses of 25, 50, and 100 mg/kg 2 hours after the onset of ischemia and its administration markedly reduced the volume of cerebral infarction and neurologic deficits. It also reduced the number of apoptotic cells, decreased the amount of cleaved caspase-3 and Bax, and increased the amount of Bcl-2 in rats exposed to IR injury in a dose-dependent manner. In addition, high-dose ginkgetin treatment (100 mg/kg) significantly increased the phosphorylations of Akt and mTOR. Blocking PI3K by LY294002 significantly decreased the antiapoptotic effect and reduced both Akt and mTOR phosphorylation levels. According to the authors, ginkgetin counteracts cerebral IR-induced injury by inhibiting apoptosis in rats, and this effect was attenuated by activation of the PI3K/Akt/mTOR pathway. The same experiment procedure and the same ginkgetin concentration was used by Pan et al. [92] who reported that ginkgetin attenuated I/R-induced autophagy activation, pyramidal neuron death in cerebral I/R, and reduced I/R-induced upregulation of p53. They concluded that ginkgetin can attenuate cerebral ischemia/reperfusion-induced autophagy and apoptosis by inhibiting the NF-κB/p53 pathway. Some other researchers [93] used oxygen glucose deprivation (OGD) cellular and MCAO animal models to study neuroprotective activity of ginkgetin reported that ginkgetin treatment converted microglia from M1 type to M2 type and inhibited neuroinflammation. Detailed study of the neuroprotective mechanism suggested that ginkgetin can inhibit neuroinflammation by promoting M2 polarization of microglia through PPARγ signaling pathway thus promoting recovery of neurological functions in an ischemic stroke.

#### *4.3. Activity against Neurotumors*

Different *in vitro* and *in vivo* studies showed that ginkgetin combats cancer progression by arresting the cell cycle, inducing *via* apoptosis, stimulating autophagy, and targeting many deregulated signaling pathways such as JAK/STAT and MAPKs (reviewed by Adnan et al. [20]). Ginkgetin was also studied as a potential agent for the treatment of neurotumores by Ye et. al. [26], who investigated the potential of natural products in the treatment of medulloblastoma (MB), a form of malignant brain tumor that occurs predominantly in infants and children and in which approximately 25% is due to upregulation of the canonical Wnt pathway, with mutations mainly in CTNNB1. They screened for antagonists of Wnt signaling from 600 natural compounds and identified ginkgetin as a potential molecule that showed marked cytotoxicity. Ginkgetin efficiently induced G2/M phase arrest in Daoy cells, reduced the expression of Wnt target genes, including Axin2, CyclinD1, and Survivin in MB cells, and decreased the phosphorylation level of β-catenin. They concluded that ginkgetin is a novel inhibitor of Wnt signaling and, as such, warrants further exploration as a promising candidate against medulloblastoma.

#### *4.4. Protective Effect against Alzheimers' Disease*

AD is caused by multiple mechanisms such as excessive accumulation of extracellular amyloid-beta 42 (Aβ42) plaques, intracellular hyperphosphorylated tau neurofibril tangles in the brain, oxidative stress due to mitochondrial dysfunction, and/or genetic as well as environmental factors [94]. Aggregation and accumulation of amyloid-β plaques and tau proteins in the brain are central features in the pathophysiology of AD and are therefore the focus of most research investigating potential therapeutics for this neurodegenerative disease [95]. Kang et al. [14] investigated whether biflavones showed protective effects against Aβ-induced cytotoxicity using SH-SY5Y (triple subcloned cell line derived from SK-N-SH neuroblastoma) cells and found that ginkgetin showed protective effects at 2 μM, with an inhibition percentage of 43.6%. In the same study they tested protective effects against neuronal cell death induced by a DNA-damaging agent, etoposide, but gingetin did not show a protective effect.

Amyloid-β-42 (Aβ42) is proteolytic derivative of the large transmembrane protein amyloid precursor protein (APP) and it plays an early and important role in all cases of AD [96]. Thus, blocking Aβ42 production by specific inhibition of key proteases required for Aβ42 formation is a major focus of AD therapy research. β-Secretase, the aspartic protease that generates the N-terminus of Aβ42, has become a major target and researchers are focused on discovering its inhibitors [96]. Sasaki et al. [97] examined the activity of twenty-one bioflavonoids against β-secretase and ginkgetin showed a significant inhibitory effect with an IC50 value of 4.18 μM. The authors indicated that the importance of the position of hydroxyl groups in two apigenin molecules for the inhibition of β-secretase and the presence of hydroxyl groups in the C3 and C8" position might enhance the inhibitory effects. Ullah et al. [98] reviewed β-secretase inhibitors from plant sources and, among them, ginkgetin was a significant inhibitor with a low IC50 value. In an in silico

study performed by Grewal et al. [99], ginkgetin showed a good binding potential on N-methyl-D-aspartate glutamate receptor (NMDA) and beta secretase-1 (BACE-1), and was suggested as a neuroprotective agent. In another study conducted by Choi et al. [100], eight amentoflavone-like bioflavonoids were tested to inhibit amyloid-beta fibrillation and to disaggregate amyloid-beta fibrils. In the study, the IC50 value of ginkgetin was 4.92 μM in the inhibition of Aβ fibrils assay. In the same study, ginkgetin exhibited a disaggregation effect on Aβ fibrils with the IC50 value of 6.81 μM.

