The Potential of Ginkgo biloba as a Source of Biologically Active Compounds—A Review of the Recent Literature and Patents

Ginkgo biloba is a relict tree species showing high resistance to adverse biotic and abiotic environmental factors. Its fruits and leaves have high medicinal value due to the presence of flavonoids, terpene trilactones and phenolic compounds. However, ginkgo seeds contain toxic and allergenic alkylphenols. The publication revises the latest research results (mainly from 2018–2022) regarding the chemical composition of extracts obtained from this plant and provides information on the use of extracts or their selected ingredients in medicine and food production. A very important section of the publication is the part in which the results of the review of patents concerning the use of Ginkgo biloba and its selected ingredients in food production are presented. Despite the constantly growing number of studies on its toxicity and interactions with synthetic drugs, its health-promoting properties are the reason for the interest of scientists and motivation to create new food products.


Introduction
The fossil remains of plants of the Ginkgoaceae family are well known to paleobotanists: representatives of this family lived 300 million years ago (in the Permian period), and they achieved the greatest importance in the Jurassic period (200 million years ago). Currently, only Ginkgo biloba L. (Figure 1) is a naturally occurring species in this group. This plant survived the mass extinctions in the Cretaceous and Paleogene periods and the Pleistocene glaciation, becoming a relict and endemic species in China [1].
Molecules 2023, 28, x FOR PEER REVIEW 2 of 53 Ginkgo biloba leaf extract is one of the best-selling herbal remedies in the world and the most-sold herbal supplement in the US and Europe. It has great therapeutic potential, including scavenging free radicals, reducing oxidative stress, as well as reducing damage to the nervous system and reducing platelet aggregation. It also has anti-inflammatory, anti-cancer and anti-aging properties. Clinical studies also confirm its beneficial effects, among others, in the treatment of central nervous system disorders-Alzheimer's disease and cognitive deficits [5].
The aim of the publication is to analyze the results of research mainly from 2015-2022 on the chemical composition of Ginkgo biloba and its biological activities, as well as toxicity and interactions with drugs. This publication contains a section devoted to the analysis of granted patents in the field of innovative possibilities of using Ginkgo biloba in the production of food and beverages.
Proanthocyanidins constitute 4-12% of ginkgo leaves, and standardized extracts contain 7% of proanthocyanidins. Although studies on the composition of these compounds are still ongoing, it has already been shown that proanthocyanidins and flavan-3-oils have antioxidant activity and the ability to scavenge free radicals. Moreover, they alleviate ischemic-reperfusion damage conditions and exhibit antihypertensive, anti-atherosclerotic and anti-aggregating, immunomodulating, antiseptic and anti-inflammatory effects [17].

Polyprenols
Polyprenols consist of 12-20 cis-and two trans-isoprene units and one form of betulaprenol and are terminated in an isoprene unit (having a hydroxyl group). Polyprenols mainly occur as a mixture of homologs in the photosynthetic organs of plants and have a similar structure and composition to dolichols [24]. Ginkgo biloba leaf polyprenols are weakly polar unsaturated polyisoprenoid alcohols found in leaf lipids. There they occur mainly in the form of polyprene acetate [25]. Ginkgo leaf polyprenols exhibit antioxidant, immunomodulating, anti-bacterial, anti-viral, antitumor and hepatoprotective properties [24][25][26].

Polysaccharides
Among the many bioactive compounds found in ginkgo, there are also polysaccharides. Purified polysaccharides are obtained from ginkgo by extraction methods (hot water extraction, ultrasound-assisted and enzymatic extraction) and by purification methods (including ion exchange chromatography and gel filtration). Large amounts of structurally diverse polysaccharides, mainly in terms of monosaccharide composition, were isolated from both leaves, sarcotesta and seeds. However, most of them consist of rhamnose (Rha), galactose (Gal), mannose (Man), xylose (Xyl), arabinose (Ara), glucose (Glu) and fucose (Fuc) with different mole fractions of the individual components [11,27]. Ginkgo seeds are a rich source of mannose (Man), and the sarcotesta contains more galactose (Ga) and glucose (Glu) compared to leaves. Interestingly, the molecular weight of Ginkgo biloba polysaccharides shows a varied distribution ranging from 1.0 kDa to 5679 kDa [27].

Alkylphenols and Alkylphenolic Acids
Alkylphenols occurring in the leaves of Ginkgo biloba can be divided into five groups: cardanols, α-hydroxycardanols, cardans, urushiols and isourushiols, and a group of alkylphenolic acids, which include ginkgolic acids. These compounds are among the toxic ingredients of Ginkgo biloba. Ginkgolic acid occupies a special place here, as it is considered to be toxic, mutagenic and sensitizing. However, despite their negative effect, a beneficial pharmacological effect on the human body was also shown for example, ginkgolic acid C17:1 in studies showed various antitumor effects [6].

