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ABSTRACT: The new age of nutraceuticals is now embracing the centuries old herbal extract of Ginkgo biloba (Mantissa Plantarum Altera, 1771, Ginkgoceae). The standardized preparation of the Ginkgo leaf extract (EGb 761) contained 2 main bioactive constituents, flavonoid glycosides (24%) and terpene lactones (6%), along with less than 5 ppm of the allergenic component, ginkgolic acid. The Ginkgo leaf extract has been reported to have neuroprotective, anticancer, cardioprotective, stress alleviating, and memory enhancing effects and possible effects on tinnitus, geriatric complaints, and psychiatric disorders. The therapeutic mechanisms of action of the Ginkgo leaf extract are suggested to be through its antioxidant, antiplatelet, antihypoxic, antiedemic, hemorrheologic, and microcirculatory actions, where the flavonoid and the terpenoid constituents may act in a complementary manner. Toxicity studies show that the Ginkgo leaf extract is relatively safe for consumption, although a few side effects have been reported, that is, intracerbral hemorrhage, gastrointestinal disturbances, headaches, dizziness, and allergic skin reactions. The use of Ginkgo leaf extract may be promising for treatment of certain conditions, although its long-term use still needs to be evaluated.
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Complementary and alternative medicine is defined as a “diagnosis, treatment and/or prevention which complements mainstream medicine by contributing to a common whole, by satisfying a demand not met by orthodoxy or by diversifying the conceptual frameworks of medicine” (Ernst 2000). Over the past 15 y, there has been a steady growing trend of these unconventional therapies throughout the globe. The European and the U.S. market alone contributed to about $7 and $5 billion per annum, respectively, in 1999 (Calixto 2000). Until 2000, estimates showed that nearly 50% to 75% of the U.S. population have tried complementary and alternative medicine (Neldner 2000).
NIH broadly classifies complementary and alternative medicine into 6 categories; Mind-Body Interventions, Bioelectromagnetics Applications, Alternative Systems of Medical Practice, Manual Healing Methods, Herbal Medicine, and Diet and Nutrition (Wootton 2005). The category of herbal medicine has grown faster than any of the other forms of alternative treatment (Ernst 2002). Traditional Chinese medicine is a subsection of herbal medicine (Wootton 2005). The 12 most commonly used and best-studied medicinal herbs are listed in O'Hara (1998).
Ginkgo biloba: botanical data
Ginkgo biloba L. (Mantissa Plantarum Altera, 1771, Ginkgoceae) belongs to the botanical family of Ginkgoceae with synonyms like Salisburia adiantifolia, Salisburia macrophylla, and Pterophylla salisburiensis. The ginkgo tree, known to be among the oldest living species on this planet, has flourished in forests for over 150 million y and hence it is called a “living fossil” (McKenna and others 2001). It is a dioecious tree with the male and female reproductive organs on separate trees. They have a large trunk with a girth of about 7 m and a height of about 30 m. Young trees are conifer like and exhibit branching dimorphism. Leaves that grow in clusters are golden yellow in fall during senescence. The leathery leaves are very uniquely shaped with 2 lobes and resemble the maidenhair fern in shape and venation. The pollination process involves the male microstrobilli bearing loosely distributed sporangiophores containing microspores with male gametophytes and the female pendulous pairs of ovules borne on the shoots. These trees begin to reproduce after about 20 y by developing naked seeds (nuts) with an outer fleshy layer (fruits). The outer fleshy layer of the fruit has a considerable amount of butanoic and hexanoic acids, which are responsible for the rotting flesh, fermented odor (McKenna and others 2001).
