23 : Recent Advances in the Reproductive and Hormonal Effects of Ginkgo biloba
Abstract
Ginkgo biloba is a medicinal plant widely used by the population. Due to its actions as an anti-inflammatory and antioxidant, G. biloba extract (GBE) has been largely used in the treatment of Alzheimer’s disease, cerebrovascular insufficiency and peripheral arterial occlusive disease. In spite of those benefits, recent findings showed that GBE may interfere with the physiology of reproduction causing reproductive toxicity. In vitro and in vivo studies showed that GBE can cause oocyte degeneration, inhibition of human sperm motility, apoptosis of mouse blastocysts, as well as, low rate of pregnancy and pre-implantation loss. Furthermore, recent studies showed that GBE has both estrogenic and anti-estrogenic effects, which could be useful as an alternative hormone replacement therapy or, on the other hand, it could act like an endocrine disruptor and interfere with the physiology of the natural hormones. This review provides a brief overview of reproductive and hormonal effects of G. biloba extract.
Key words: Ginkgo biloba, Toxicity, Reproduction, Hormones, Estrogen, Endocrine disruptors
1. Universidade Federal de Juiz de Fora, Centro de Biologia da Reprodução, Caixa Postal 328, CEP 36001-970, Juiz de Fora, Minas Gerais, Brazil.
* Corresponding author : E-mail : luvalenteb@yahoo.com.br
Introduction
Medicinal plants use has risen exponentially in recent years, attributable primarily to an augment in the proportion of the population seeking alternative therapies rather than prescription medications (Eisenberg et al., 1998). The increased intake of those substances was followed by an enlargement of news media coverage of health issues through newspapers, magazines, and journal articles, with both informative and commercial goals. Despite substantial growth in the use of alternative medicines and therapies, much of the information the public receives about it, is inaccurate or incomplete, usually not reporting potential health hazards (Bonevski et al., 2008). An alternative to minimize the risks would be to increase the number of researches on alternative compounds in order to form a reliable database for patients, physicians and other health professionals (Joos et al., 2008). One of the most sold and consumed herbal medicines worldwide is Ginkgo biloba (Kleijnen & Knipschild, 1992; Mar & Bent, 1999; Sierpina et al., 2003; Chan et al., 2007). In spite of being widely used in the treatment of several pathologies, little is known about its action on reproductive physiology.
History and Botany
Ginkgo biloba is the most ancient living tree on earth, originally from Paleozoic Era, around 225 million years ago (Blumenthal, 2003; Zhou & Zheng, 2003). It is the only surviving member of the Ginkgoaceae family, Ginkgoales order and Gymnospermae family (Who, 1999; Nakanishi, 2005). The Ginkgo takes its name from ‘ginkyo’ in Japanese and ‘yinhsing’ in Chinese; both words translate to ‘silver apricot’, and the notation biloba refers to the morphology of leaves, which are divided into two distinct lobes (Fig 1) (Nakanishi, 2005).
Fig 1a. Ginkgo biloba leaf. It is about 3 inches across with a notch dividing into 2 lobes (thus biloba). Numerous veins radiate out of the base with no midrib. Available in: <http://zo-d.com/stuff/gardening/ginkgo-biloba-leaves.html>
Fig 1b. Ginkgo Fossil - British Columbia, Canada. Also known as maidenhair-tree, Ginkgo biloba’s leaf shape and other vegetative organs are identical to fossils found in the United States, Europe and Greenland. Available in: <http://forestry.about.com/ od/forestphotogalleries/ig/Ginkgo-Biloba/Ginkgo-Fossil.htm>
The tree Ginkgo biloba (Fig 2) is native from China, but it has been diffused, with ornamental purpose, to other countries, such as Australia, Japan, United States and other European countries, and it is commercially cultivated in France and United States. The maidenhair tree, as it is also known, is often called a ‘living fossil’, being able to live among 2000 to 4000 years, to reach more than 35 meters high and presents a diameter varying from 3 to 4 meters (Who, 1999).
Fig 2. The tree Ginkgo biloba. Available in: < http://forestry.about.com/od/ forestphotogalleries/ig/Ginkgo-Biloba/Moses-Cone-Ginkgo.htm >
The tree Ginkgo biloba is also known for its great genetic resistance, being highly resistant to insects, microorganisms and environmental toxins (Smith & Luo, 2004; Nakanishi, 2005). That resistance was proved in the Japanese city Hiroshima, since G. biloba was the first plant to grow after the atomic bomb explosion (Tesch, 2003).