Other
Sixty-eight chemical compounds have been identified in the composition of essential oils obtained from Ginkgo biloba leaves [30], among which the largest percentage was sesquiterpenes (42.11%) [7].
Ginkgo seeds and leaves are a source of vitamins B, C and E. Among the minerals, a relatively high content of zinc, iron, sodium, magnesium, potassium, calcium, carbon and nitrogen was found. According to research by Pereira et al. (2013) [20], among the macronutrients, carbohydrates (72.98 g/100 g dw) have the largest share in Ginkgo biloba leaves. The protein content is 12.27 g/100 g dw, the ash content is 12.27 g/100 g dw, and the fats have the smallest share-4.75 g/100 g dw. The content of free sugars such as fructose, glucose and sucrose was 1.42; 0.78; 0.23 g/100 g dw, respectively, a total of 2.43 g/100 g dw free sugars.

Structure and Biosynthesis of Ginkgolides and Bilobalides
Ginkgolides have a similar molecular formula. Some have the notation C 20 H 24 O (A, B, C, M, J, P, Q, N), and some C 20 H 22 O (K, L) ( Table 2) [34,40,43,44]. These compounds have a rare group in natural products. These are six 5-membered rings, including a spiro [4,4] carbocyclic nonane ring, three lactones, a tetrahydrofuran ring, and a tert-butyl moiety. All ginkgolides have a similar structure and differ in substituents R1, R2 and R3, which are permutations of H or OH [114].
Bilobalide was first isolated by a Major in 1967 [115]. It is characterized by a tendency to isomerization under mild acylation conditions to form diacyl derivatives of the spiro compound. Bilobalide, also belonging to the diterpenoids, also have a tert-butyl group.
Although the health-promoting properties of the terpenoids found in ginkgo have been thoroughly investigated, their biosynthesis is still not fully understood. One of the enzymes most often involved in ginkgolides biosynthesis is Gb LPS (levopimaradiene synthase), a diterpene synthase that catalyzes the synthesis of levopimaradiene (2)-possibly the precursor to all ginkgolides. Ginkgolides and bilobalides share a common three-step biosynthetic pathway. The first step involves the biosynthesis of two simple five-carbon units that build the isoprene skeleton of isopentyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In the second step, there is a repetitive condensation of IPP and DMAPP towards farnesyl precursors (FPP) and geranylgeranyl diphosphate (GGPP). This is followed by late cyclization and oxidation steps catalyzed by terpenoid synthases and cytochrome P450 (CYP-450) dependent monooxygenases, which define the specific carbon backbone and oxidation pattern of the product [114].
The cytosolic pathway of mevalonic acid in the cytosol, from 3-acetyl-CoA to IPP, is responsible for the synthesis of sesquiterpenoids and sterols, while for the formation of monoterpenoids, diterpenoids components-the plastid pathway of methylerythritol 4-phosphate producing IPP and dimethylallyl diphosphate from pyruvaldehydeglyceraldehyde-3-glycerate [116]. This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.      Antioxidant supernatant obtained after mixing the fermented seed powder and saline • fermentation increased the antioxidant activity • Initial decrease in antioxidant activity (2nd day of fermentation), then a marked increase (maximum value-4th day of the process), and then another decrease • Reduction of the content of harmful ginkgolic acids (decrease by 45%) [94] hydroethanolic leaf extract and ingredients: flavone, ginkgolide, procyanidins, and organic acids • DPPH scavenging ability was highest with procyanidins and lowest with ginkgolide. Flavone ability was lower than procyanidins and higher than organic acids. • scavenging capacity of ABTS was the highest in the case of flavone and the lowest in the case of ginkgolide, while procyanidins-lower than that of flavone and higher than that of organic acids • antioxidation shows synergistic effects • the highest scavenging of ABTS and DPPH radicals was obtained in a solution of flavone: procyanidins in the proportion of 1: 9 [21] Leaf extract (EGb 761) • EGb 761 exhibits antioxidant activity • enhances the action of drugs used in diseases of the nervous system [74]     Substances isolated from fresh male flowers: in combination with gemcitabine: inhibiting cell proliferation; inhibiting tumor growth; increasing cell apoptosis; no effect when gemcitabine and extract are used separately • suppression of the effect of gemcitabine in the following areas: increase in NF-kB activity and PAFR and phosphorylated NF-kB/p65 expression • suppression of PAFR expression • no effect on the IC50 of gemcitabine in IκBα-SR [86] Leaf extract IDN 5933 • there were no adverse clinical effects leading to liver or thyroid tumors • liver injury markers remained constant • there was no difference in the expression of the c-myb, p53, and ctnnb1 genes [80] Amentoflavone • strong reduction in the synthesis of tumor necrosis factor alpha (TNF-), interleukin-1 (IL-1) and IL-6 in cells infected with Streptococcus suis [57]     reducing the anxiety index and increasing latency to immobility [90] Extract (GbE) • beneficial changes in kidney function associated with reduction of renal glomerular hypertrophy; reduction of the kidney/body weight ratio and reduction of albuminuria • inhibition of the reduction of synaptopodine and nephrine • increase in HO-1 expression in the kidney • reduction of the accumulation of TNF-α, IL-6, fibronectin and lipids in the renal glomeruli • reducing the uptake of low-density oxidized lipoprotein and reducing the production of ROS in podocytes with high glucose levels [55]

