How to Extract Ginkgo Flavone?
The ginkgo tree, also known as the white fruit tree, has existed for tens of millions of years. It has a long history in China, and is therefore also known as a “living fossil” [1]. The main medicinal chemical component of the ginkgo tree is ginkgo flavone, and there are different types of ginkgo flavone depending on the species, origin, harvest time and age of the tree [2-3]. Ginkgo flavonoids are widely used in various fields of modern medicine because of their good antioxidant, antiviral, antitumor, cardiovascular protection, and lipid-regulating properties [4-5].
1 Ginkgo flavonoids
Ginkgo is one of the oldest tree species in China. According to the “Dietary Herbal”, ginkgo leaves can be used to treat palpitations, coughing and shortness of breath. Traditional Chinese medicine says that ginkgo biloba leaves have the effect of activating blood circulation and removing blood stasis, and relieving menstrual pain and activating collaterals [6]. The chemical composition of ginkgo biloba leaf extract is very complex. The main active ingredients are flavonoids. Ginkgo flavonoids are mainly derivatives of chromane and chromone, including biflavones, bilobetin, quercetin, catechin, etc. [7], and their corresponding structures are shown in Figure 1.
Ginkgo flavonoids are fat-soluble substances that are generally poorly or insoluble in water, but are easily soluble in organic solvents such as methanol, ethanol, chloroform and ethyl acetate. Since flavonoids generally have phenolic hydroxyl groups [8], they are acidic in nature and can be dissolved in dilute alkaline solutions. Flavonoid glycosides can be linked to sugars to form glycosides. After glycosidation, the proportion of polar groups increases, and water solubility increases. Therefore, they are soluble in hot water, methanol, ethanol and other polar solvents, but are difficult to dissolve in organic solvents with lower polarity such as cyclohexane and petroleum ether [9].
2 Extraction and purification of ginkgo flavonoids
Ginkgo flavones are one of the main medicinal ingredients in ginkgo leaves. In addition, ginkgo leaves also contain terpene lactones and other ingredients [10]. According to the physical and chemical properties of flavonoids, flavonoids are generally extracted using methods such as resin adsorption, supercritical extraction and solvent methods [11].
2.1 Resin adsorption method
The macroporous resin adsorption method has relatively high selectivity for ginkgo flavonoids, so the purity of the obtained ginkgo flavonoids is relatively high. Currently, polystyrene, activated carbon, polyamide adsorption resins, macroporous resins, and silica gel adsorption resins 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 on the resin to form a coordination adsorbent, which significantly improves the adsorption selectivity of ginkgo flavonoids. Since the structure of ginkgo flavonoids contains polar groups such as phenolic hydroxyl groups, a certain amount of anhydrous ethanol can be added to the solvent to increase its solubility. This method is used to extract ginkgo flavonoids in non-aqueous systems, and the purity has been greatly improved [13].
In addition, the polyamide resin adsorption method is also a relatively good method. This method uses polyamide to form hydrogen bonds with ginkgo flavones to adsorb and extract the compounds. The adsorption capacity varies depending on the number of phenolic hydroxyl groups in the flavones [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. Therefore, this method is not very selective, and the purity of the ginkgo flavone obtained is also lower than that of the coordination adsorption [15].
2.2 Supercritical extraction method
Supercritical fluid extraction has high selectivity, good solubility, and no solvent residue, so the purity of the product obtained is also relatively high. Supercritical extraction has relatively mild operating conditions and can be used to extract heat-labile active ingredients. The critical temperature, critical pressure, CO2 flow rate, cosolvent, etc. all have a certain effect on supercritical extraction, so certain compounds can be separated and purified by changing the critical temperature, critical pressure, extraction medium flow rate, cosolvent type, etc. [16]. When using this method to extract ginkgo flavonoids, it should be noted that ginkgo flavonoids contain a lot of phenolic hydroxyl groups and are polar in nature. The extraction medium is non-polar, and the solubility of ginkgo flavonoids in the extraction medium is relatively low. A cosolvent needs to be added to increase the solubility. The cosolvent that has the least effect on the extraction of ginkgo flavonoids and is relatively cheap and readily available is ethanol [17]. Therefore, ethanol can be used as a cosolvent, and supercritical fluid extraction can be used with CO2 as the extraction medium to extract ginkgo flavonoids [18].
2.3 Solvent extraction method
Solvent extraction is a commonly used method for the isolation and extraction of some compounds. Some studies have shown [19] that flavonoids can be extracted by heating with water as the extracting agent. Although this method is simple to operate, it has low selectivity, resulting in the low purity of the extracted compounds. Generally, after extracting the target product using this method, it needs to be refined using methods such as column chromatography.
In addition, according to the weakly acidic nature of ginkgo flavonoids, they can be extracted with an organic solvent and then purified using an alkaline solvent-acid precipitation method. Then, depending on their polarity, they can be further purified using a more selective column chromatography method, or they can form hydrogen bonds, coordination bonds, covalent bonds and other special chemical bonds or forces with certain substances [20].