The term Ginkgo was first used by the German physician and botanist Engelbert Kaempfer in 1712, but Linnaeus provided the terminology Ginkgo biloba in 1771 (Nakanishi, 2005).
Standardized Extract
The extracts preparations of Ginkgo biloba (GBE) are usually accomplished with either fresh or dried leaves and using acetone and water for the final product (Van Beek, 2002). The standardized extract is commonly administered via oral under liquid or tablet forms (Who, 1999).
The standard extract usually contains from 22 to 27% of flavonol glycosides (flavonols: quercetin, kaempferol (Fig 3) and isorhamnetin; biflavones: bilobetin, ginkgetin, isoginkgetin and sciadopitysin) and from 5 to 7% of diterpene lactones (ginkgolides: A, B, C and J; sesquiterpene lactone: bilobalide) (Who, 1999; Van Beek, 2002, 2005; Xie et al., 2006; Ding et al., 2008; Xie et al., 2008). The alkylphenols are also present in GBE, predominantly ginkgolic acids, and ginkgols. Due to the allergenic, cytotoxic, mutagenic and neurotoxic properties of these substances, their concentration in extracts is limited to 5 parts per million, an amount that does not cause toxicity (Who, 1999; Baron-Ruppert & Luepke, 2001).
Fig 3. Chemical structures of quercetin and kaempferol, the main flavonols found in GBE
GBE flavonols are absorbed and metabolized, and its metabolites are detected in urine, faeces and blood of Wistar rats (Pietta et al., 1995) as well as in human urine after GBE extract oral intake (Pietta et al., 1997).
Properties and mechanisms of action
Ginkgo biloba has been used as a medicinal plant through centuries. The first written record of clinical use of Ginkgo comes from more than 5000 years ago, in ancient China, where Chen Noung (2767 to 2687 AD) reported the medicinal properties of the plant (Diamond et al., 2000). There are reports that Ginkgo biloba was used in the treatment of senility in aging members of the Chinese royal court in 1578 AD (Nakanishi, 2005). The indications included the treatment of heart and lungs maladies through G. biloba steam inhalation or by tea intake (Diamond et al., 2000). The tea was used for treating asthma, bronchitis (Kleijnen & Knipschild, 1992), cough and enuresis (Smith & Luo, 2004).
Nowadays, the Ginkgo biloba leaf extract has been widely used by population for the treatment of several diseases (Mahadevan & Park, 2008). The popular use of GBE include the treatment of inflammation, allergy (Blumenthal, 2003), bronchitis, chronic rhinitis, arthritis, edema and as a vermifuge and on labor induction (Who, 1999). Recent studies indicate neuroprotective properties (Calapai et al., 2000; Ahlemeyer & Krieglstein, 2003; Li et al., 2003; Paganelli et al., 2006), anticarcinogenic (Defeudis et al., 2003; Kim et al., 2005; Chang et al., 2006; Ye et al., 2007; Dias et al., 2008; Zhang et al., 2008), hepatoprotective (Welt et al., 2004; Naik & Panda, 2008) and cardioprotective activities (Pietri et al., 1997; Rioufol et al., 2003; Yao et al., 2004; Rodriguez et al., 2007; Mahadevan et al., 2008; Panda & Naik, 2008; Wu et al., 2008).
Currently, the main indications for the use of Ginkgo biloba are the following pathologies: ´cerebral insufficiency’ (confusion, reduced memory, distraction, dizziness, buzz, headache, low energy, depression, fatigue, anxiety and decreased physical activity); Alzheimer’s disease; peripheral vascular diseases (intermittent claudication, deep venous thrombosis) and central vascular diseases (Who, 1999; Blumenthal, 2003; Chan et al., 2007).
The use of GBE is due mainly to: its activity on inhibiting the platelet activating factor (Koch, 2005) and on the important antioxidant property (Naik & Panda, 2008; Panda & Naik, 2008). In the former activity, Ginkgo biloba prevents platelet aggregation and causes an increased blood flow (Blumenthal, 2003; Koch, 2005). As an antioxidant, GBE induces a decrease of reactive oxygen species (Blumenthal, 2003) besides attenuating membrane lipid peroxidation (Panda & Naik, 2008).
Furthermore, other GBE activities which could be related to the clinical effects are: inhibition of nitric oxide synthesis (Calapai et al., 2000); increase in serotonine release and reuptake; inhibition of amyloid beta deposition (Blumenthal, 2003); increased neuronal plasticity (Williams et al., 2004); augment of glucose uptake and consume by the brain (Dubey et al., 2004).
