Self-assembly of CuAuTA nanozymes for intelligent detection of ginkgolic acids.

Toxic ginkgolic acids (GAs) are a challenge for Ginkgo biloba-related food. Although a detection method for GAs is available, bulky instruments limit the field testing of GAs. Herein, by assembling gold nanoclusters with copper tannic acid (CuTA), CuAuTA nanocomposites were designed as peroxidase mimics for the colorimetric determination of GAs. Compared with single CuTA, the obtained CuAuTA nanocomposites possessed enhanced peroxidase-like properties. Based on the inhibitory effect of GAs for the catalytic activity of CuAuTA nanozymes, CuAuTA could be utilized for the colorimetric sensing of GAs with a low limit of quantitation of 0.17 µg mL-1. Using a smartphone and the ImageJ software in conjunction, a nanozyme-based intelligent detection platform was developed with a detection limit of 0.86 µg mL-1. This sensing system exhibited good selectivity against other potential interferents. Experimental data demonstrated that GAs might bind to the surface of CuAuTA, blocking the catalytically active sites and resulting in decreased catalytic activity. Our CuAuTA nanozyme-based system could also be applied to detect real ginkgo nut and ginkgo powder samples with recoveries of 93.12-111.6% and relative standard deviations less than 0.3%. Our work may offer a feasible strategy for the determination of GAs and expand the application of nanozymes in food safety detection.

Effects of processing methods on quality, antioxidant capacity, and cytotoxicity of Ginkgo biloba leaf tea product.

BACKGROUND: Ginkgo biloba leaves contain beneficial flavonoids, bilobalide (BB), and ginkgolides. However, the toxic ginkgolic acid (GA) limit its application. In this study, various traditional processing methods were used to prepare G. biloba leaf tea (GBLT), including white tea, black tea, dark tea, green tea, and freeze-dried as control, followed by investigations of their effects on quality, antioxidant capacity, bioactive components, and cytotoxicity of the tea products. RESULTS: Results showed that different processing methods significantly impact the tea products' quality indexes and the principal component analysis (PCA) and hierarchical cluster analysis (HCA) corroborated it. White tea had the highest total sugar (TS) and GA content and the most potent cytotoxicity on HepG2 cells. However, TS and GA content and the cytotoxicity of GBLT markedly decreased during fermentation and fixation. Moreover, white tea possessed higher total phenolic content (TPC), total flavonoid content (TFC), and more vigorous antioxidant activities than green tea, black tea, and dark tea. Terpene trilactones value was stable, but different catechins contents fluctuated according to the manufacturing process of different GBLTs. Among the four GBLTs, dark tea combining fixation and fermentation had the lowest GA content and cytotoxicity, less bioactive components reduction, appropriate quality, and stronger flavor. CONCLUSION: These findings demonstrate that fixation and fermentation help reduce GAs during the manufacturing of GBLT. However, their ability to retain bioactive substances needs further optimization in future studies. © 2023 Society of Chemical Industry.

Synergistic effect of Hsp90 inhibitor ginkgolic acids C15꞉1 combined with paclitaxel on nasopharyngeal carcinoma. / Hsp90æŠ‘åˆ¶å‰‚ç™½æžœé ¸ä¸Žç´«æ‰é†‡çš„ååŒæŠ—é¼»å’½ç™Œä½œç”¨.

