Research Advances in Extraction and Functions of Ginkgo Flavonoids

Jumin XIE, Mingchun QUAN, Zhenhong SU, Xiaolin XIE, Dawei FANG

1. Medical School of Hubei Polytechnic University, Huangshi 435003, China; 2. Hubei Key Laboratory of Renal Disease Occurrence and Intervention, Hubei Polytechnic University, Huangshi 435003, China; 3. Department of Medical, Huangshi Psychiatric Hospital, Huangshi 435003, China; 4. General Surgery Department, the Second People’s Hospital of Huangshi, Huangshi 435000, China

Abstract Ginkgo flavonoids are one of the most effective medicinal components extracted from leaves of Ginkgo biloba L. According to the physical and chemical properties of ginkgo flavonoids, they can be separated and purified by resin adsorption, supercritical extraction and solvent methods. Previous studies have proved that ginkgo flavonoids have pharmacological functions in promoting blood circulation, anti-oxidation, anti-aging, improving memory, anti-tumor and liver protection, etc. Ginkgo flavonoids have been used clinically to treat angina pectoris, coronary heart disease, myocardial ischemia, hypertension, hyperlipidemia, diabetes, alzheimer’s disease, atherosclerosis, gastric cancer and ovarian malignancy and other difficult miscellaneous diseases.

Key words Ginkgo flavonoids, Pharmacological functions, Separation and purification, Disease treatment

1 Introduction

GinkgobilobaL. tree is also called the white fruit tree. It has existed since thousands of years ago and has a long history in China, so it is also called "living fossil"[1]. The main medicinal chemical component ofG.bilobaL. trees is ginkgo flavonoids, and different types of ginkgo flavonoids are obtained according to the tree species, place of production, picking time and age of the tree[2-3]. Because of good antioxidant, antiviral, anti-tumor, cardiovascular system protection, and blood lipid regulation functions, ginkgo flavonoids are widely used in various fields of modern medicine[4-5].

2 Ginkgo flavonoids

G.bilobaL. tree is one of the oldest tree species in China. According to records ofDieteticMateriaMedica,G.bilobaL. leaves can be used for treating palpitations and cough. According to the theory of traditional Chinese medicine, it is said thatG.bilobaL. leaves have the effects of promoting blood circulation and removing blood stasis, and unblocking the meridian and activating collaterals[6]. The chemical components of extracts fromG.bilobaL. leaves are very complicated. The most important active component is flavonoids. Ginkgo flavonoids are mainly derivatives of chroman and chromone, including diflavones, bilobetin, quercetin, catechins,etc.[7], their corresponding structure are shown in Fig.1.

Fig.1 Structural formulas of ginkgo flavonoids

Ginkgo flavonoid aglycone is a fat-soluble substance, generally insoluble or insoluble in water, and easily soluble in organic solvents such as methanol, ethanol, chloroform and ethyl acetate. Because flavonoids generally have phenolic hydroxyl groups[8], flavonoids have acidic properties and are soluble in dilute alkali solutions. Flavonoid aglycones can be linked with sugars to form glycosides. After glycoside formation, the proportion of polar groups increases and water solubility increases. Then flavonoid aglycones are soluble in hot water, methanol, ethanol and other polar solvents, but hard to dissolve in organic solvents with low polarity such as cyclohexane and petroleum ether[9].

3 Extraction and purification of ginkgo flavonoids

Ginkgo flavonoids are one of the main medicinal components inG.bilobaL. leaves, andG.bilobaL. leaves also contain terpene lactones and other components[10]. According to the physical and chemical properties of flavonoids, resin adsorption, supercritical extraction and solvent methods are generally used to extract flavonoids[11].