History and traditional uses of Ginkgo biloba
The ginkgo tree is the only surviving member of Ginkgoaceae family, class of Ginkgoatae, rediscovered in Asian graced temple gardens by Kaempfer in 1670. The class of Ginkgoatae consists of approximately 15 genera, and among these, Ginkgo, Baiera, and Ginkgoites are the most important (Bilia 2002). The name ginkgo comes from the Chinese words sankyo or yin-kuo, which means a hill apricot or silver fruit, due to their apricot shaped mature fruits and yellow color (McKennaand others 2001). Englbert Kaempfer, a German surgeon, first used the term “Ginkgo” in 1712, but it was Linnaeus who termed it Ginkgo biloba in 1771 (Gertz and Kiefer 2004).
Both the leaves and the nuts of this tree have been in use for the past several centuries in traditional Chinese medicine. In fact, the nuts are known to have a longer history of usage, being first mentioned in herbals in the Yuan dynasty [1280 to 1368 AD], published in 1350 AD (Goh and Barlow 2002). For over 5000 y, the seeds (nuts) have been known to treat pulmonary disorders (like asthma, cough, and enuresis), alcohol abuse, and bladder inflammation while the leaves have been mainly used to treat heart and lung dysfunctions and skin infections (Mahady 2002; Smith and Luo 2004). However, it was only in the last 20 to 30 y that the use of the ginkgo leaf and its standardized extract formulation, EGb 761, originated in Germany, and now is the most used form of supplement for cognitive ailments in the United States (Smith and Luo 2004).
Other uses of this tree include the fruit, prepared by fermentation and cooking, being a delicacy in weddings and feasts (McKenna and others 2001; Bilia 2002). The roasted or boiled ginkgo seeds are also considered a gourmet delicacy in Japan, China, Korea, and Malaysia. The tree is also grown in many parts of Europe and the United States mainly for its ornamental value. It grows well in most places due to such properties as pest, pollution, and disease resistance (McKenna and others 2001).
Ginkgo leaf extract
Since the constituent composition of ginkgo leaf undergoes fluctuating changes with respect to their origin, species, and time of harvest like any other plant, it is essential to standardize the Ginkgo leaf extract to obtain consistent bioactivities. The culturing, harvesting, and extraction of the ginkgo leaves are rigorously standardized and controlled based on its known active components (Smith and Luo 2004). Typically, the leaves are collected during summer and fall between the months of July and September when they are still green. These leaves are then dried and analyzed for the presence of pollutants and toxic substances like heavy metals and aflatoxins. The crude dried leaves are then extracted using an acetone:water mixture (35 to 67:1). Standardization of Ginkgo leaf extract can be done by measuring flavonoids and terpenoids (Bilia 2002). The standardized extract preparation of the ginkgo leaf, EGb 761, developed by Beaufor-Ipsen Pharma (Paris, France) and Dr. Willmar Schwabe Pharmaceuticals (Karlsruhe, Germany), contains 24% flavonoid glycosides, 6% terpene lactones, and less than 5 ppm ginkgolic acid (the constituent proposed to have allergenic properties) (Smith and Luo 2004). Recently, a new method to evaluate Ginkgo leaf extract using HPLC-fingerprinting has been suggested as a better method to monitor various preparation of the Ginkgo leaf extract (Sun and Liu 2007).
Active components of Ginkgo biloba leaf
The 2 main pharmacologically active groups of compounds present in the Ginkgo leaf extract are the flavonoids and the terpenoids (Smith and Luo 2004).