3 Pharmacological effects of ginkgo flavonoids
3.1 Promotes blood circulation
Ginkgo biloba flavonoids have a blood circulation-promoting effect and can inhibit platelet aggregation caused by platelet-activating factor (PAF) [21]. High concentrations of PAF not only damage nerve cells, reduce blood flow to the brain, and trigger inflammation [22], but also accelerate aging. Ginkgo biloba flavonoids can significantly increase high-density lipoprotein (HDL) levels, reduce plasma cholesterol, regulate blood lipids, reduce blood viscosity, and improve blood circulation. Studies have shown [23] that ginkgo biloba flavonoids can reduce the size and extent of myocardial infarction to a certain extent, and have a good therapeutic effect on relieving acute myocardial ischemia caused by pituitary posterior lobe hormone.
3.2 Antioxidant and anti-aging
Studies have shown that ginkgo flavonoids have strong antioxidant properties. They can not only scavenge oxygen free radicals and increase the activity of superoxide dismutase in the body, but also prevent lipid peroxidation. Ginkgo flavonoids inhibit the synthesis of NADPH family oxidases such as NOX2 and NOX4, thereby inhibiting the production of free radicals and playing an antioxidant role [24].
Brain cell damage caused by attacks from oxygen free radicals is one of the factors that accelerates aging and the onset of Alzheimer's symptoms [25]. Lipid oxidation is another important factor that accelerates human aging. Lipid oxidation is a free radical chain reaction, and the secondary metabolites it produces damage cell membranes to a certain extent [26]. Ginkgo biloba flavonoids have the effect of scavenging free radicals. They can supply hydrogen to lipid radicals, thereby slowing lipid oxidation [27]. On the other hand, flavonoids can chelate metal ions, thereby promoting the decomposition of lipid peroxides and thus inhibiting peroxide damage to cells [28]. Nitric oxide is an important signaling molecule that plays a dual role in the central nervous system. When the nitric oxide content in brain tissue is too high, it can have a cascading amplifying effect on the production of oxygen free radicals. Ginkgo biloba flavonoids can reduce nitric oxide production, reduce free radical damage to cell membranes, inhibit apoptosis of nerve cells, and slow down aging.
3.3 Anti-tumor
Tumor cells proliferate rapidly and have high nutritional requirements, so the blood vessels around tumor tissue are relatively abundant. Vascular endothelial growth factor (VEGF), produced by paracrine secretion, can stimulate the proliferation, migration and angiogenesis of vascular endothelial cells, and plays an important role in the angiogenesis of tumor tissue [30]. After intervention with ginkgo flavonoids in tumor tissue, VEGF expression levels decreased, inhibiting the formation of new blood vessels around the tumor tissue and thus achieving the goal of inhibiting the tumor [31]. The epidermal growth factor receptor (EGFR) also plays an important role, regulating the proliferation and apoptosis of tumor cells and contributing to the formation of new blood vessels in tumors [32]. In addition, the AKT protein, which is involved in cell proliferation, can regulate the cell cycle and promote the division and proliferation of tumor cells after phosphorylation [33]. Studies have shown [31] that protein molecules involved in cell proliferation, such as EGFR, are highly expressed in tumor tissue. After intervention with ginkgo flavonoids, the expression level of EGFR decreased significantly, and the proliferation and migration of tumor cells were also inhibited.
3.4 Liver protection
There are various factors in life that can cause liver damage. Some studies have shown [34] that ginkgo flavonoids can activate various types of cell regulatory factors, increase the expression levels of alanine aminotransferase and aspartate aminotransferase, and reduce the production of free radicals, thereby providing a certain degree of protection for 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 all increased, as were the expression levels of some cytokines involved in inflammatory responses. Therefore, ginkgo flavonoids can regulate the levels of liver fat and inflammatory factors, thereby reducing liver damage [35].
4 Discussion and outlook
Ginkgo flavonoids are the main chemical constituents of ginkgo biloba that exert medicinal value. They have antioxidant, vasodilatory, and anti-tumor effects, and are used in clinical treatments for liver damage, Alzheimer's disease, myocardial ischemia, gastric cancer, and other diseases.
Although ginkgo flavonoids have relatively few side effects, long-term use can also inhibit the function of blood coagulation factors, leading to adverse consequences such as coagulation disorders and internal bleeding. Therefore, it is important to note that the duration of use should not be too long [36]. Although the functions of ginkgo flavonoids have been clearly defined, the mechanisms of some functions are still unclear and require further research. Only by clearly understanding the specific mechanisms of action of each function can more accurate and safer methods of drug use be provided for clinical practice. This paper reviews the extraction methods and pharmacological effects of ginkgo flavonoids, providing a certain reference and guidance for the extraction and application of active pharmaceutical ingredients in traditional Chinese medicine.
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