OBJECTIVES: Nasopharyngeal cracinoma is a kind of head and neck malignant tumor with high incidence and high mortality. Due to the characteristics of local recurrence, distant metastasis, and drug resistance, the survival rate of patients after treatment is not high. Paclitaxel (PTX) is used as a chemotherapy drug in treating nasopharyngeal carcinoma, but nasopharyngeal carcinoma cells are easy to develop resistance to PTX. Inhibition of heat shock protein 90 (Hsp90) can overcome common signal redundancy and resistance in many cancers. This study aims to investigate the anti-tumor effect of ginkgolic acids C15꞉1 (C15:1) combined with PTX on nasopharyngeal carcinoma CNE-2Z cells and the mechanisms. METHODS: This experiment was divided into a control group (without drug), a C15:1 group (10, 30, 50, 70 µmol/L), a PTX group (5, 10, 20, 40 nmol/L), and a combination group. CNE-2Z cells were treated with the corresponding drugs in each group. The proliferation of CNE-2Z cells was evaluated by methyl thiazolyl tetrazolium (MTT). Wound-healing assay and transwell chamber assay were used to determine the migration of CNE-2Z cells. Transwell chamber was applied to the impact of CNE-2Z cell invasion. Annexin V-FITC/PI staining was used to observe the effect on apoptosis of CNE-2Z cells. The changes of proteins involved in cell invasion, migration, and apoptosis after the combination of C15꞉1 and PTX treatment were analyzed by Western blotting. RESULTS: Compared with the control group, the C15꞉1 group and the PTX group could inhibit the proliferation of CNE-2Z cells (all P<0.05). The cell survival rates of the C15꞉1 50 µmol/L combined with 5, 10, 20, or 40 nmol/L PTX group were lower than those of the single PTX group (all P<0.05), the combination index (CI) value was less than 1, suggesting that the combined treatment group had a synergistic effect. Compared with the 50 µmol/L C15꞉1 group and the 10 nmol/L PTX group, the combination group significantly inhibited the invasion and migration of CNE-2Z cells (all P<0.05). The results of Western blotting demonstrated that the combination group could significantly down-regulate Hsp90 client protein matrix metalloproteinase (MMP)-2 and MMP-9. The results of double staining showed that compared with the 50 µmol/L C15꞉1 group and the 10 nmol/L PTX group, the apoptosis ratio of CNE-2Z cells in the combination group was higher (both P<0.05). The results of Western blotting suggested that the combination group could decrease the Hsp90 client proteins [Akt and B-cell lymphoma-2 (Bcl-2)] and increase the Bcl-2-associated X protein (Bax). CONCLUSIONS: The combination of C15꞉1 and PTX has a synergistic effect which can inhibit cell proliferation, invasion, and migration, and induce cell apoptosis. This effect may be related to the inhibition of Hsp90 activity by C15꞉1.

Medicinal Values and Potential Risks Evaluation of Ginkgo biloba Leaf Extract (GBE) Drinks Made from the Leaves in Autumn as Dietary Supplements.

Ginkgo tea and ginkgo wine are two familiar Ginkgo biloba leaf extract (GBE) drinks in the form of dietary supplements (DS) used for healthcare in east Asia. Nevertheless, a comprehensive evaluation of their safety and efficacy is still lacking. In this study, GBE drinks were prepared from naturally newly senescent yellow leaves (YL) and green leaves (GL) in autumn. Their total flavonoids, antioxidant capacity and prescribed ingredients were investigated. In brief, the proportions of total flavonoids, total flavonol glycosides (TFs), total terpene trilactones (TTLs) and ginkgolic acids in the GBE drinks all did not meet the standards of worldwide pharmacopoeias. Specifically, the levels of TFs in the ginkgo tea prepared from YL were significantly higher than that prepared from GL. Further analyses revealed a substandard ratio of isorhamnetin/quercetin and an accumulation of leaf-age-related compounds, which were both unqualified. The proportions of specific TTLs varied between the ginkgo tea and ginkgo wine, although no significant differences were detected in terms of the total levels of TTLs. Noticeably, numerous biflavones and thousands of times over the limiting concentration of ginkgolic acids, including newly identified types, were only detected in ginkgo wine. Finally, the use of the GBE drinks as DSs was comprehensively evaluated according to the acceptable daily intake. This study showed the limited healthcare effects of GBE drinks despite their powerful antioxidant capacity.

Anti-Cancer Properties of Ginkgolic Acids in Human Nasopharyngeal Carcinoma CNE-2Z Cells via Inhibition of Heat Shock Protein 90.