Adverse Effects
Although Ginkgo biloba extract is generally well tolerated, it is contraindicated to people who are allergic to the phytotherapic, as well as to patients using anticoagulants or anyone that will be submitted to a surgery, in order to avoid hemorrhage (Who, 1999; Blumenthal, 2003). Care should also be taken regarding drugs interactions (Gaudineau et al., 2004), since GBE might potentialize the action of antiplatelet agents, such as aspirin and of anticoagulants as warfarin, and also thiazide-like diuretics (Blumenthal, 2003).
Some adverse effects related after GBE use are gastrointestinal disorders, as diarrhea, nausea and vomit, and vascular disorders, as headache, allergic cutaneous reaction, vertigo and stroke (Who, 1999; Ernst, 2002; Blumenthal, 2003).
Reproductive Toxicology
Currently, Ginkgo biloba use is contraindicated during pregnancy and lactation, due to the lack of studies with GBE during these periods (Who, 1999; Blumenthal, 2003; Dugoua et al., 2006). Some in vitro and in vivo studies were accomplished in order to assess the reproductive toxicity of GBE.
Male reproductive toxicity
Al-Yahya et al. (2006) evaluated Ginkgo biloba extract for its effects on reproductive toxicity in male Swiss albino mice. The treatment of mice with growing doses of GBE (25, 50 and 100 mg/kg/day) for 90 days by oral gavage, caused significant increase in the mean weight of caudae epididymis at 50 and 100 mg/kg, and of prostate at 100 mg/kg of GBE. Although the percent motility, sperm count and morphology of spermatozoa were not affected; chromosomal studies demonstrated increased aneuploidy and other chromosomal aberrations in animals receiving the highest dose of GBE. After mating these mice with reproductive female, it was found a decreased rate of pregnancy and it caused embryonic losses before implantation at 100 mg/kg of GBE. As those results indicate the possibility of occurring malformations and infertility after GBE intake, it is recommended caution on Ginkgo biloba use (Al-Yahya et al., 2006).
Toxicity during fertilization period
The toxicity of Ginkgo biloba extract on reproductive cells were evaluated through the incubation of hamster oocytes in 0.1 and 1 mg/ml of GBE solutions during one hour and later, those oocytes have been inseminated by human spermatozoa. Spermatozoa were also incubated in the same GBE solutions during seven days in 23 °C, with later analysis of DNA integrity. Although GBE did not cause damage to spermatozoa DNA, it produced a numerically lower percentage of sperm penetration and also a decreased capacitation index, as well as it caused oocyte degeneration (Ondrizek et al., 1999a). In another experiment, the same authors, when analyzing the effect of GBE on spermatozoa kinetics, have found decrease and even inhibition of motility when spermatozoa were incubated in 1.2 mg/ml during 24 hours (Ondrizek et al., 1999b).
Another sign of cellular toxicity of GBE was demonstrated by Li et al. (1997). In this work, they studied the effect of the flavonoids kaempferol and quercetin, substances present in Ginkgo biloba exctract, on the activity of hyaluronidase and on cumulus oophorus penetration by monkey sperm. This study demonstrated that when the male gametes are incubated in GBE solution, both kaempferol and quercetin inhibit the activity of hyaluronidase from monkey spermatozoa. It also inhibits sperm penetration into hamster cumulus, being able to interfere in the reproductive process (Li et al., 1997).
Embryonic toxicity
In order to evaluate the toxicity of GBE in embryo, mice embryos were collected and cultivated in solutions containing 5 and 10 mM ginkgolide A or ginkgolide B, the major components of Ginkgo biloba extracts. The results indicated that ginkgolide treatment of mouse embryo induces apoptosis, decreases cell numbers and interferes in the development of embryo cultivated with both concentrations of ginkgolide A and B (Chan, 2005). In another study, the embryonic toxicity of ginkgolide B was assessed in vitro and in vivo. Treatment of embryonic stem cells with ginkgolide B induced apoptosis via reactive oxygen species generation, c-Jun protein phosphorylation and loss of mitochondrial membrane potential. Furthermore, it was showed that ginkgolide B induced a decrease in the number of cells, together with an increased level of apoptosis in cells of mice embryo. Besides the in vitro toxicity, when embryo were treated with ginkgolide B and transferred to uterus of mice, there was a decreased number of implantations and of live fetuses, an augment of resorptions, and a diminished fetal weight. Furthermore, it was demonstrated that ginkgolide B intake can cause harm to embryo, since the embryo collected from animals that received diet supplemented with this substance also showed decreased cell proliferation and increased apoptosis (Chan, 2006).