Type of Activity Substance Result Source
Anti-obesity, anti-atherogenic and anti-diabetic Ginkgo biloba seeds (GBS) • regulation of glucose and lipid metabolism • lowering fasting blood glucose and serum insulin levels • improvement of glucose and insulin tolerance • antioxidant and anti-inflammatory effect [91] vinegar obtained from fermented coats of ginkgo seeds

Type of Activity Substance Result Source
Anti-obesity, anti-atherogenic and anti-diabetic Ginkgolide B • effect on metabolic disorders in mice with obesity induced by a high-fat diet • activation of hPXR transactivity • decrease in body weight and serum triglyceride (TG) levels • reduction of fatty liver (improvement of lipid accumulation) • increase in mRNA expression of target PXR genes in the liver [74] Extract GbE • reduction of food consumption (as a source of energy) • reduction of weight gain • increase in the expression of Adipo R1 and IL-10 genes • increase in IR and Akt phosphorylation • reduction of NF-κB p65 phosphorylation and TNF-α levels [88] Neuroprotective and anti-neurodegenerative

Extract EGb
• beneficial effect in Alzheimer's patients, especially women • decreased production of TNF-α, IFN-γ and IL-10 • increase in the production of IL-15 and IL-1β • lowering the expression of SOCS1, SOCS3, IRF-3, IRF-7, tetherin, NFKB1, p65 and MxA genes [106] Extract EGb 761 • alleviation of cognitive impairment occurring in mild and moderate forms of Alzheimer's disease [103]  • with simultaneous administration with donepezil: increased pro-cholinergic and antioxidant effect, which leads to a marked improvement in memory (cognitive functions) • no changes in plasma or uptake by brain of donepezil or bilobalide [74] Extracts (GB, EGb 761) Tablets • beneficial effect on the functioning of memory, including cognitive functions in neurodegenerative diseases (Alzheimer's and Parkinson's) and in cancer • improved blood supply to the brain, which improves the ability to concentrate • beneficial effect on executive functions and non-verbal memory • soothing mood changes and reducing stress [105] Extract EGb 761 • treatment of neurodegenerative diseases by reducing cell apoptosis as a result of: decrease in acetylation of p53 lysine 382, increase in the potential of the mitochondrial membrane, reduction of the BAX/Bcl-2 ratio • reduction of cleavage of PARP [Poly (ADP-ribose) polymerase] [102] Ginkgolides • disruption of the signaling pathways of the nuclear factor NF-κB and MAPK kinase responsible for inflammation occurring in neurological diseases • reduction of inflammation as a result of inhibition of signal transduction by altering the activity of the PAF activating factor [53] Extract EGb • improvement of cognitive memory function in mild symptoms of Alzheimer's dementia [3] Extract EGb 761 • reduction of inflammatory processes in primary microglial cells occurring in Alzheimer's disease by: inhibition of prostaglandin E2 release reduction of mPGES-1 protein synthesis change in the levels of proinflammatory cytokines decrease in cytosolic activity of phospholipase A2 [101] • vascular protection: favorable morphological changes of the vessels in radial peripapillary capillary of the eyes (increased density) [109] Ginkgo leaf tablets • effective control of the rate of retinopathy and disease progression (type 2 diabetes) • improvement in the rate of remission (extension of the asymptomatic period) [107] Extract EGb 761 • prevention of hearing impairment when taking cisplatin during chemotherapy (confirmed by comparison of the amplitude of the distortion products of otoacoustic emissions and the signal-to-noise ratio) [108] Cardiovascular protection Ginkgolide B • decreased expression of cyclooxygenase-2 and etalloproteinase-1 in RAW264.7 macrophages (contributes to the protection against atherosclerosis) [48]

Pharmacological Activities
Ginkgo biloba has been used for years as a herbal plant supporting memory processes. Initially, it was studied in terms of neuroprotective and anti-neurodegenerative effects. However, as a result of the analysis of the composition of the extracts obtained from the leaves and the influence of these extracts or their selected components, it was found that this plant has a wide multidirectional effect on the functioning of the organism. As a result of studies conducted on animals and on human tissue lines, it has been shown that ginkgo extracts and tablets and their selected ingredients exhibit anti-inflammatory, anti-bacterial, antioxidant, anti-cancer, anti-obesity, anti-diabetic, anti-atherogenic, cardioprotective and oto-protective effects ( Table 2).