Flavonoids, also called phenylbenzopyrones or phenylchromones, are a group of low molecular weight substances that are widely spread in the plant kingdom. Flavonoids present in the Ginkgo leaf extract are flavones, flavonols, tannins, biflavones (amentoflavone, bilobetol, 5-methoxybilobetol, ginkgetin, isoginkgetin and sciadopitysin), and associated glycosides of quercitin and kaempferol attached to 3-rhamnosides, 3-rutinosides, or p-coumaric esters (McKenna and others 2001). The flavonoid content in the Ginkgo leaf is known to vary between seasons; greater amounts are found in fall than in spring (McKenna and others 2001). These compounds are known to act mainly as antioxidants/free radical scavengers, enzyme inhibitors, and cation chelators (DeFeudis and Drieu 2000). In general, the bioavailability of flavonoids is relatively low due to limited absorption and rapid elimination (Goh and Barlow 2004). Flavonoids in the glycosidic form are poorly absorbed in the intestine; only in the aglycone form can they be absorbed directly (Goh and Barlow 2004). Unabsorbed flavonoids that reach the colon may be subject to metabolism by bacterial enzymes, and then absorbed (DeFeudis and Drieu 2000). Once absorbed, flavonoids reach the liver where they are metabolized to conjugated derivatives (DeFeudis and Drieu 2000). It is known that the biological activities of flavonoid metabolites are not always the same as those of the parent compound (Manach and others 2004).
Two types of terpenoids are present in Ginkgo as lactones (nonsaponifiable lipids present as cyclic esters): ginkgolides and the bilobalide (Smith and Luo 2004). Ginkgolides are diterpenes with 5 types A, B, C, J, and M, where types A, B, and C account for around 3.1% of the total Ginkgo leaf extract (DeFeudis and Drieu 2000). Bilobalide, a sesquiterpene trilactone, accounts for the remaining 2.9% of the total standardized Ginkgo leaf extract (Smith and Luo 2004).
There are no adequate studies determining the dose of Ginkgo extract needed to achieve beneficial effects, although the recommended dose of standardized extract, EGb 761, is 40 to 60 mg, 3 to 4 times daily based on clinical trials (Mahady 2001). For chronic conditions the German commission recommends a minimum 8-wk intake in order to observe the beneficial effects of the Ginkgo leaf extract (McKenna and others 2001).
Pharmacological effects of Ginkgo biloba
Ginkgo leaf extract has shown beneficial effects in treating neurodegenerative diseases like Alzheimer's, cardiovascular diseases, cancer, stress, memory loss, tinnitus, geriatric complaints like vertigo, age-related macular degeneration, and psychiatric disorders like schizophrenia (Ramassamy and others 2007). These multifaceted activities of the Ginkgo leaf extract may work through various mechanisms of action. The suggested mechanisms of the Ginkgo leaf extract are its antioxidant effect, anti-platelet activating factor (Anti-PAF) activity for cardio and cerebral vascular diseases, inhibition of beta amyloid peptide (Aβ) aggregation to reduce Alzheimer's progression, and decreased expression of peripheral benzodiazepine receptor (PBR) for stress alleviation and stimulation of endothelium derived relaxing factor to improve blood circulation (Amri and others 1996; Pietri and others 1997a; DeFeudis and Drieu 2000; Smith and Luo 2004).
Antioxidant effects The underlying principle behind the therapeutic action of the Ginkgo leaf extract on chronic ailments (such as neurodegenerative diseases, cardiovascular diseases and cancer) has focused on its antioxidant properties. The 2 proposed mechanisms of action are (1) directly scavenging free radicals and (2) indirectly inhibiting formation of free radicals. The Ginkgo leaf extract can scavenge reactive oxygen species (ROS) such as hydroxyl radicals (OH˙), peroxyl radical (ROO˙), superoxide anion radical (O2−˙), nitric oxide radical (NO˙), hydrogen peroxide (H2O2), and ferryl ion species (Mahady 2002; DeFeudis and others 2003). The Ginkgo leaf extract can also enhance activities of antioxidant enzymes such as superoxide distmutase (SOD), glutathione peroxidase, catalase, and/or heme-oxygenase-1, thereby indirectly contributing as an antioxidant (Song and others 2000; DeFeudis and others 2003). It has been suggested that ginkgo leaf extract increases expression of mitochondrial enzymes like NADH dehydrogenases, which can influence ROS generation in the mitochondria. This is a protection against uncoupling of oxidative phosphorylation, thereby increasing ATP levels regulating energy metabolism (Janssens and others 1995; Tendi and others 2002). In comparison to other antioxidants, the Ginkgo leaf extract (EGb 761) is known to be regulatory and adaptive, either dilating or contracting blood vessels, or controlling neurochemicals or neuroendocrine indicators according to the circumstances (Smith and Luo 2003). The main constituents implicated in all these actions are the flavonoids (quercitin and kaempferol) and the terpenoids (ginkgolides and bilobalide) (Bastianetto and others 2000; DeFeudis and others 2003; Smith and Luo 2004), where each contributes their antioxidant property differently. The flavonoids are known to exert their effects through inhibition of the cyclooxygenase-2 enzyme, which is a part of prostaglandin synthesis, and its inhibition is known to reduce colon carcinogenesis. The bilobalide increase the activities of the antioxidant enzymes (SOD and catalase) and improve cell viability (Watanabe and others 2000; DeFeudis and others 2003).
