salicylates and ginkgolic-acid

The Ginkgo biloba has astonished scholars globally with enormous bioactives, with sales exceeding $10 billion since 2017. The Ginkgo biloba seed (GBS) is an essential part of culinary culture. Nevertheless, toxins in fresh Ginkgo biloba seed (GBS) have limited GBSs' daily consumption. Ginkgotoxin and ginkgotoxin-5-glucoside cause poisoning, tonic-clonic convulsions, and neurotoxic effects. Ginkgolic acid causes cytotoxicity and allergies. Allergic glycoprotein in GBS causes nausea, seizures, dyspnea, mydriasis, vomiting, and bellyache. The amygdalin-derived hydrocyanic acid cause dizziness, vomiting, cramping, and sleeping disorders. Food products are frequently exposed to various processing techniques to increase food safety and functionality. As a result, this review focused on the technologies that have been used to minimize toxins in GBS. In addition, a comparison of these techniques was made based on their benefits, drawbacks, feasibility, pharmacological activities, and future direction or opportunities to improve current ones were provided.

Topics: Cyanides; Ginkgo biloba; Glucosides; Glycoproteins; Humans; Hypersensitivity; Plant Extracts; Pyridoxine; Salicylates; Seeds

Ginkgo biloba is among the most favourite and best explored herbal drugs. Standardized extracts of Ginkgo biloba represent the only herbal alternative to synthetic antidementia drugs in the therapy of cognitive decline and Alzheimer's diseases. The clinical efficiency of such standardized Ginkgo biloba extracts (GBE) is still controversial, but authors of numerous international clinical studies recommended the use of GBE in the described therapies.Extracts of Ginkgo biloba are a mixture of substances with a wide variety of physical and chemical properties and activities. Numerous pharmacological investigations lead to the conclusion that the terpene trilactones (TTL) and the flavonoids of GBE are responsible for the main pharmacological effects of the extract in the therapy of cognitive decline. Therefore, the quality of GBE products must be oriented on a defined quantity of TTL and flavonoids. Furthermore, because of their toxic potential the amount of ginkgolic acid should be less than 5 ppm.However, data on pharmacokinetics and bioavailability, especially related to the central nervous system (CNS), which is the target tissue, are relatively rare. A few investigations characterize the TTL and flavonoids of Ginkgo biloba pharmacokinetically in plasma and in the brain. Recent investigations show that significant levels of TTL and Ginkgo biloba flavonoids cross the blood-brain barrier and enter the CNS of rats after oral application of GBE. Knowledge about the pharmacokinetic behaviour of these substances is necessary to discuss the pharmacological results on a more realistic basis.

Topics: Animals; Flavonoids; Ginkgo biloba; Humans; Legislation, Drug; Phytochemicals; Plant Extracts; Plants, Medicinal; Pyridoxine; Salicylates; Terpenes

Ginkgo biloba has a very high medicinal value. The flavonol glycosides and terpene lactones contained in G. biloba extract (GBE) have such pharmacological effects as antioxidant, anti-platelet aggregation and memory improvement, enhancement of immune function. However, the ginkgolic acid (GA) contained in GBE is proved to be highly allergenic and cytotoxic, even minimal residual could also cause severe adverse effects. To minimize the potential safety hazards of ginkgo leaf preparations, this study focuses on GA's chemical structure, adverse effects, toxicity and genesis mechanism, desorption and attenuation in the hope of providing a new thought for studies on safety of Ginkgo biloba preparations.

Topics: Animals; Ginkgo biloba; Humans; Salicylates

The latest progress in research on constituents and pharmacological activities of sarcotestas of Ginkgo biloba has been studied. The main constituents in sarcotestas of G. biloba include flavones, ginkgolides, alkylphenols, polysaccharides and amino acids, etc. They show the following activities, such as bacteriostatic, bactericidal and pesticidal activities, antitumor and mutagenic, carcinogenic effects, antianaphylaxis and allergenic activity, effects on immunologic function, scavenging free radical, antisenile action, etc. The problems at present and the reseach direction for the future on sarcotestas of G. biloba have been put forward.

Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents, Phytogenic; Flavonoids; Free Radical Scavengers; Fruit; Ginkgo biloba; Ginkgolides; Humans; Plants, Medicinal; Salicylates

Topics: Adult; Anti-Bacterial Agents; Bacteria; Humans; Infant, Newborn; Molecular Docking Simulation; Salicylates; Streptococcus agalactiae

With the increasing reports of community-acquired and nosocomial infection caused by multidrug-resistant Gram-positive pathogens, there is an urgent need to develop new antimicrobial agents with novel antibacterial mechanisms. Here, we investigated the antibacterial activity of the natural product ginkgolic acid (GA) (15:1), derived from Ginkgo biloba, and its potential mode of action against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus. The MIC values of GA (15:1) against clinical E. faecalis and S. aureus isolates from China were ≤4 and ≤8 μg/mL, respectively, from our test results. Moreover, GA (15:1) displayed high efficiency in biofilm formation inhibition and bactericidal activity against E. faecalis and S. aureus. During its inhibition of the planktonic bacteria, the antibacterial activity of GA (15:1) was significantly improved under the condition of abolishing iron homeostasis. When iron homeostasis was abolished, inhibition of planktonic bacteria by GA (15:1) was significantly improved. This phenomenon can be interpreted as showing that iron homeostasis disruption facilitated the disruption of the functions of ribosome and protein synthesis by GA (15:1), resulting in inhibition of bacterial growth and cell death. Genetic mutation of ferric uptake regulator (Fur) led to GA (15:1) tolerance in

Topics: Animals; Anti-Bacterial Agents; Enterococcus faecalis; Female; Ginkgo biloba; Gram-Positive Bacterial Infections; Homeostasis; Humans; Iron; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Plant Extracts; Salicylates; Staphylococcus aureus

Ginkgolic acid (C13:0) (GA), isolated from Ginkgo biloba, is a potential therapeutic agent for type 2 diabetes. A series of GA analogs were designed and synthesized for the evaluation of their structure-activity relationship with respect to their antidiabetic effects. Unlike GA, the synthetic analog

Topics: Diabetes Mellitus, Type 2; Glucose; Humans; Insulin; Insulin Resistance; Muscle Fibers, Skeletal; Palmitates; Salicylates; Signal Transduction; Structure-Activity Relationship

Idiopathic pulmonary fibrosis (IPF) is a refractory chronic respiratory disease with progressively exacerbating symptoms and a high mortality rate. There are currently only two effective drugs for IPF; thus, there is an urgent need to develop new therapeutics. Previous experiments have shown that ginkgolic acid (GA), as a SUMO-1 inhibitor, exerted an inhibitory effect on cardiac fibrosis induced by myocardial infarction. Regarding the pathogenesis of PF, previous studies have concluded that small ubiquitin-like modifier (SUMO) polypeptides bind multiple target proteins and participate in fibrosis of multiple organs, including PF. In this study, we found altered expression of SUMO family members in lung tissues from IPF patients. GA mediated the reduced expression of SUMO1/2/3 and the overexpression of SENP1 in a PF mouse model, which improved PF phenotypes. At the same time, the protective effect of GA on PF was also confirmed in the SENP1-KO transgenic mice model. Subsequent experiments showed that SUMOylation of SMAD4 was involved in PF. It was inhibited by TGF-

Topics: Animals; Bleomycin; Epithelial-Mesenchymal Transition; Humans; Idiopathic Pulmonary Fibrosis; Mice; Salicylates; Smad4 Protein; Sumoylation; Transforming Growth Factor beta1

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Ginkgo biloba; HSP90 Heat-Shock Proteins; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Salicylates; Tumor Cells, Cultured

Ginkgo biloba seeds are well known for the significant curative effects on relieving cough and asthma. However, the development of products from ginkgo seeds still falls behind at present, resulting in a great waste of ginkgo seeds' resource. In this work, submerged fermentation of ginkgo seed powder using Eurotium cristatum was studied to investigate its feasibility as a new processing method.. To promote the growth of E. cristatum, the optimum fermentation medium was 80.0 g L. Submerged fermentation using E. cristatum could significantly enhance the functional value and safety of ginkgo seed powder, and had great potential to become a novel processing method for the development of ginkgo seeds fermented products. © 2020 Society of Chemical Industry.