Ginkgo biloba L. has been used in traditional Chinese medicine (TCM) for thousands of years. However, the anti-cancer properties of ginkgolic acids (GAS) isolated from G. biloba have not been investigated in human nasopharyngeal carcinoma cells. In this study, GAS exhibited an inhibitory effect on the ATPase activity of heat shock protein 90 (Hsp90) and anti-proliferative activities against four human cancer cell lines, with IC50 values ranging from 14.91 to 23.81 µg·mL-1. In vivo experiments confirmed that GAS inhibited tumor growth in CNE-2Z cell-xenografted nude mice with low hepatotoxicity. We further demonstrated that GAS suppressed migration and invasion and induced the apoptosis of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (MMP-2, MMP-9, Her-2, c-Raf, Akt, and Bcl-2). Together, GAS are new Hsp90 inhibitors by binding to Hsp90 (hydrogen bond and hydrophobic interaction). Thus, GAS from G. biloba might represent promising Hsp90 inhibitors for the development of anti-nasopharyngeal carcinoma agents.

Ginkgolic acid induces interplay between apoptosis and autophagy regulated by ROS generation in colon cancer.

Presently, developing effective anti-colon cancer drugs still remains to be important. Ginkgolic acids (GA), as a botanical drug extracted from the seed coat of Ginkgo biloba L., possess various bioactive properties. Our findings, for the first time, indicated that GA suppressed colon cancer cell proliferation, migration and invasion. GA led to cell death through G0/G1 phase arrest. In addition, apoptosis was significantly induced by GA treatment. The intrinsic apoptosis pathway was included, proved by the release of cytochrome c (Cyto-c) from the mitochondria into the cytosol. GA-induced autophagy was supported by the dose-dependent increase of LC3BII, autophagy-related gene-5 (ATG-5) and Beclin-1. Notably, silencing ATG-5 further reduced the cell viability and enhanced apoptosis in GA-treated colon cancer cells, indicating that GA-induced apoptosis rather than autophagy contributes to colon cancer cell death. And mammalian target of rapamycin complex 1 (mTORC1) was dose-dependently reduced by GA, evidenced by the reduction of p-mTOR, p-p70 ribosomal S6 kinase (p70s6k) and p-pras40. Moreover, GA markedly resulted in reactive oxygen species (ROS) generation, along with increased H2O2 and O2-. However, blocking ROS generation using its scavenger, NAC, significantly recovered GA-induced cells death, supported by the increase of cell viability, and the decrease of apoptosis. The expressions of autophagy- and cell cycle arrest-related molecules, as well as mTORC1 were also reversed by N-acetyl-l-cysteine (NAC) in GA-treated cells. In vivo, GA reduced tumor growth without toxicity to animals. In conclusion, our study illustrated that GA caused G0/G1 phase arrest and triggered intrinsic apoptosis and autophagy modulated by ROS generation in human colon cancer, elucidating that GA might be considered as a potential agent for colon cancer therapy.

Application of coordination agent in high-speed counter-current chromatography for the preparative separation and isolation ginkgolic acids from the sarcotesta of Ginkgo biloba L.

An efficient coordination high-speed counter-current chromatography method for the preparative separation of ginkgolic acids from the sarcotesta of Ginkgo biloba L was developed. The type, concentration, and mechanism of the coordination agent were investigated. Following the use of four types of metal salts including silver nitrate, copper chloride, ferric chloride, and aluminium nitrate, n-heptane/ethyl acetate/methanol/acetic acid 5:4:1:1, v/v with 0.20 mol/L silver nitrate as the coordination agent was chosen as the optimum two-phase solvent system. Five main ginkgolic acids including C13:0, C15:0, C15:1, C17:1, and C17:2 were successfully separated with purities greater than 98%. The sample loading was 500 mg, the flow-rate was 2.0 mL/min, rotation speed was 800 rpm and temperature was 20°C. The structures of the separated ginkgolic acids were identified by comparison with standard samples and electrospray ionization mass spectrometry. The introduction of coordination chemistry in high-speed counter-current chromatography is novel and effective for the preparative separation and isolation of ginkgolic acids from the sarcotesta of Ginkgo biloba L and could also be applied to separate compounds which form coordination bonds in other complex natural products.