3.1 Resin adsorption methodMacroporous resin adsorption method has relatively high selectivity for ginkgo flavonoids, so the purity of ginkgo flavonoids obtained is higher. At present, polystyrene, activated carbon, polyamide adsorption resin, macroporous resin and silica gel adsorption resin are commonly used to extract, separate and purify ginkgo flavonoids[12]. According to the structural characteristics of ginkgo flavonoids, some metal ions can be loaded in the resin to form a coordinated adsorbent, so that the adsorption selectivity for ginkgo flavonoids is significantly improved. Since the structure of ginkgo flavonoids contains polar groups such as phenolic hydroxyl groups, a certain amount of absolute ethanol can be added to the solvent to increase its solubility. This method is used to extract ginkgo flavonoids in a non-aqueous system, and the purity is greatly improved[13].

In addition, the polyamide resin adsorption method is also a better method. This method uses hydrogen bond association adsorption between polyamide and ginkgo flavonoids to separate and extract compounds, which show different degrees of adsorption capacity according to the number of phenolic hydroxyl groups of the flavonoids[14]. This method is widely used in industrial production, but due to the complex composition of traditional Chinese medicine, impurities may also contain phenolic hydroxyl groups, so the selectivity of this method is not very high, and the purity of ginkgo flavonoids obtained is also lower than that of complex adsorption[15].

3.2 Supercritical fluid extraction methodThe supercritical fluid extraction method has high selectivity, good solubility, and no solvent residue, so the purity of the product obtained is relatively high. The supercritical extraction operating conditions are relatively mild, and the thermally unstable active components can be extracted by this method. Critical temperature, critical pressure, CO2flow rate, co-solvent,etc. have certain effects on supercritical extraction. Therefore, certain compounds can be separated and purified by changing the critical temperature, critical pressure, extraction medium flow rate, and co-solvent type[16]. When using this method to extract ginkgo flavonoids, it should be noted that ginkgo flavonoids contain more phenolic hydroxyl groups and have a certain polarity. The extraction medium is non-polar, and the solubility of the extraction medium to ginkgo flavonoids is relatively low, so it is necessary to add a co-solvent. Ethanol is the co-solvent that increases the solubility, has the least impact on the extraction of ginkgo flavonoids and is cheaper and available[17]. Therefore, ethanol can be used as a co-solvent and CO2as the extraction medium to extract ginkgo flavonoids using supercritical fluid extraction method[18].

3.3 Solvent extraction methodSolvent extraction is a commonly used method in the separation and extraction of some compounds. A study indicates that using water as an extracting agent and heating to reflux can extract flavonoids[19]. Although this method is simple to operate, the selectivity is low, the extracted compounds are low in purity. Generally, after this method is used to extract the target product, it needs to be refined by methods such as column chromatography.

In addition, according to the weakly acidic nature of ginkgo flavonoids, it can be first extracted with organic solvents and then purified by alkali-dissolved acid precipitation. Then, depending on its polarity, it may form hydrogen bonds or coordination bonds with certain substances, covalent bonds and other special chemical bonds or forces, use column chromatography with higher selectivity to make further purification[20].

4 Pharmacological effects of ginkgo flavonoids

4.1 Promoting blood circulationGinkgo flavonoids have the effect of promoting blood circulation and can inhibit platelet aggregation caused by platelet activating factor (PAF)[21]. High concentration of PAF not only damages nerve cells, reduces blood flow in the brain, triggers inflammation[22], but also accelerates aging. Ginkgo flavonoids can significantly increase high-density lipoprotein (HDL) content, lower plasma cholesterol, regulate blood lipids, reduce blood viscosity, and improve blood circulation. According to some study[23], ginkgo flavonoids can reduce the area and degree of myocardial infarction to a certain extent, and have a good therapeutic effect on alleviating acute myocardial ischemia caused by pituitary gland.

4.2 Anti-oxidation and anti-agingStudies have shown that Ginkgo flavonoids have a strong antioxidant effect, can not only scavenge oxygen free radicals and increase the activity of superoxide dismutase in the human body, but also play an anti-lipid peroxidation function. Ginkgo flavonoids inhibit the synthesis of NADPH family oxidases such as NOX2 and NOX4, thereby inhibiting the generation of free radicals and playing an antioxidant role[24].