Topics: Antioxidants; Eurotium; Fermentation; Fermented Foods; Food Microbiology; Ginkgo biloba; Lovastatin; Powders; Pyridoxine; Salicylates; Seeds

Ginkgo biloba seeds are used as a functional food across Asia. However, the presence of toxic compounds has limited their application. In this study, freeze drying, infrared drying, hot-air drying and pulsed-vacuum drying were used to dry G. biloba seeds. A comprehensive analysis was performed on their product quality, antioxidant activities, bioactive and toxic components.. Results showed that the drying methods had a significant influence on product quality with freeze drying being superior due to the minimal microstructural damage, followed by infrared drying and pulsed-vacuum drying. Infrared-dried product possessed the strongest antioxidant activities and higher bioactive compound content than hot-air-dried and pulsed-vacuum-dried product. Toxic compounds in fresh G. biloba seeds (ginkgotoxin, ginkgolic acid and cyanide) were reduced markedly by drying. Ginkgotoxin was reduced fourfold, and the contents of acrylamide, ginkgolic acid and cyanide in dried G. biloba seeds were reduced to the scope of safety. Amongst the four drying methods, infrared drying had the shortest drying time, and its product showed higher quality and bioactive compound content, and stronger antioxidant activities.. These findings will offer salient information for selecting a drying method during the processing of ginkgo seeds. Infrared drying could be considered as a multiple-effect drying method in the processing of ginkgo seeds. © 2020 Society of Chemical Industry.

Topics: Antioxidants; Cyanides; Desiccation; Food Handling; Ginkgo biloba; Pyridoxine; Quality Control; Salicylates; Seeds

The imbalance of SUMOylation is related to different cancers, including gastric cancer (GC). Ginkgolic acid (GA) inhibits the growth and invasion of many cancer cells, and it has been reported to restrain SUMOylation. However, the role of GA in GC and whether it functions through SUMOylation remains to be clarified. Our research revealed that GA (15:1) inhibited cell proliferation, migration, epithelial-mesenchymal transition (EMT) and overall protein SUMOylation in BGC823 and HGC27 cells. In addition, knockdown of SUMO1 (small ubiquitin-like modifier) instead of SUMO2/3 played a similar role to GA in cell behaviors. Besides, nuclear IGF-1R (insulin-like growth factor 1 receptor) expression was markedly upregulated in GC cells compared to normal gastric epithelial cells. GA prevented IGF-1R from binding to SUMO1, thereby suppressing its nuclear accumulation. Further research found that IGF-1R directly bound to SNAI2 (snail family zinc finger 2) promoter. The interference of IGF-1R downregulated the mRNA and protein levels of SNAI2, while the overexpression of SUMO1, IGF-1R and UBC9 (SUMO-conjugating enzyme) played the opposite role. Furthermore, the co-transfection of SUMO1, UBC9 and IGF-1R vectors or the overexpression of SNAI2 reversed the inhibitory effects of GA on cell proliferation, migration and EMT. Finally, GA impeded the growth of GC xenografts and decreased the expression of nuclear IGF-1R and SNAI2 in vivo. In conclusion, these findings demonstrated that GA hindered the progression of GC by inhibiting the SUMOylation of IGF-1R. Thus, GA might be a promising therapeutic for GC.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation; Humans; Mice; Mice, Nude; Receptor, IGF Type 1; RNA Interference; RNA, Small Interfering; Salicylates; Small Ubiquitin-Related Modifier Proteins; Snail Family Transcription Factors; Stomach Neoplasms; SUMO-1 Protein; Sumoylation; Transplantation, Heterologous

3-Chymotrypsin-like protease (3CL

Topics: Antiviral Agents; Biflavonoids; Coronavirus Protease Inhibitors; COVID-19 Drug Treatment; Flavones; Ginkgo biloba; Humans; Molecular Structure; Phytotherapy; Plant Extracts; Plant Leaves; Salicylates; SARS-CoV-2; Virus Replication

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Cell Line, Tumor; Cell Survival; Endometrial Neoplasms; Female; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Salicylates; Signal Transduction; TOR Serine-Threonine Kinases

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; NF-kappa B; Proto-Oncogene Proteins c-akt; Salicylates