Rapid determination of ginkgolic acids in Ginkgo biloba kernels and leaves by direct analysis in real time-mass spectrometry.

A novel method based on direct analysis in real time integrated with mass spectrometry was established and applied into rapid determination of ginkgolic acids in Ginkgo biloba kernels and leaves. Instrument parameter settings were optimized to obtain the sensitive and accurate determination of ginkgolic acids. At the sample introduction speed of 0.2 mm/s, high intensity of [M-H]- ions for ginkgolic acids were observed in the negative ion mode by utilization of high-purity helium gas at 450°C. Two microliters of methanol extract of G. biloba kernels or leaves dropped on the surface of Quick-Strip module was analyzed after solvent evaporated to dryness. A series of standard solutions of ginkgolic acid 13:0 in the range of 2-50 mg/L were analyzed with a correlation coefficient r = 0.9981 and relative standard deviation (n = 5) from 12.5 to 13.7%. The limit of detection was 0.5 mg/L. The results of direct analysis in real time-mass spectrometry were in agreement with those observed by thermochemolysis gas chromatography. The proposed method demonstrated significant potential in the application of the high-throughput screening and rapid analysis for ginkgolic acids in dietary supplements.

Concurrent supercritical fluid chromatographic analysis of terpene lactones and ginkgolic acids in Ginkgo biloba extracts and dietary supplements.

Supercritical fluid chromatography was used to resolve and determine ginkgolic acids (GAs) and terpene lactones concurrently in ginkgo plant materials and commercial dietary supplements. Analysis of GAs (C13:0, C15:0, C15:1, and C17:1) was carried out by ESI (-) mass detection. The ESI (-) spectra of GAs simply displayed only the [M-H](-) pseudo-molecular ions, and selected ion monitoring (SIM) for those ions was used for the quantification. Analysis of terpene lactones (ginkgolides A, B, C, J and bilobalide) was complicated by in-source collision-induced dissociation (IS-CID) in the ESI source. Thus, MS analysis could be influenced by the fragmentation pattern produced by the IS-CID. However, it was established that the fragmentation pattern, measured by ion survival yield (ISY), was independent of analyte concentration or matrix at a fixed cone voltage in the ESI source. Therefore, MS with SIM mode was applicable for the analysis of these analytes. The reported method provided consistent and sensitive analysis for the analytes of interest. The LOQs and LODs were determined to be below 100 and 40 ng/mL for GAs and 1 µg/mL and 400 ng/mL for terpene lactones, respectively. Intra- and inter-day precisions were found to be satisfactory with RSDs being below 5.2 %. Analyte recoveries ranged from 87 to 109 %. The developed method was successfully applied to the analysis of 11 ginkgo plant samples and 8 dietary supplements with an analysis time of less than 12 min.

Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis.

Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet-B (UV-B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV-B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV-B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV-B radiation exposure activated antioxidants and stress-responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV-B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV-B irradiation.

Modeling the Antiviral Activity of Ginkgo biloba Polyphenols against Variola: In Silico Exploration of Inhibitory Candidates for VarTMPK and HssTMPK Enzymes.