Brain cells are damaged by the attack of oxygen free radicals, which is one of the factors that accelerate aging and the symptoms of Alzheimer’s disease[25]. Lipid oxidation is another important factor that accelerates human aging. Lipid oxidation is a free radical chain reaction, and the secondary metabolites produced by it can damage the cell membrane to a certain extent[26]. Ginkgo flavonoids have the effect of scavenging free radicals and can donate hydrogen to lipid free radicals, thereby slowing the oxidation of lipids[27]. On the other hand, flavonoids can chelate with metal ions, thereby promoting the decomposition of lipid peroxides, thereby inhibiting the damage of peroxides to cells[28]. As an important signal transmission molecule, nitric oxide has a dual role in the central system. When the content of nitric oxide in brain tissue is too high, it will play a cascade amplification effect on the generation of oxygen free radicals. Ginkgo flavonoids reduce the production of nitric oxide, reduce the damage of free radicals to cell membranes, inhibit the apoptosis of nerve cells, and slow down aging[29].

4.3 Anti-tumorTumor cells proliferate faster and have higher nutritional requirements, so the blood vessels surrounding the tumor tissue are relatively abundant. Vascular endothelial growth factor (VEGF) produced by paracrine can stimulate the proliferation, migration and angiogenesis of vascular endothelial cells, which plays an important role in tumor tissue angiogenesis[30]. The use of ginkgo flavonoids to interfere with tumor tissues decreased the expression of VEGF and inhibited the formation of new blood vessels around tumor tissues, thereby achieving the tumor suppression[31]. Epidermal growth factor receptor (EGFR) also plays an important role, it regulates the proliferation and apoptosis of tumor cells, and has a certain contribution to tumor angiogenesis[32]. In addition, phosphorylation of AKT protein related to cell proliferation can regulate the cell cycle and promote the division and proliferation of tumor cells[33]. A study[31]shows that EGFR and other protein molecules related to cell proliferation are highly expressed in tumor tissues. After the intervention of ginkgo flavone, the expression level of EGFR decreased significantly, and the proliferation and migration of tumor cells were also inhibited.

4.4 Liver protectionIn daily life, there are various factors that can cause liver damage. A study[34]shows that ginkgo flavonoids can activate various cell regulatory factors, increase the expression levels of alanine aminotransferase and aspartate aminotransferase, reduce the generation of free radicals, and thus exert a certain protective effect on liver cells. After intervention with ginkgo flavonoids in rats with non-alcoholic fatty liver disease, the expression levels of triglycerides, low-density lipoprotein-cholesterol, and free fatty acids in the liver were increased, and the expression levels of some cytokines involved in inflammation were also increased. Therefore, ginkgo flavonoids can regulate the levels of liver fat and inflammatory factors, thereby reducing the liver damage[35].

5 Discussion and prospects

Ginkgo flavonoids are main chemical components ofG.bilobaL. that exerts its medicinal value. It has anti-oxidation, dilation of blood vessels, and anti-tumor effects. These effects are used in the clinical treatment of liver injury, Alzheimer’s disease, myocardial ischemia, gastric cancer, and other diseases.

Although ginkgo flavonoids have fewer side effects, long-term use will also inhibit the function of coagulation factors, and cause coagulation disorders to cause internal bleeding and other undesirable consequences, so be careful not to take too long[36]. Although the functions of ginkgo flavonoids have been clear, the mechanism of some functions is still unclear. Thus, it is still necessary to make further research. Only a clear understanding of the specific mechanism of action of each function can provide more accurate clinical methods for safe medication. In this paper, we summarized the extraction methods and pharmacological effects of ginkgo flavonoids, to provide certain reference and guidance for the extraction and application of active pharmaceutical components in traditional Chinese medicine.