However, proanthocyanidins (present at about 7% in Ginkgo leaf extract) present in the whole leaf extract bind to proteins and inactivate antioxidant enzymes such as catalase, glutathione peroxidase, and lactate dehydrogenase (Pietri and others 1997a). Hence, the presence of these proanthocyanidins may hinder the antioxidant effects of the Ginkgo leaf extract.
Aβ is a polypeptide with 39 to 43 amino acid residues and a major component of senile plaques and vascular amyloid deposits of the brains of patients suffering from Alzheimer's disease. Ginkgo leaf extract is known to inhibit the formation of Aβ from β-amyloid precursor protein (APP), a crucial process in the pathogenesis of Alzheimer's disease (Yao and others 2004). Formation of amyloid precursor protein has been indirectly linked to high cholesterol levels (Koudinov and Koudinova 2001; Wolozin 2002; Puglielli and others 2003). It has been postulated that the inhibition of Aβ is through the Ginkgo leaf extract's ability to compete with free cholesterol for interaction with Aβ and thereby decrease their aggregation (Yao and others 2004). Alternatively, the Ginkgo leaf extract inhibits ROS accumulation induced by Aβ (particularly flavonol quercitin) and also reduces neuron apoptosis, where apoptosis is considered to be one of the main causes for neurodegenerative diseases (Bastianetto and others 2000; Ahlemeyer and Krieglstein 2003; Ergun and others 2005; Ramassamy and others 2007) and thus help to relieve Alzheimer's disease. Ginkgolide B and bilobalide are reported to inhibit apoptosis induced by staurosporine (alkaloid anticancer drug) and serum deprivation (Ahlemeyer and Krieglstein 2003). Bilobalide also prevented DNA fragmentation due to hydroxyl radical β-amyloid and hydrogen peroxide (Ahlemeyer and Krieglstein 2003). In addition, Ginkgo leaf has also been reported to improve cerebral blood flow by stimulating norepinephrine secretion (Yang and others 2005) and increased the life span in a particular study of rats treated chronically with EGb 761, due to its antioxidant action in reducing oxidative stress and free radical production (Winter 1998).
Ginkgo leaf extract is known to improve memory complaints as well. Walesiuk and others (2005) used EGb 761 at a dose of 100 mg/kg and found improvements of spatial and nonspatial memory tested in rats using the maze and object recognition test. Not only was EGb761 responsible for memory response improvement but was also reported to improve response retrieval.
Cardioprotective effects Ischemia, impaired blood circulation, is a common underlying condition of cardiovascular and cerebral vascular diseases. During an ischemic attack, there is an increased release of free radicals and lipid peroxidation causing tissue damage and resulting in chronic diseases (Mahady 2002). Cardioprotective effects of Ginkgo leaf extract are through antioxidant, antiplatelet activity and increased blood flow through release of nitric oxide and prostaglandins (Pietri and others 1997a, 1997b; Mahady 2002).