BACKGROUND: The aim of this study is to use modeling methods to estimate the antiviral activity of natural molecules extracted from Ginkgo biloba for the treatment of variola which is a zoonotic disease posing a growing threat to human survival. The recent spread of variola in nonendemic countries and the possibility of its use as a bioterrorism weapon have made it a global threat once again. Therefore, the search for new antiviral therapies with reduced side effects is necessary. METHODS: In this study, we examined the interactions between polyphenolic compounds from Ginkgo biloba, a plant known for its antiviral activity, and two enzymes involved in variola treatment, VarTMPK and HssTMPK, using molecular docking. RESULTS: The obtained docking scores showed that among the 152 selected polyphenolic compounds; many ligands had high inhibitory potential according to the energy affinity. By considering Lipinski's rules, we found that Liquiritin and Olivil molecules are the best candidates to be developed into drugs that inhibit VarTMPK because of their high obtained scores compared to reference ligands, and zero violations of Lipinski's rules. We also found that ginkgolic acids have good affinities with HssTMPK and acceptable physicochemical properties to be developed into drugs administered orally. CONCLUSION: Based on the obtained scores and Lipinski's rules, Liquiritin, Olivil, and ginkgolic acids molecules showed interesting results for both studied enzymes, indicating the existence of promising and moderate activity of these polyphenols for the treatment of variola and for possible multi-targeting. Liquiritin has been shown to exhibit anti-inflammatory effects on various inflammation- related diseases such as skin injury, hepatic inflammatory injury, and rheumatoid arthritis. Olivil has been shown to have antioxidant activity. Olivil derivatives have also been studied for their potential use as anticancer agents. Ginkgolic acids have been shown to have antimicrobial and antifungal properties. However, ginkgolic acids are also known to cause allergic reactions in some people. Therefore, future studies should consider these results and explore the potential of these compounds as antiviral agents. Further experimental studies in-vitro and in-vivo are required to validate and scale up these findings.

Development, Validation, and Application of High-Performance Liquid Chromatography with Diode-Array Detection Method for Simultaneous Determination of Ginkgolic Acids and Ginkgols in Ginkgo biloba.

Ginkgo biloba leaves (GBLs), which comprise many phytoconstituents, also contain a toxic substance named ginkgolic acid (GA). Our previous research showed that heating could decarboxylate and degrade GA into ginkgols with high levels of bioactivity. Several methods are available to measure GA in GBLs, but no analytical method has been developed to measure ginkgols and GA simultaneously. Hence, for the first time, an HPLC-DAD method was established to simultaneously determine GA and ginkgols using acetonitrile (0.01% trifluoroacetic acid, v/v) as mobile phase A and water (0.01% trifluoroacetic acid, v/v) as mobile phase B. The gradient elution conditions were: 0-30 min, 75-90% phase A; 30-35 min, 90-90% phase A; 35-36 min, 90-75% phase A; 36-46 min, 75-75% phase A. The detection wavelength of GA and ginkgol were 210 and 270 nm, respectively. The flow rate and injection volume were 1.0 mL/min and 50 µL, respectively. The linearity was excellent (R2 > 0.999), and the RSD of the precision, stability, and repeatability of the total ginkgols was 0.20%, 2.21%, and 2.45%, respectively, in six parallel determinations. The recoveries for the low, medium, and high groups were 96.58%, 97.67%, and 101.52%, respectively. The limit of detection of ginkgol C13:0, C15:1, and C17:1 was 0.61 ppm, 0.50 ppm, and 0.06 ppm, respectively. The limit of quantification of ginkgol C13:0, C15:1, and C17:1 was 2.01 ppm, 1.65 ppm, and 0.20 ppm, respectively. Finally, this method accurately measured the GA and ginkgol content in ginkgo leaves and ginkgo tea products (ginkgo black tea, ginkgo dark tea, ginkgo white tea, and ginkgo green tea), whereas principal component analysis (PCA) was performed to help visualize the association between GA and ginkgols and five different processing methods for GBLs. Thus, this research provides an efficient and accurate quantitative method for the subsequent detection of GA and ginkgols in ginkgo tea.

Ginkgolic acids induce liver injury in mice through cell cycle arrest and immune stress under specific condition.

