molecules Review Advances in Supercritical Carbon Dioxide Extraction of Bioactive Substances from Different Parts of Ginkgo biloba L. Ruihong Li 1,2,† , Ziming Xia 2,†, Bin Li 2, Ying Tian 2, Guangjie Zhang 2, Min Li 2,* and Junxing Dong 2,* 1 School of Pharmacy, Henan University, Kaifeng 475000, China; [email protected] 2 Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing 100850, China; [email protected] (Z.X.); [email protected] (B.L.); [email protected] (Y.T.); [email protected] (G.Z.) * Correspondence: [email protected] (M.L.); [email protected] (J.D.); Tel.: +86-010-6693-2294 (M.L.); +86-010-6693-1314 (J.D.) † These authors contributed equally to this work. Abstract: Ginkgo biloba L. has always been a popular area of research due to its various active ingredients and pharmacological effects. Ginkgo biloba is rich in ginkgo flavonoids, ginkgolides, and ginkgolic acid, with anti-inflammation, antioxidation, neuroprotection, anti-platelet agglutination, hypolipidemic effect, anti-cancer, and anti-radiation properties. There are many methods to extract and separate the active components of ginkgo. Among them, supercritical carbon dioxide fluid extraction (SFE-CO2) is known for its green, clean, and environment-friendly properties. In this paper, the pharmacological activities, the active components, and structures of different parts of ginkgo, the extraction methods of its effective ingredients, and the application of the SFE-CO2 method for the extraction and separation of active ingredients in Ginkgo biloba from leaves, seeds, pollen, and roots were reviewed, in order to make best use of ginkgo resources, and provide support and references for the development of SFE-CO2 of active components from Ginkgo biloba. Citation: Li, R.; Xia, Z.; Li, B.; Tian, Y.; Zhang, G.; Li, M.; Dong, J. Advances Keywords: Ginkgo biloba L.; active ingredients; supercritical CO2 fluid extraction in Supercritical Carbon Dioxide Extraction of Bioactive Substances from Different Parts of Ginkgo biloba L.. Molecules 2021, 26, 4011. https:// 1. Introduction doi.org/10.3390/molecules26134011 Ginkgo biloba L., also called maidenhair tree, is a rare species of Mesozoic relict and the sole species in the class Ginkgopsida, also known as the “living fossil” [1,2]. Ginkgo biloba Academic Editor: Alessandra Gentili is rich in a variety of natural active ingredients and has a wide range of pharmacological activities [3], which plays an important role in food [4,5], health care [6], medicine [7,8], Received: 16 May 2021 and other fields. The traditional methods of extracting active substances from Ginkgo biloba Accepted: 26 June 2021 include solvent extraction [9], enzymatic hydrolysis [10], and resin extraction [10]. Com- Published: 30 June 2021 pared with supercritical carbon dioxide fluid extraction (SFE-CO2), these methods have many disadvantages, such as low yield, the excessive residue of harmful substances, and Publisher’s Note: MDPI stays neutral long extraction. As a new extraction and separation method, SFE-CO has aroused increas- with regard to jurisdictional claims in 2 published maps and institutional affil- ing concern due to less use of organic solvents, short extraction time, and high yield. In iations. recent years, several studies [11,12] also showed that the yield of bioactive components in Ginkgo biloba will be higher if SFE-CO2 is applied to its extraction and separation. How- ever, no review is available on the SFE-CO2 of active components from different parts of Ginkgo biloba. Therefore, this paper reviewed the biological activities of different parts of Ginkgo biloba and the application of SFE-CO2 in different parts of ginkgo, attempting Copyright: © 2021 by the authors. to provide support and reference for the further development of SFE-CO technology of Licensee MDPI, Basel, Switzerland. 2 active components from Ginkgo biloba. This article is an open access article distributed under the terms and 2. Biological Activity of Ginkgo biloba conditions of the Creative Commons Attribution (CC BY) license (https:// Modern pharmacological studies revealed the various biological activities of ginkgo, creativecommons.org/licenses/by/ such as anti-inflammatory, antioxidation, neuron protection, anti-platelet aggregation, 4.0/). Molecules 2021, 26, 4011. https://doi.org/10.3390/molecules26134011 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 17 2. Biological Activity of Ginkgo biloba Molecules 2021, 26, 4011 2 of 16 Modern pharmacological studies revealed the various biological activities of ginkgo, such as anti-inflammatory, antioxidation, neuron protection, anti-platelet aggregation, hypolipidemic effects, anti-cancer, anti-radiation properties, etc. The main biological ac- hypolipidemic