Anti-Microbial Activity
The use of ginkgo for anti-bacterial purposes has been the subject of research for a long time. Initially, the effectiveness of extracts prepared from various parts of plants was analyzed-e.g., fruit, leaves and roots, as well as selected components of these extracts, e.g., ginkgo acids or free phenolic acids for the few taxa of bacteria. The most frequently tested microorganisms were Escherichia coli and Staphylococcus aureus. In these studies, inhibition of the activity of selected bacterial taxa was demonstrated, and the results became the basis for further research on anti-bacterial activity. In recent years, ginkgetin [129,133], amentofavone [132], ginkgolic acid C15:1 monomer [134] and polyprenol [135] and the effectiveness of leaf extracts obtained with the use of various solvents (water, ethanol, chloroform and methanol) [136][137][138][139]. The group of microorganisms has been significantly expanded, including taxa of gram-positive and gram-negative bacteria [125,126,134,136,138,140,141], intestinal microflora typical of the tested mammalian organisms [137,139], as well as human pathogenic fungi (e.g., Candida albicans used in the study by) [141]. In all the studies carried out, different effects on the activity of microorganisms were shown, and the strength of the effect depends on the tested pathogen and the dose/amount of the substance used. Moreover, higher efficiency of alcoholic extracts than water extracts was found [136].

Antioxidant Activity
The study of antioxidant activity was carried out on the supernatant obtained from fermented ginkgo seeds, leaf extracts obtained with the use of various solvents, as well as their individual components (including polysaccharides and their monomers, organic acids, procyanidins, flavone and ginkgolide). These analyzes were carried out using various methods (e.g., DPPH, ABTS, scavenging hydroxyl radicals or superoxide anion methods), which does not allow for a direct comparison of the results of all these studies. However, all studies showed the antioxidant activity of the tested substrates, often assessed as high or very high [21,126,131,138,[141][142][143][144][145][146][147][148][149]. However, it was noted that this activity varies depending on the date of leaf harvest: it is the highest in the case of raw material harvested in autumn [68]. Both commercial EGb761 extracts [143], as well as methanol and ethanol extracts prepared under laboratory conditions, are highly active. In the latter case, extracts made in alcohol at a concentration of 40% and 70% are more effective compared to extracts made in the presence of alcohol with a concentration of 96% [144]. Among the tested extract components, procyanidins and flavones contained in ginkgo leaves showed the highest antioxidant activity [21].

Antitumor Activity
The research on antitumor activity was carried out using the tissue culture method, which consisted of treating selected tumor cell lines (Table 2) with selected substances. When analyzing the results of studies published mainly in 2015-2022, it can be concluded that work on the use of ginkgo extracts or their selected ingredients is widely conducted, but it should be intensified. In total, the effect of the extract and seven selected substances found in ginkgo was analyzed and tested on at least 22 cancer cell lines; however, usually, the effect of the selected substance on only 2-3 tumor cell lines was analyzed. Nevertheless, the discussed studies demonstrated cytotoxicity or inhibition of selected development phases of neoplastic cells that inhibit their proliferation, both when using extracts obtained from leaves [42,[150][151][152][153][154][155][156][157], as well as selected components isolated from them: ginkgolide B [158][159][160] and polysaccharides isolated from leaves [13], bilobol isolated from the fruit [161] and chemical components isolated from fresh male flowers, incl. amentoflavone and its derivatives [134,160], bilobetin, isoginkgetin and sciadopitysin [160]. Very often, the effect of substance application was the improvement of parameters indicating the stage of disease development, which resulted in a slowdown or complete inhibition of tumor development ( Table 2).

Anti-Obesity, Anti-Atherogenic and Anti-Diabetic
During the research, a positive, antiatherogenic, anti-obesity and anti-diabetic effect of ginkgo on the metabolism of mammalian organisms was observed. The effects of both leaf extracts [130,139,[162][163][164] and seeds [165], vinegar obtained from fermenting seeds [166], leaves [148,167], well as ginkgolide B [123,168] and ginkgolide C [169] were investigated. During the research, positive changes in body weight (decrease) were observed [163,164,170], and a reduction in adipose tissue mass [167][168][169] was caused by the reduction of fat cells (adipocytes) [169,170]. Blood tests showed positive changes in the fat profile [145,164], including lowering cholesterol and triglycerides [167,168], and the regulation of lipid metabolism, glucose and insulin [166,167] had a positive effect on the condition and functioning of the kidneys [130]. Additionally, a study by Wang et al. (2022) [139] showed that the oral administration of Ginkgo biloba leaf extract is effective in relieving hypercholesterolemia, systemic inflammation and atherosclerosis, and these effects are associated with the modulation of the taxonomic composition of intestinal microbes, protection of the integrity of the intestinal mucosa, and improvement of microbial metabolic phenotypes (Figure 2).