Ginkgo biloba L. is a valuable plant, which can be used for medicine, food and ornamental purposes. Despite the above benefits, the components of ginkgolic acids (GA) in ginkgo are considered to cause allergies, embryotoxicity, liver damage and some other adverse reactions. However, the mechanism of GA induced liver injury is still unclear. In this study, we developed an acute liver injury model induced by GA in mice, and investigated the mechanism of GA induced liver injury from the perspectives of oxidative stress, steatosis, apoptosis, and immune response. Intraperitoneal injection of GA (400 mg/kg) can cause liver damage. The levels of serum transaminase, oxidation and triglycerides were increased, liver fibrosis, hepatocyte apoptosis, G2/M phase arrest of the hepatic cell cycle and monocyte infiltration in the liver were detected in GA-treated mice. Flow cytometry analysis of cells separated from the spleen showed that the proportion of Th1 and Th17 cells were increased, and the proportion of Th2 cells were decreased in GA-treated mice. The rise in Th1/Th2 ratio and Th17 cell ratio usually cause inflammatory problems. At the same time, cleaved Caspase-8 and Caspase-3 were detected in hepatocytes, indicating that GA may induce apoptosis through FADD pathway. Although GA is capable of causing the above problems, the inflammation and damage in liver tissue are not severe and there are certain individual differences. Our study reveals the potential hepatotoxicity of GA in ginkgo and its mechanism of action, providing a new perspective for the intervention and prevention of ginkgo toxicity.

Thermal fixation technologies affect phenolic profile, ginkgolides, bilobalide, product quality, and ginkgolic acids in Ginkgo biloba leaf tea.

Ginkgo biloba leaves (GBLs) contain high phytoconstituents, but ginkgolic acids (GAs, the main toxic compound in GBLs) have limited its applications. Processing Ginkgo biloba dark tea (GBDT) using fixation technology could decrease the toxic compounds; retain flavonoids, ginkgolides, and bilobalide; and improve the product quality. For the first time, various thermal fixations (hot air fixation [HAF], iron pot fixation [IPF], and boiled water fixation [BWF]) followed by rolling, fermentation, and drying were applied to produce GBDT. A comprehensive analysis of the toxicants (GAs), main bioactive compounds (ginkgolides and bilobalide, flavonoids, antioxidants, and phenolic profiles), and product qualities (moisture content, reducing sugar [RS], free amino acids [FAAs], enzyme activity, color properties, antioxidant capacity, etc.) were evaluated. The results revealed that thermal fixations BWF and HAF significantly reduced the GA contents (41.1%-34.6%). Most terpene lactones showed significant differences in control, IPF, and HAF. The HAF had lower total flavonoid content (TFC) than BWF and IPF. The control group (unfixated) had the highest toxic components (GA), terpene trilactones, and TFC compared with various fixations. Adding different fixations to rolling, fermentation, and drying had various impacts on GBDT, and principal component analysis supported the results. Among four thermal fixations, HAF yielded the best results in RS, FAA, total phenolic content, and antioxidant activities, while IPF had the highest TFC. BWF had the lowest content for GA. In conclusion, HAF (6) was chosen as the best technique for producing GBDT since it preserved GBDT's bioactive components while lowering its toxic components.

Combinative effect of pulsed-light irradiation and solid-state fermentation on ginkgolic acids, ginkgols, ginkgolides, bilobalide, flavonoids, product quality and sensory assessment of Ginkgo biloba dark tea.

Pulsed light (PL) is a prospective non-thermal technology that can improve the degradation of ginkgolic acid (GA) and retain the main bioactive compounds in Ginkgo biloba leaves (GBL). However, only using PL hasn't yet achieved the ideal effect of reducing GA. Fermentation of GBL to make ginkgo dark tea (GDT) could decrease GA. Because different microbial strains are used for fermentation, their metabolites and product quality might differ. However, there is no research on the combinative effect of PL irradiation fixation and microbial strain fermentation on main bioactive compounds and sensory assessment of GDT. In this research, first, Bacillus subtilis and Saccharomyces cerevisiae were selected as fermentation strains that can reduce GA from the five microbial strains. Next, the fresh GBL was irradiated by PL for 200 s (fluences of 0.52 J/cm2), followed by B. subtilis, S. cerevisiae, or natural fermentation to make GDT. The results showed that compared with the control (unirradiated and unfermented GBL) and the only PL irradiated GBL, the GA in GDT using PL + B. subtilis fermentation was the lowest, decreasing by 29.74%; PL + natural fermentation reduced by 24.53%. The total flavonoid content increased by 14.64% in GDT using PL + B. subtilis fermentation, whose phenolic and antioxidant levels also increased significantly. Sensory evaluation showed that the color, aroma, and taste of the tea infusion of PL + B. subtilis fermentation had the highest scores. In conclusion, the combined PL irradiation and solid-state fermentation using B. subtilis can effectively reduce GA and increase the main bioactive compounds, thus providing a new technological approach for GDT with lower GA.