Pietri and others (1997b) showed that consumption of Ginkgo leaf extract prior to cardiac surgery helped in reducing reperfusion induced lipid peroxidation and prevented ascorbate depletion, tissue necrosis, and cardiac dysfunction. Moreover, they also showed that ginkgolide B reduces 50% to 60% of the postischemic production of ROS (Pietri and others 1997b). Pietri and others (1997a) also showed that reperfused hearts treated with terpene constituents alone recovered functionally better than those treated with EGb 761 (Pietri and others 1997a). This leads to the conclusion that terpene constituents decreased myocardial vulnerability to ischemic reperfusion.
The Ginkgo leaf extract is also known to improve coronary blood flow through antiplatelet activity (by ginkgolide B) and by improving contractile functions which are due to increased release of catecholamines from endogenous liver tissue reserves by flavonoids (quercitin, kaempferol, and isorhamnetin) (Mahady 2002).
Anticancer effects Cancer is a disease characterized by uncontrolled division of cells and the ability of these cells to invade other tissues. The disease is of mutifactorial origin that involves changes in gene expressions and aberrations in the cell signaling pathways. Ginkgo leaf extract is known to exhibit a chemopreventive action at various levels with antioxidant, antiangiogenic properties, and influence gene expression (Sagar and others 2006). The Ginkgo leaf extract's antioxidant ability contributes to improving cellular tolerance to oxidative stress (Smith and Luo 2004) as well as to reduce angiogenesis, which is blood vessel formation required for tumor metastasis (Monte and others 1994; DeFeudis and others 2003; Kim and others 2006; Sagar and others 2006). The nitric oxide (NO) involved in cancer progression also appears to be resolved through the terpenoids of the Ginkgo leaf extract by altering the expression of NO synthase enzymes (DeFeudis and others 2003). In addition, Ginkgo leaf extract is known to influence the expression of genes involved in cell proliferation, cell differentiation, and apoptosis at the mRNA levels in breast and bladder cancer models (Gohil and others 2000; Papadopoulos and others 2000; DeFeudis and others 2003), thus providing anticancer effects.
Effects on stress modification, mood, and memory Anxiety syndromes such as stress, moods, and depression are becoming common in the modern world. Complementary and alternative medicine is becoming popular as a prophylactic and/or therapeutic treatment for these symptoms. Stress involves a rise in the levels of glucocorticoids, and a subsequent memory dysfunction, increased anxiety, decreased immunity, gastrointestinal tract disturbances, myocardial infarction, or effects such as increased vigilance (Walesiuk and others 2005). Since mood and emotion are related to stress, the alleviating effects of Ginkgo leaf extract may result in improving mood, thus resulting in antidepressant activity (DeFeudis and Drieu 2004).
Ginkgolides A and B decreased the ligand binding capacity, protein, and mRNA expression of peripheral benzodiazepine receptor (PBR) which led to decreased corticosteroid synthesis and subsequently the circulating levels of glucocorticoids (Amri and others 1996). The memory enhancing effects of Ginkgo leaf extract through prevention of neuron degeneration are discussed in the previous section on prevention of neurodegenerative diseases.
Effects on tinnitus, geriatric, and psychiatric disorders Tinnitus, or “ringing in the ears,” is a common condition observed in almost 10% of the population (Drew and Davies 2001). One of the common causes for tinnitus is the inadequate blood supply to the inner ear (Ernst and Stevinson 1999). Thus, Ginkgo leaf extract was thought to have some potential beneficial effects in treating tinnitus.
Age-related macular degeneration is thought to be one of the common causes of age-related visual loss, possibly due to oxidative damage to the retina. Ginkgo has been reported to be effective against senile macular degeneration due to its free radical scavenging effect (Diamond and others 2000). Vertigo, which involves a sensation of movement when no movement is occurring, is another disorder which ginkgo has been found to be effective against (Issing and others 2005). Schizophrenia is a mental disorder involving impairments in the perception or expression of reality and by significant social or occupational dysfunction. The condition is characterized by excessive free radical formation in the brain. A clinical trial carried out by Atmaca and others (2005) showed a positive effect in treating schizophrenia patients through increase in the levels of antioxidant enzymes like SOD, catalase, and glutathione peroxidase.