Toxicity during organogenesis and fetogenesis
There are signs that GBE could be toxic if administered during the period of organogenesis and fetogenesis. Fetuses from Wistar, whose mothers were treated with 7 and 14 mg/kg body weight/day of G. biloba, by gavage, from the 8th to 20th day of pregnancy, had decreased liver and body weights. However the extract did not cause maternal toxicity (Pinto et al., 2007).
The results presented here reinforce the possible occurrence of infertility, embryo death and malformations following the use of Ginkgo biloba extract. Herewith, pregnant women should avoid consuming GBE until more studies are accomplished to better evaluate the effects of the extract.
Hormonal Effects
Ginkgo biloba has been widely used by population for treating hormonal disturbs, menopausal symptoms (Kam et al., 2002; Gold et al., 2007) and even to help in the treatment of sexual dysfunctions (Ashton et al., 2000; Waynberg & Brewer, 2000; Rowland & Tai, 2003). Although important clinical studies have not been accomplished yet, there are some evidences to support the use of Ginkgo biloba to treat the previously cited disturbs.
In vitro estrogenic effect
The estrogenic action of Ginkgo biloba was suspected since studies demonstrated that quercetin and kaempferol, the main flavonols found in GBE showed estrogenic effects, which were determined in one in vitro assay, through the binding of flavonols to the human recombinant estrogen receptor, and also through the induction of cell proliferation in a human breast cancer (MCF-7 cells) (Han et al., 2002). Then, in 2004, Oh & Chung proved the estrogenic effect of Ginkgo biloba and of its major flavonols (quercetin, kaempferol and isorhamnetin). The substances present in the extract bound directly to the estrogen receptor, showing higher affinity to beta receptor when compared to the alpha receptor, and also inhibited the binding of estradiol to both alpha and beta receptors, what indicates a possible competition for the same receptors. In this experiment, another proof of estrogenic effect of Ginkgo biloba was the induction of cell proliferation in breast cancer cells (MCF-7) and of pS2 gene expression in these cells, which are regulated by the estrogen response pathway (Oh & Chung, 2004). The same authors have demonstrated later that Ginkgo biloba, besides presenting estrogenic effect, also exhibits antiestrogenic effect. It was observed that the low concentrated extract induces cell proliferation in MCF-7 cells, but as the concentration of the extract is increased, it inhibits cell proliferation, such effect is similar to that of tamoxifen, an estrogen antagonist. Moreover, the authors proved that high concentration of GBE inhibit the pS2 gene expression, which responds to estrogenic stimulus and inhibits the aromatase enzyme, which is responsible for the conversion of androgens to estrogens in several human tissues (Oh & Chung, 2006). These results indicate the possibility of GBE use in the treatment of menopause symptoms and also as an adjuvant therapy in estrogen-dependent cancers.
In vivo estrogenic effect
GBE was given to animals in order to evaluate a possible estrogenic effect. Lee et al. (2000) administered solutions bearing 1.4 and 2.8 mg of GBE to female ovariectomized mice, via intraperitoneal injection, during 15 days. There was no significant difference in endometrial thickness, neither in mammary diameter, but the treatment caused changes in vaginal epithelium compatible with the estrogenic phase, what was probably induced by the estrogenic effect of the extract (Lee et al., 2000).
Another sign of estrogenic action was found in a study with rats. In this experiment, the treatment with GBE was similar to estrogen preventing cognitive dysfunction in ovariectomized females, which were submitted to restraint stress. The treatment also reduced the loss of hippocampal CA3 neurons of the animals, suggesting estrogenic effect (Takuma et al., 2007). Currently it is known that the estrogenic effect of GBE is mainly due to the action of its major components: quercetin and kaempferol. The estrogenic effect of kaempferol was mainly detected in immature female mice. Immature female mice, which received 100 mg/kg of kaempferol via oral gavage during 4 days, did not present alterations in uterine weight; however, it was found to significantly increase the overall uterine concentration of estrogen receptor alpha, what could indicate an increased sensibility of the uterus to circulating estrogens (Breinholt et al., 2000). Moreover, the administration of kaempferol only or together with 17-b-estradiol to immature and ovariectomized rats, during four days, significantly increased both absolute and relative uterine weights, suggesting a synergistic effect of the two substances; such effect was found only in immature animals. The analysis of vaginal smears from immature rats revealed an increased cornification in animals receiving kaempferol only or in combination with estrogen, suggesting that it potentialized the uterotrophic effect of 17-b-estradiol (Stroheker et al., 2003).
Effect on bone metabolism
The action of Ginkgo biloba and its components have also been studied for its abilities to interfere with the bone metabolism, since it presents estrogenic effect, it could be useful in the treatment of osteoporosis.