Novel mechanisms for the synthesis of important secondary metabolites in Ginkgo biloba seed revealed by multi-omics data.

Although the detailed biosynthetic mechanism is still unclear, the unique secondary metabolites of Ginkgo biloba, including ginkgolic acids (GAs) and terpene trilactones, have attracted increasing attention for their potent medicinal, physiological and biochemical properties. In particular, GAs have shown great potential in the fields of antibacterial and insecticidal activities, making it urgent to elucidate their biosynthetic mechanism. In this study, we systematically revealed the landscape of metabolic-transcriptional regulation across continuous growth stages of G. biloba seeds (GBS) based on multi-omics mining and experimental verification, and successfully identified all major types of GAs and terpene trilactones along with more than a thousand kinds of other metabolites. The phenological changes and the essential gene families associated with these unique metabolites were analyzed in detail, and several potential regulatory factors were successfully identified based on co-expression association analysis. In addition, we unexpectedly found the close relationship between large introns and the biosynthesis of these secondary metabolites. These genes with large introns related to the synthesis of secondary metabolites showed higher gene expression and expression stability in different tissues or growth stages. Our results may provide a new perspective for the study of the regulatory mechanism of these unique secondary metabolites in GBS.

Reducing toxic constituents of ginkgolic acid content and improving bioactive flavonoid content from Ginkgo biloba leaves by high-temperature pretreatment processing.

High-temperature pretreatment was developed in this article to remove the main toxic constituents of ginkgolic acids (GAs) from Ginkgo biloba leaves (GBLs) and improve the bioactive flavonoid content by water extraction. To optimize the effects of high-temperature pretreatment process parameters on removing toxic GAs to a limited level and improving the content of bioactive flavonoids, a Box-Behnken design (BBD) combined with response surface methodology (RSM) was also conducted. The results showed that the content of GAs could be reduced to 4.11 ppm and the highest content of flavonoids could reach 3.51% under the optimized conditions of high-temperature pretreatment process of 177°C with water extraction at 96°C at a liquid-to-solid ratio of 56:1. The content of toxic GAs substantially decreased by 83.50% while the content of bioactive flavonoids increased by 44.30% compared with the conventional water extraction method. Moreover, the new process was more efficient, environmentally friendly, and could get avoid a subsequent multi-step process of removing toxic GAs. The crude extracts were then purified by macroporous resin to obtain the 60% ethanol fraction. After purification, the flavonoid content increased to 43.50% while the GAs were not detected. The main compounds of 60% ethanol fraction were identified by UPLC-QTOF-MS/MS. Antioxidant activities including reducing powder, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and OH· scavenging assays all showed that the 60% ethanol fraction was better than the butylated hydroxytoluene (BHT) standard.

Pharmacological Activities of Ginkgolic Acids in Relation to Autophagy.

Plant-derived natural compounds are widely used as alternative medicine in healthcare throughout the world. Ginkgolic acids, the phenolic compounds isolated from the leaves and seeds of Ginkgo biloba, are among the chemicals that have been explored the most. Ginkgolic acids exhibit cytotoxic activity against a vast number of human cancers in various preclinical models in vitro and in vivo. Additionally, the pharmacological activities of ginkgolic acids are also involved in antidiabetic, anti-bacteria, anti-virus, anti-fibrosis, and reno/neuroprotection. Autophagy as a highly conserved self-cleaning process that plays a crucial role in maintaining cellular and tissue homeostasis and has been proven to serve as a protective mechanism in the pathogenesis of many diseases, including neurodegenerative diseases, cancer, and infectious diseases. In this review, we surveyed the pharmacological activities of the major three forms of ginkgolic acids (C13:0, C15:1, and C17:1) that are linked to autophagic activity and the mechanisms to which these compounds may participate. A growing body of studies in last decade suggests that ginkgolic acids may represent promising chemical compounds in future drug development and an alternative remedy in humans.