Studies show that a relatively low risk is associated with the consumption of Ginkgo leaf products. Occasional adverse effects with excessive consumption of Ginkgo leaf extract have been reported which include gastrointestinal disturbances, headaches, dizziness, excessive bleeding, allergic skin reactions, and occasional anaphylaxis-like reactions (only with intravenous administration) (Kleijnen and Knipschild 1992; Skogh 1998; Vale 1998; Benjamin and others 2001; De Smet 2002). Long-term safety of Ginkgo leaf extract is not clear.
Chinese and Japanese cuisines often involve ginkgo nut consumption as a part of their diet. A particular case study reported frequent vomiting and clonic convulsions in a 36-y-old woman with no prior history of epilepsy after consuming approximately 70 to 80 nuts over 1 meal. 4'-Methoxypyridoxine, one of the components present in the nuts, is suspected to cause convulsions by indirectly affecting an enzyme glutamate decarboxylase, resulting in a decrease of the γ-aminobutyric acid (GABA) level in the brain (Miwa and others 2001).
Hemorrhage or excessive bleeding caused by Ginkgo leaf extract is due to its inhibitory effects on PAF. At levels of 120 to 240 mg/d EGb 761 does not have significant effects on PAF antagonistic action; however, dosages greater than 100 times are reported to cause hemorrhage in rabbits and humans (Koch 2005). Due to its effect on PAF, Ginkgo has been known to interact with anticoagulant drugs (Lu and others 2006; Aruna and Naidu 2007), although Jiang and others (2005) did not observe any significant effects of Ginkgo in clotting status in healthy subjects. Ginkgo leaf extract also interacts with antidepressants (that is, Trazodone), antiepileptic, antidiabetic, diuretics, and nonsteroidal anti-inflammatory drugs, as well as other herbal drugs (Matthews 1998; Uchida and others 2006; Tang and others 2007). These interactions are believed to be affected mainly by the flavonoidic glycosides and the terpenoids by selectively inhibiting particular enzymes, including cytochrome P450 (Gaudineau and others 2004). However, others reported no effect on clearance of cytochrome P450 substrates by Ginkgo leaf extract (Markowitz and others 2003; Greenblatt and others 2006; Mohutsky and others 2006). The apparent discrepancy has been suggested to be due to age-dependent responses (Gurley and others 2005).
The other components of Ginkgo leaf extract are the ginkgolic acids (alkyl phenols), which are considered to be toxic. They are bilobol, cardanols, cardols, and ginkgol and are known to cause gastrointestinal and allergic reactions. All commercial preparations of Ginkgo leaf extract must contain 5 ppm or less of ginkgolic acids to minimize these adverse reactions of Ginkgo leaf extract use (McKenna and others 2001).
German authorities report no side effects on pregnant and lactating women; however, data with respect to effects on fertility, lactation, and pregnancy, particularly near labor, are inadequate to be conclusive (McKenna and others 2001; Dugoua and others 2006).
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The standardized Ginkgo leaf extract preparation has been found to exhibit multifaceted therapeutic effects that include effects on neurodegenerative diseases, cancer, cardiovascular diseases, tinnitus, geriatric complaints, and psychiatric disorders. The main underlying mechanism of action in all these cases has been the antioxidant properties of the extract. There are other principles of action that include PAF antagonism, modulation of the peripheral benzodiazepine receptor, and endothelium relaxing factor improving the circulatory properties of blood. Thus, Ginkgo leaf extract has been shown to be a promising herbal dietary supplement with proven therapeutic benefits. However, its long-term safety needs to be properly addressed.