Zeng et al. [101] studied ginkgetin therapeutic potential against AD using a transgenic mouse model of AD, PS1dE9/APPS mice. Prior to the onset of AD-type neuropathology, mice were randomly assigned to four diet groups: ginkgetin group, curcumin group, normal diet group, with wild-type littermates used as a control group. All animals were fed with the above diets for 9 months. The mean daily food consumption of the mice was 0.08– 0.12 g/g body weight and the corresponding daily ginkgetin and curcumin were about 200 and 80 mg/kg/day based on a previous report indicating lack of toxicity. The equivalent consumption in a 60 kg human is about 0.91 g/day for ginkgetin and 0.35 g/day for curcuminas. In their experiments they showed that ginkgetin effectively reduced the Aβ levels in the brain and blood, decreased cerebral microhemorrhage, lowered astrogliosis, and ameliorated inflammation in APP/PS1 transgenic model, which indicates in vivo therapeutic potential of ginkgetin against AD. However, as the authors stated, pathophysiology mechanisms of Aβ clearance need further research.

It has been noted that the development of amyloid-β plaques occurs about 10–20 years before the manifestation of AD symptoms, thus the earlier interventions are necessary to address presymptomatic AD. Studies suggesting that amyloid-β peptides may play a role in innate immunity as antimicrobial peptides indicate that the buildup of amyloid-β plaques may be a response to the presence of viruses and bacteria [94]. This has led to the establishment of the antimicrobial hypothesis for AD and the use of antimicrobial and antiviral drugs as potential therapeutics targeting the root cause of AD. Biflavonoids are in the focus of the sciences as a potent antimicrobial, expecially antiviral agents [13,94] where ginkgetin stands out as a compound with antiviral capabilities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) [25], cytomegalovirus (HCMV) [25], influenza A virus [32], and SARS-CoV-2 virus [59,60,102]. Ginkgetin also shows antifungal activity against *Alternaria alternate, Cladosporium oxysporum*, and *Fusarium culmorum* [35], and antibacterial activity against *Streptococcus suis* [103] (Figure 6).

**Figure 6.** Antimicrobial activity of ginkgetin.

At this point, researchers have not linked any specific bacterium or virus alone to the development of AD. Thus, a number of viruses and bacteria may be involved in the progression of neurodegenerative diseases independently or simultaneously with other pathogens. Given the good antimicrobial, especially antiviral, activity of ginkgetin, its potential role in mechanisms related to the antimicrobial hypothesis for AD is worthy of future research.

Considering all these results, it is obvious that ginkgetin has potential for the treatment of AD, but further studies should be performed to confirm this activity, especially in a clinical trial. Possible mechanisms of ginkgetin related to the protection of AD are shown in Figure 7.

**Figure 7.** Biological activity of ginkgetin related to protection against AD.

#### *4.5. Protective Effect against Parkinson' Disease*

PD is reported to be the second most common neurodegenerative disorder after AD [104]. Therefore, great efforts have been made to search for new molecules that could be effective in the treatment of PD. Although there are several in silico and in vitro models for finding new active molecules, our literature search did not yield any results related to ginkgetin. The animal model commonly used is a model where PD is induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is the gold standard for researchers in order to induce all aspects of PD hallmarks in animal model of the disease [104]. Wang et al. [105] investigated the neuroprotective ability of ginkgetin in vivo in a model of PD induced by MPTP. Animals received ginkgetin (80 mg/100 g body weight) via the stomach for 5 days and then were injected intraperitoneally with MPTP (20 mg/kg) once daily for 5 days. The authors showed that ginkgetin significantly improved sensorimotor coordination in a mouse model PD by dramatically inhibiting the decline in tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. They reported that ginkgetin can strongly chelate iron ions, thereby inhibiting the increase in intracellular labile iron pool by downregulating L-ferritin and upregulating transferrin receptor 1, suggesting that the neuroprotective mechanism of ginkgetin against neurological damage induced by MPTP is via the regulation of iron homeostasis. In another animal study [106], mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (25 mg/kg) and probenecid (250 mg/kg) for five consecutive days to induce PD. Ginkgetin (5, 10, 20 mg/kg) and bromocriptine (10 mg/kg), which is used to treat PD, were administered orally to PD mice for 26 days, including a five-day pretreatment period. The authors reported that in MPTP-induced PD mice, movements and muscle

functions improved with ginkgetin. The number of tyrosine hydroxylase-positive cells was reduced and later recovered without degeneration. The level of glial fibrillary acidic protein (GFAP) decreased, while the level of brain-derived neurotrophic factor (BDNF) increased significantly after treatment with ginkgetin. In summary, the authors concluded that ginkgetin effectively protects dopaminergic neurons by reducing oxidative damage, activating microglia, and increasing neurotrophic potential, indicating that it is a potential candidate for the treatment of PD.

#### **5. Conclusions**

Ginkgetin is the first biflavonoid isolated from ginkgo, after which it was named. All biflavonoids belong to the flavonoid group, well-studied specialized metabolites from plants, but they are much less studied compared to monomeric flavonoids. In this review, we have summarized the available data on the neuroprotective potential of ginkgetin. The available data are in vitro studies or in vivo animal studies, and as far as we know, there have been no clinical studies performed as yet. There is evidence of protection against neuronal damage caused by ischemic stroke, neurotumors, AD, and PD, but further studies and clinical trials should explain the mechanisms of action and the effective and safe concentration of ginkgetin for clinical use. The focus of future research should be primarily on the potential to cross the blood-brain barrier, as there is currently a lack of information in this regard.

**Author Contributions:** Conceptualization, D.Š.; writing—original draft preparation, D.Š., ˙ I.˙ I.T.Ç., H.P.D. and G.Z.; writing—review and editing, D.Š., ˙ I.˙ I.T.Ç., H.P.D. and G.Z.; visualization, D.Š.; supervision, D.Š.; project administration, D.Š.; funding acquisition, D.Š. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been supported by Croatian Science Foundation project "Biflavonoids role in plants: *Ginkgo biloba* L. as a model system" under the project no. UIP-2019-04-1018.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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