effects, anti-cancer, anti-radiation properties, etc. The main biological tivitiesactivities of Ginkgo of Ginkgobiloba are biloba asare follows as follows (Figure (Figure 1): 1): FigureFigure 1. Bioactivity 1. Bioactivity of Ginkgo of Ginkgo biloba biloba L. L. 2.1. Anti-Inflammatory Effect 2.1. Anti-Inflammatory Effect Li et al. [13] found that the ethyl acetate and chloroform parts of the ginkgo male flower Liextract et al. could[13] found significantly that downregulatethe ethyl acetat inflammatorye and chloroform cytokines parts of nitric of oxidethe ginkgo (NO), TNF- male flowerα ,extract IL-6, PEG could2, iNOS significantly mRNA, and downregulate COX-2 mRNA ininflammatory a dose-dependent cytokines manner, of which nitric made oxide (NO), themTNF- theα, IL-6, anti-inflammatory PEG2, iNOS mRNA, active parts and of COX-2 ginkgo mRNA male flowers. in a dose-dependent Li et al. [14] found manner, that whichginkgolide made them A the could anti-inflammat reduce the inflammatoryory active parts response of ginkgo induced ma byle lipopolysaccharideflowers. Li et al. [14] foundin that vivo ginkgolideand in vitro A. Zhang could et reduce al. [15] the found inflammatory that Ginkgo biloba responseextract induced EGB761 could by lipopoly- inhibit saccharideinflammation in vivo and by regulating in vitro. Zhang inflammatory et al. [15] cytokines found and that mediators Ginkgo bilobain vivo extractand in EGB761 vitro. could Theinhibit main inflammation active ingredient by flavonoidregulating glycosides inflammatory could inhibit cytokines the release and mediators of inflammatory in vivo cytokines in LPS induced RAW264.7 macrophages. and in vitro. The main active ingredient flavonoid glycosides could inhibit the release of inflammatory2.2. Antioxidant cytokines Effect in LPS induced RAW264.7 macrophages. Ginkgo extract is rich in flavonoids, which could be used as a natural antioxidant. 2.2. AntioxidantIt could play Effect an antioxidant activity by scavenging free radicals, superoxide anions, and GinkgoNO, as wellextract as inhibitis rich freein flavonoids, radical reaction which and could lipid peroxidationbe used as a [ 16natural,17]. Mart antioxidant.ínez-Solís It could etplay al. [18an] foundantioxidant that flavonoids activity and by terpenoidsscavenging in ginkgofree radicals, leaf extracts superoxide could be usedanions, to pre- and vent and treat retinal diseases related to oxidative stress. Liu et al. [19] found the activities NO, as well as inhibit free radical reaction and lipid peroxidation [16,17]. Martínez-Solís of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) could be improved et al. [18]by ginkgolide found that while flavonoids the concentration and terpenoids of malondialdehyde in ginkgo (MDA)leaf extracts in the myocardial could be used tissue to preventof myocardialand treat retinal ischemia-reperfusion diseases related (MI/R) to ox rats.idative Ren stress. et al. [Liu20] isolatedet al. [19] polysaccharide found the ac- tivitiescompounds of superoxide GBPS-2 dismutase and GBPS-3 (SOD) from and ginkgo glutathione leaves, which peroxidase could significantly (GSH-Px) scavenge could be improvedsuperoxide by ginkgolide radicals andwhile 3-ethylbenzothiazoline-6-sulphonic the concentration of malondialdehyde (ABTS) radicals, (MDA) asin well the asmy- ocardialhydroxyl tissue freeof myocardial radicals and DPPHischemia-reperfu free radicals.sion (MI/R) rats. Ren et al. [20] isolated polysaccharide compounds GBPS-2 and GBPS-3 from ginkgo leaves, which could sig- 2.3. Neuroprotective Effect nificantly scavenge superoxide radicals and 3-ethylbenzothiazoline-6-sulphonic (ABTS) radicals, asGinkgolide well as hydroxyl B could protect free radicals the nervous and systemDPPH by free inhibiting radicals. platelet-activating factors, reducing the concentration of calcium ions, and downregulating the level of NO [16]. Meanwhile, ginkgo leaf extract could also enhance the local cerebral blood flow to reduce cerebral ischemia and hypoxia [17]. The accumulated amyloid β protein (Aβ) in the brain is a key feature in Alzheimer’s disease. Sasaki et al. [21] found that kayaflavone and sciadopitysin showed a higher inhibitory effect on Aβ42 cytotoxicity in rat adrenal Molecules 2021, 26, 4011 3 of 16 pheochromocytoma (PC12) culture. Li et al. [22] found that ginkgo diterpenoid lactones could reduce the cerebral infarction area and improve the neurological deficit and brain edema remarkably. Kaur et al. [23] investigated the protective effect of Ginkgo biloba leaf extract (GBE) on hippocampal neuronal injury induced by methyl tin chloride (TMT) and suggested that GBE can protect hippocampal neuronals by downregulating the NO level. 2.4. Antiplatelet Aggregative Effect Platelet-activating factor (PAF)