Neuroprotective and Anti-Neurodegenerative
In studies on the influence of ginkgo on the functioning of the nervous system, mainly ginkgo leaf extracts were used [3,140,[171][172][173][174][175][176][177][178][179][180][181][182], less often tablets [176,177]. The use of all these types of preparations was conducive to the reduction of inflammatory processes within the nervous system [128,170], the number of nerve cells subject to damage and apoptosis [169,173] and the improvement of blood circulation within the cerebral vessels [171]. The therapies resulted in the improvement of memory, especially cognitive memory [3,[168][169][170][171][172], which was of great importance for limiting and inhibiting neurodegenerative changes, especially in Alzheimer's and Parkinson's diseases. It is also important that these preparations enhance the effect of a drug traditionally used in the treatment of dementia symptoms occurring in neurodegenerative diseases-donepezil [140].

Neuroprotective and Anti-Neurodegenerative
In studies on the influence of ginkgo on the functioning of the nervous system, mainly ginkgo leaf extracts were used [3,140,[171][172][173][174][175][176][177][178][179][180][181][182], less often tablets [176,177]. The use of all these types of preparations was conducive to the reduction of inflammatory processes within the nervous system [128,170], the number of nerve cells subject to damage and apoptosis [169,173] and the improvement of blood circulation within the cerebral vessels [171]. The therapies resulted in the improvement of memory, especially cognitive memory [3,[168][169][170][171][172], which was of great importance for limiting and inhibiting neurodegenerative changes, especially in Alzheimer's and Parkinson's diseases. It is also important that these preparations enhance the effect of a drug traditionally used in the treatment of dementia symptoms occurring in neurodegenerative diseases-donepezil [140].

Protection of Other Organs
Research has shown that both EGb extract [179] and ginkgo leaf tablets [180,181] have a protective effect on the sensory organs. This is of great importance, especially for patients suffering from type 2 diabetes, because it allows to delay or completely inhibit damage to blood vessels in the eye's retina leading to visual disturbances [179], as well as contributing to at least partial regeneration and improvement of the condition of these vessels [181]. In addition, the substances contained in the ginkgo leaf extract protect the hearing organ against damage caused by taking cisplatin during chemotherapy implemented during the treatment of neoplastic diseases [180]. One of the components found in ginkgo leaves, ginkgolide B, also has a protective effect on blood vessels, preventing the occurrence of atherosclerotic lesions [123].

Protection of Other Organs
Research has shown that both EGb extract [179] and ginkgo leaf tablets [180,181] have a protective effect on the sensory organs. This is of great importance, especially for patients suffering from type 2 diabetes, because it allows to delay or completely inhibit damage to blood vessels in the eye's retina leading to visual disturbances [179], as well as contributing to at least partial regeneration and improvement of the condition of these vessels [181]. In addition, the substances contained in the ginkgo leaf extract protect the hearing organ against damage caused by taking cisplatin during chemotherapy implemented during the treatment of neoplastic diseases [180]. One of the components found in ginkgo leaves, ginkgolide B, also has a protective effect on blood vessels, preventing the occurrence of atherosclerotic lesions [123].