Ginkgolic Acids Confer Potential Anticancer Effects by Targeting Pro- Inflammatory and Oncogenic Signaling Molecules.

BACKGROUND: Medicinal plants and herbal preparations in the form of traditional medicines have been used in healthcare worldwide. The extracts of Ginkgo biloba L. seeds and leaves contain a complex mixture of numerous components, such as flavonol glycosides, terpene lactones, and a group of alkylphenols (anacardic or ginkgolic acids, cardanols and cardols) that have been a part of traditional Chinese medicine. These extracts are also sold as dietary supplements worldwide. G. biloba extract (EGb 761 and LI 1370) represent the standard form of G. biloba extract. Six different 6-alkylsalicylic acids (syn. ginkgolic acids) with alkyl substituents (C13:0, C15:0, C15:1, C17:0, C17:1, and C17:2) have been identified. OBJECTIVE: The aim of this review is to unravel scientific evidence on anti-inflammatory and anticancer activities of ginkgolic acids to understand its therapeutic potential against inflammatory and oncologic diseases. METHODS: A structured literature search was independently performed by the authors on PubMed, ScienceDirect, Scopus, and Web of Science. Accordingly, this review article critically analyses available scientific evidence on anti-inflammatory and anticancer activities of ginkgolic acids. Moreover, the review only included articles written in the English language. RESULTS: Several forms of ginkgolic acids, especially C13:0, C15:0 and C17:1, isolated from the leaves of G. biloba exhibited cytotoxic activity against a variety of human cancers by suppressing various pro-inflammatory signaling cascades and oncogenic transcription factors through multiple modes of action in various in vitro and in vivo preclinical models. Ginkgolic acids have also been reported to be potent post-translational small ubiquitin-related modifiers (SUMO)ylation inhibitors. CONCLUSION: In this review, we present updated information on the anti-inflammatory and anticancer properties of ginkgolic acids both in vitro and in vivo. Although ginkgolic acids show significant therapeutic potential in inflammatory and oncologic diseases, more investigations regarding the safety and efficacy of these natural agents are warranted before the clinical transition.

Effects of preheating and drying methods on pyridoxine, phenolic compounds, ginkgolic acids, and antioxidant capacity of Ginkgo biloba nuts.

Although ginkgo nuts are very nutritious and loaded with numerous bioactive compounds, the nuts contain significant levels of unwanted compounds (ginkolic acids) which are toxic to consumption. To reduce or eliminate these toxic compounds without impacting the nutritional value and the bioactivity of the final product, an appropriate processing technology is needed. Thus, the effect of preheating (90 and 120°C) prior to drying (freeze drying: FD, hot air drying: HAD, and HAD in tandem with FD: HAD-FD) was evaluated on ginkgolic acids, pyridoxine analogues, phenolic compounds, and antioxidant properties of ginkgo nuts. Our results pointed out a significant decrease (below 50%) of ginkgolic acids in ginkgo nuts samples processed at 90°C compared to the control. The major compounds found after treatments were respectively, kaempferol (36.66-354.38 µg/g), quercetin (9.04-183.71 µg/g), and caffeic acid (19.66-106.88 µg/g). Principal component analysis (PCA) revealed that preheating at 90°C prior to HAD-FD would be a proper and reasonable approach for preserving the bioactive compounds and antioxidant capacity of ginkgo nuts (EC50 ranged from 2.25 to 4.60 mg/mL) while significantly reducing their content in toxic compounds.