An in vitro study was accomplished to investigate the estrogenic effect of quercetin and kaempferol on osteoblasts. Ostoblasts were incubated in different concentrations of substances and it was found an increased production of alkaline phosphatase and also of cell proliferation. Those results were similar to the ones found when the same cells were treated with 17-b-estradiol. It was also demonstrated that when ICI 182780, an antagonist of estrogen, was present in cell cultures, it inhibited the production of alkaline phosphatase, confirming that the estrogenic effect of flavonols was the responsible for the previously cited results (Prouillet et al., 2004). The estrogenic effect of kaempferol on bone metabolism was also validated by Trivedi et al. (2008). They found and increased mineralization in in vitro cultures of osteoblasts and also a higher bone mineral density in rats treated with 5 mg/kg/day of kaempferol (Trivedi et al., 2008).
Besides of its major component kaempferol, the whole G. biloba extract, was analyzed regarding its estrogenic effect on bone metabolism. Osteoblasts were incubated in different concentrations of GBE (25, 50, 100, 150, 200 and 250 mg/ml) and it was found an increased production of alkaline phosphatase at the doses of 50, 100 and 150 mg/ml. On the in vivo study, female rats were ovariectomized and after 10 weeks, they received 25, 50, 100 and 200 mg/kg/day of GBE via oral gavage. The animals that received 100 and 200 mg/kg/day of GBE presented decreased osteoclast resorptive activity (Oh et al., 2008).
Effect on sexual behavior
In addition to the estrogenic effect, Ginkgo biloba seems to interfere with other hormones. Another study evaluated the sexual behavior of male rats treated with GBE (10, 50, or 100 mg/kg/day), via oral gavage, during 28 days. The sexual behavior was analyzed after 7, 14, 21 and 28 days of treatment, as well as were obtained the weight of reproductive organs, the number of spermatozoa, the serum levels of testosterone and prolactin and the cerebral levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). The treatment with GBE did not cause alterations in rat’s organs weights. However, there was increased intromission frequency in animals of treated with 50 mg/kg after 28 days, and also in rats that received 100 mg/kg/day of GBE after 14 and 21 days. Moreover, animals treated with 50 mg/Kg of the extract showed an increase in ejaculation frequency after 14, 21 and 28 days of treatment. Regarding the hormonal serum levels, the treatment did not change the testosterone level, however, the prolactin level of animals which received 50 mg/kg/day of GBE was significantly reduced when compared to control group. In the same group, it was noted a significant increase in cerebral dopamine level, without a concomitant increase of its metabolite DOPAC. The results indicate that the treatment with Ginkgo biloba can improve sexual dysfunction through the decrease of prolactin serum levels. In the same experiment, the authors also evaluated a culture of Leydig cells at different concentrations of GBE (50, 100, 250, 500, or 1000 mg/ml), finding a significant increase in the production of testosterone by the Leydig cells at 1000 mg/ml of GBE (Yeh et al., 2008).
Human studies
Paulus et al. (2002) carried out a clinical trial with 45 women, which had been diagnosed with reduced uterine arterial blood flow and who had no success in infertility treatments. After the treatment with 243 mg/day of GBE, patients showed a thickened endometrium, without a concomitant increase in uterine and ovarian blood flow (Paulus et al., 2002), suggesting the possible estrogenic action of G. biloba.
Moreover, the extract was physiologically and psychologically tested in the treatment of sexual dysfunctions in women. Meston et al. (2008) analyzed the effects of both short- and long-term GBE administration. In the first part of the experiment, 99 women received 300 mg of GBE, and then after 90 minutes the level of sexual arousal was physiologically measured, through vaginal photoplethysmography, and subjectively measured, through a questionnaire. It was found that GBE induced a small but significant facilitatory effect on physiological sexual arousal, but not subjective sexual arousal. The long-term study assessed women who were divided into four groups, receiving placebo, 300 mg of GBE, sex therapy or sex therapy plus GBE. GBE treatment alone was not superior to placebo on improving sexual function. However, when combined with sex therapy GBE treatment significantly increased sexual desire and contentment beyond placebo, being therefore, able to be used in the treatment of women with sexual dysfunctions (Meston et al., 2008).
There are few studies currently regarding the hormonal and reproductive effects of GBE in human. Therefore, more clinical trials are necessary to determine the action of Gingko biloba extract on the organism.
Acknowledgements
This work was supported by REDE Mineira de Bioterismo – FAPEMIG, MG, Brazil and REDE Mineira de Farmacologia e Toxicologia – FAPEMIG, MG, Brazil.
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