Toxicity
Toxic components in Ginkgo biloba include alkylphenols. Their classification is presented in Section 5 (Phytoconstituents of the plant). These compounds are a mixture of several 2-hydroxy-6-alkylbenzoic acids. One of the toxic components is ginkgolic acid, designated as C13:0, C15:1 and C17:1 [182].
Ginkgo biloba standardized extract EGb761 is classified as a therapeutic agent for the treatment of the central system, the main one in the treatment of dementia, but also helpful in the treatment of Alzheimer's and Parkinson's. It is credited with relieving symptoms, memory functions and handling, dizziness, migraines, or tinnitus. To be able to attribute the pro-health effect of EGb761, it should contain 22-27% of flavonoids and 5-7% of terpenoids and less than 5 ppm ginkgo biloba acid (i.e., 0.0005% ginkgo biloba acid in the preparation) [6,32].
The seeds of this plant are used in Asian cuisine for the production of stuffing, soups, desserts, meat and vegetarian dishes, and the roasted seeds are a popular delicacy. While eating cooked ginkgo seeds is safer than eating them raw, they can be toxic if consumed in large amounts or over a long period of time, especially in children. Ginkgo seeds contain a toxic component of MPN (4-methoxypyridoxin) called ginkgotoxin, and in addition, ginkgo seed tests contain large amounts of alkylphenol (over 4% ginkgolic acid)-eating more than 10-20 nuts a day may pose a health risk [32]. However, it has been shown that the concentration of ginkgotoxin in the protein of ginkgo seeds increases during the growing season and reaches its maximum in early August, but then its content drops sharply. Canned and cooked seeds now contain only about 1% of ginkgotoxin present in raw seeds, which can be attributed to their water solubility. On the other hand, the content in roasted seeds is slightly lower than in raw seeds because the compound is thermally stable [6,183]. For this reason, when using Ginkgo biloba extract preparations, it is important to ensure the safety of patients.
According to the research of Gawron-Gzella et al. (2012) [184], the pharmacopoeial requirements for ginkgo leaf extract refer to the number of bilobalides and the sum of A, B and C ginkgolides, while the manufacturers of preparations indicate the content of total terpene lactones on the labels. Research shows that many manufacturers do not always keep the declared total of terpene lactones (6%), and the preparations do not always contain the correct portion of bilobalides and the sum of ginkgolides. The content of ginkgolic acid, with the applicable norm below 5 ppm, in dietary supplements was very often overstated, sometimes even 1600 times. As a result of consuming such a large amount of ginkgolic acid, problems with the digestive system (nausea, vomiting, diarrhea), headaches and dizziness, palpitations, anxiety, weakness or skin allergy. In the case of people with blood clotting problems and/or taking non-steroidal anti-inflammatory drugs, antiplatelet or anticoagulant medications, it can lead to internal hemorrhage [185]. Ginkgotoxin poisoning can also occur. The content of ginkgotoxin in seeds ranges from 170 to 404 µg/g. Concentrations above 170 µg/g cause toxicity and manifest as seizures, loss of consciousness and leg paralysis [186]. It is important that such poisoning can be prevented with vitamin B6: administration of a dose of 30 mg of pyridoxal 5 -phosphate (corresponding to 2 mg/kg of body weight) causes the symptoms of poisoning to cease [187].
The results of Boeteng & Yang (2021) [24] showed that the number of toxic compounds in fresh Ginkgo biloba seeds (ginkgotoxin, ginkgolic acid and cyanide) was significantly reduced during seed drying. The ginkgotoxin content was reduced by a factor of four, and the amounts of acrylamide, ginkgolic acid and cyanide in the dried seeds were reduced to a safe level (safety range). Of the four drying methods tested, radiant drying turned out to be the most effective: it lasted the shortest, and the obtained product showed the highest quality and content of bioactive compounds, as well as the strongest antioxidant activity.
Recently, the attention of scientists has been attracted by the possibility of using alkylphenols for medical purposes, which in appropriate doses, have beneficial effects, including anti-cancer and anti-bacterial properties [134].
A study by Borenstein et al. (2020) [187] has been shown to inhibit Herpes simplex type 1 virus multiplication, human cytomegalovirus genome replication and Zika virus infection. In addition, it inhibits the synthesis of all three classes of HIV, Ebola, Influenza A, and Epstein-Barr virus fusion proteins. The results also indicate that inhibition of virion entry by blocking the initial fusion event following ginkgolic acid administration postinfection suggests a possible secondary mechanism targeting protein and DNA synthesis. This is confirmed by the strong action of this acid, effective even after the infection process has taken place. The results also indicate the possibility of using it in the treatment of acute infections (e.g., caused by coronavirus, Ebola virus, Zika, influenza A and measles), as well as active local lesions (e.g., caused by HSV-1, HSV-2 and varicella viruses-VZV shingles).
The publication of Omidkhoda et al. (2019) [188] discusses the protective effect of using Ginkgo biloba leaf extract in case of poisoning caused by various factors: natural toxins (scorpion venom, lipopolysaccharides, aflatoxin B1, lysophosphatidylcholine, pentacyclic triterpenoids, cassava, cotton seed pigment called gossypol), chemical toxins (metals): aluminum, lead, cadmium, mercury; heavy metals contained in aqueous waste, fluorine, triethyltin, ethanol, carbon tetrachloride, pesticides, chemotherapeutic drugs, cigarette smoke, naphthalene or monosodium glutamate) and radiation. The beneficial effect of the extract on the poisoned organism is probably related to the high antioxidant activity of the extract (manifested by the reduction of lipid peroxidation and restoration of reduced dehydrogenases, glutathione peroxidase, superoxide dismutase and catalysis) and its anti-inflammatory effect.
According to the results of in vitro studies, biflavonoids (ginkgetin, isogingetin, amentoflavone, sciadopitysin and bilobetin) can also be toxic to the body. They were observed to be cytotoxic to human proximal tubular cells and to be less toxic to healthy human liver cells. In addition, activated apoptosis was associated with biflavonoid-induced nephrotoxicity. These data suggest that these biflavonoids exhibit potential hepatic and renal toxicity [24].
The supplement market should be more regulated so as not to lead to accidental poisoning. In addition, the production should be strictly regulated to ensure that such a supplement will not contain nutrients or be contaminated. Products, such as infusions, are a safe products as a kind of supplementation with a balanced diet. Despite the risks, special care should be taken by pregnant women and children.

Interactions of Ginkgo biloba Extracts with Drugs
Ginkgo biloba, as a raw material belonging to phytotherapeutic drugs, shows mainly antioxidant and neuroprotective properties. It contains pharmacologically active ingredients that may be useful in treating many diseases, but due to its antiplatelet effects, it may interact with other antiplatelet drugs (warfarin, aspirin) or herbal preparations with similar antiplatelet effects (garlic or ginseng) [189]. According to Kedzia and Alkiewicz (2006) [190], ginkgo preparations in combination with aspirin may cause hematomas in the anterior chamber of the eye and with paracetamol-subdural hematomas.
Research by Bogacz et al. (2016) [189] proved that extracts from this plant could modulate the expression of cytochrome P450 enzymes and, thus, influence transcription factors, thanks to which they can participate in the metabolism of xenobiotics (drugs, procarcinogens, vitamins and food components).
Ginkgo preparations may also accelerate the metabolism of omeprazole and esomeprazole, primarily by influencing the mechanism of CYP2C19 induction and consequently reducing the effectiveness of these drugs in preventing upper gastrointestinal bleeding. In addition, they increase the risk of bleeding while taking SSRIs or SNRIs [191]. Single cases of coma in humans have been shown to be caused by the concomitant intake of Ginkgo biloba preparations with trazodone, and cases of priapism have been observed as a result of an interaction between Ginkgo biloba and risperidone. It was also noted that the use of Ginkgo biloba may reduce the concentration and effectiveness of valproate and reduce the anxiolytic and hypnotic effects of benzodiazepines [192,193]. Woroń & Siwek (2018) [194] proved that the combination of ginkgo biloba with dormitive and/or anxiolytics or with fluoxetine caused side effects in the form of dizziness, somnolence and hypotension.
In the case of non-standardized extracts prepared in accordance with the European Pharmacopoeia, the effect of the extract remains uncertain. A significant drug interaction potential cannot be ruled out in the case of poorly standardized ginkgo leaf extracts used in many dietary supplements. A review of research to date shows that Ginkgo biloba extracts are very reactive. Therefore, patients should be checked for health prior to administration, and any possible signs of drug interactions should be carefully considered [6].

Patents
Ginkgo biloba has been used in Chinese medicine for centuries, but recently, the leaves and fruits of this plant have become objects of interest in the pharmaceutical, food and cosmetic industries. The greatest industrial interest in Ginkgo biloba leaves and fruits occurred at the end of the last century when several hundred patents were issued, mainly in Japan, China and the USA. However, at the beginning of the 21st century, more than a thousand patents were published each year, with the largest number of publications appearing in 2017 (over 7000); in the following years, the number decreased, with 391 patents published in 2021 and 275 in 2022 (state on 7 November 2022). Generally, referring to the Espacenet patent database (European Database Espacenet), during the last three decades, more than 29 thousand patents appeared all over the world. Most of them concern the application of ginkgo extracts in medicine, methods of extraction or preparation of tablets or pills.
Until 1990 of the 44 patents listed for the entry "Ginkgo biloba" in the Espacenet patent database (European Database Espacenet), many patents were related to cultivation and chemicals, as well as drugs, for example, anti-vomiting preparations or anti-inflammatory medicines, which contain ginkgo extract.
The new inventions concerned mainly extraction methods allowing to obtain extracts with a reduced content of toxic compounds, such as 4 -o-methyl pyridoxine and biflavones, alkyl polyphenols or ginkgolic acid. Extracts were usually obtained from ginkgo leaves using organic solvents, e.g., acetone or methanol, which were removed in the next steps of processing, and the resulting concentrate was dissolved in water, ethanol or other solvents, with the steps of purification and filtration to remove alkyl polyphenols [208]. The patented production process of extract EGb 761 ® involves extraction of a mixture of leaves from China, France, and the USA with 60% (m/m) aqueous acetone, acetone removal by evaporation, cooling with stirring the aqueous solution to precipitate chlorophylls, biflavones and most of the ginkgolic acid [23,202,209]. Extracts prepared using organic solvents are treated with lead compound (e.g., lead acetate) or insoluble polyamide than the filtrated solution is extracted with an aliphatic solvent, the aqueous-alcoholic solution is concentrated, ammonium sulfate is added, the solution is extracted with methylethylketone and ethanol. The concentrated, filtrated and dried extract contains less than 20 ppm, preferably less than 10 ppm and in particular less than 2 ppm 4 -O-methyl pyridoxine and/or less than 20 ppm, preferably less than 10 ppm and in particular less than 5 ppm biflavones [202,210].
Inventors from China in 2017 [211] patented a new method of obtaining flavonoids from ginkgo leaves by using fermentation with Aspergillus niger, which allows reducing the amount of undesired ginkgo acid in the extract. Moreover, the obtained extract is free of organic solvents. According to this procedure, cleaned and crushed leaves are mixed with water (40-60%) and sterilized. In the next step, the mixture is inoculated with Aspergillus niger, then enzymes are added, and the fermentation is carried out for 3-6 h at 25-30 • C. Subsequently, further protease is added, and the reaction lasts for the next 3-4 h, followed by 10 min long heat termination, centrifugation, extraction with ethanol, concentrating, and finally freeze-drying.
The raw material subjected to extraction was mainly Ginkgo biloba leaves, fresh or dried. They were also used to make mixtures for the production of infusions, e.g., a mixture of ginkgo leaves and other herbal materials [212,213] or beer [214].
In recent years, the subject of patents has also been products made from ginkgo nuts (also called ginkgo fruits or ginkgoes), e.g., shortbread [215], vinegar [216], beverages [217,218] or wines [219][220][221][222]. In some formulas, ginkgo powder obtained earlier from ginkgo nuts was used [223,224]. An important element was to obtain products with improved taste, devoid of bitterness. Due to the insufficient taste of the obtained products or the content of undesirable ingredients, there were searched formulas containing the addition of other valuable ingredients, e.g., wine or drink in which ginkgo fruits were used together with the other raw materials like sorghum, wheat, sugar, saffron etc. [225][226][227]. Reduction of bitterness was also caused by fermentation caused by yeast in wine or beer production, as well as Aspergillus niger or Lactobacillus strains [213,[228][229][230][231].
Although the application of Ginkgo biloba in food was always motivated by its healthpromoting properties, in recent years, this appeared to be the main reason for forming new products, and inventors evidenced their health-promoting activities [212,[232][233][234].
Moreover, there have been patented many devices to facilitate the extraction, harvesting of ginkgo fruits and pre-treatment of ginkgo nuts.

Conclusions
Ginkgo biloba is a very popular raw material used not only in medicine but also in industrial technology. The content of ginkgolides, bilobalides, flavonoids and other bioactive ingredients contributes to its wide application. Currently, ginkgo is a herbal dietary supplement (EGb761); it is also used in complementary medicine and is an additive to cosmetics [198]. Products containing it are gaining popularity all over the world. Its primary action focuses on alleviating and/or preventing CNS dysfunction by regulating the level of cytokinins, antioxidant enzymes, kinases and receptors and modifying the activity of the PAF activating factor. Currently, more and more studies are carried out on the health-promoting properties of ginkgolic acids: their anti-cancer, neuroprotective and anti-bacterial activity is being tested. High hopes are associated with the possible medical use of ginkgolic acid. Although in 2000 years old traditional Chinese medicine, Ginkgo biloba seeds are used in the treatment of cough, asthma, tuberculosis, bladder infections, flatulence and diarrhea, however, such use of Ginkgo biloba, along with the development of knowledge about its active ingredients, brings a lot of concerns. The largest of these are the interactions between biologically active substances contained in Ginkgo biloba and drugs. Until now, not all the mechanisms by which the use of non-standardized extracts of Ginkgo biloba leaves can be used are known to cause excessive activity or inhibition of the drug's action.
In conclusion, Ginkgo biloba is still an interesting research object for scientists dealing with, among others, medicine and food production. New products containing extracts or fractions of Ginkgo biloba fruit or leaves are being developed. So far, patented food products are not popular on the European food market, with some exceptions-products containing the addition of dried ginkgo leaves or Ginkgo biloba extracts, such as health beverages, are valued. Of great importance, there is the possibility of using the active substances contained in the leaves and seeds for the production of the so-called superfoods, other than dried leaf infusions. Focusing on the purification of Ginkgo biloba extracts can contribute to increasing the offer and improving the quality of dietary supplements and medicines. In addition, the development of a method for the isolation of specific Ginkgo biloba metabolites can provide compounds used for food enrichment.
It is worth noting that expanding the market with Ginkgo biloba products may have a positive impact on the development of Europe's agricultural economy. This will result in no need to import the plant, e.g., from Asian countries, which seems to be a greener solution.
Author Contributions: All three authors are responsible for the concept of the work. P.B. is responsible for the chapters on the introduction, phytonutrients of ginkgo (including the table), structure and biosynthesis of ginkgolides and bilobalides, toxicity and interactions of extracts with drugs, as well as graphics and photos in the publication. I.A. is responsible for the part on pharmacological activities (including the table) and for the substantive control. K.F. is responsible for the patent part. All authors have read and agreed to the published version of the manuscript.