The retinoic-acid inducible gene (RIG)-I is a cytoplasmic pattern recognition receptor that senses single-stranded (ss) or double-stranded (ds) RNA. RIG-I also senses AT-rich dsDNA, poly(dA:dT), through the action of an RNA polymerase III-transcribed RNA intermediate. Upon the binding of an RNA ligand, RIG-I binds to the mitochondrial antiviral-signaling protein (MAVS) and induces the formation of filamentous aggregates of MAVS, leading to the formation of a signaling complex that drives Type I interferon (IFN) responses. In the current study, we investigated the issue of whether the SUMOylation of MAVS induced by poly(dA:dT) affects the aggregation of MAVS in the RIG-I/MAVS pathway in human keratinocytes. Our results show that the poly(dA:dT)-induced secretion of IFN-β was dependent on RIG-I and MAVS. The inhibition of SUMOylation by Ginkgolic acid or Ubc9 siRNA was found to inhibit the poly(dA:dT)-induced secretion of IFN-β, suggesting that the SUMOylation is required for the poly(dA:dT)-activated RIG-I/MAVS pathway, which drives the secretion of IFN-β. In addition, treatment with poly(dA:dT) enhanced the formation of polymeric chains of small-ubiquitin like modifiers (SUMO)3, but not SUMO1 and SUMO2, on MAVS. Our results also show that the conjugation of SUMO3 to MAVS induced by poly (dA:dT) enhanced the aggregation of MAVS. These collective results show that the formation of SUMO3-conjugated chains of MAVS induced by poly (dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS which, in turn, drives the secretion of IFN-β in human keratinocytes.

Topics: Adaptor Proteins, Signal Transducing; Cell Line; DEAD Box Protein 58; Humans; Interferon-beta; Keratinocytes; Ligands; Poly dA-dT; Protein Aggregates; Protein Domains; Receptors, Immunologic; RNA, Small Interfering; Salicylates; Sequence Deletion; Sumoylation; Ubiquitin-Conjugating Enzymes; Ubiquitins

Two-step targeted 2D planar chromatographic method (2DTLC) was used in the determination of ginkgolic acids in pharmaceuticals and dietary supplements. The choice of the extraction method and the separation technique was guided by the formulation type (capsule, tablet, tincture) with expected low amounts of ginkgolic acids in the analyzed herbal samples. Separation of ginkgolic acids C15:1 and C17:1 on HPTLC RP18 WF254s was preceded by its separation from the sample matrix on TLC Si60 F254s. Mobile phases consisted of acetonitrile/water/formic acid (80:20:1, V/V/V) and n-heptane/ethyl acetate/formic acid (20:30:1, V/V/V), resp. Identification of separated compounds was based on 2D-TLC co-chromatography with reference substances and off-line 2D-TLC x HPLC-DAD-ESI-MS analysis. Quantification of ginkgolic acids C15:1 and C17:1 was conducted densitometrically. Among the analyzed products, the presence of ginkgolic acids was confirmed only in herbal drugs containing 60 % ethanolic tinctures of Ginkgo biloba leaves. The use of TLC in the quantification of ginkgolic acids C15:1 and C17:1 in ginkgo extracts was described for the first time.

Topics: Chromatography, High Pressure Liquid; Dietary Supplements; Ginkgo biloba; Plant Extracts; Salicylates

Ginkgo biloba leaf extract contains many active ingredients that are beneficial for health. However, ginkgolic acid, one of the major components found in G. biloba extract, may cause serious allergic and toxic side effects. The purpose of this study is to immobilize the laccase system on the electrospun nylon fiber mat (NFM) to hydrolyze the ginkgolic acid in G. biloba leaf extract efficiently.. Novel electrospinning technology successfully produced high-quality nanoscopic fiber mats made of a mixture of multi-walled carbon nanotube and nylon 6,6. Laccase that was immobilized onto the NFM exhibited much higher efficiency in the catalyzation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) than nylon 6,6 pellets. After being immobilized onto the NFM, the pH and temperature stability of laccase were significantly improved. The NFM-immobilized laccase could maintain more than 50% of its original activity even after 40 days of storage or 10 operational cycles. The kinetic parameters, including rate constant (K), the time (τ50) in which 50% of ginkgolic acid hydrolysis was reached, the time (τcomplete) required to achieve complete ginkgolic acid hydrolysis, Km and Vmax were determined, and were 0.07 ± 0.01 min. The result successfully demonstrated the strong potential of using novel electrospun nanofiber mats as enzyme immobilization platforms, which could significantly enhance enzyme activity and stability. © 2020 Society of Chemical Industry.

Topics: Enzymes, Immobilized; Ginkgo biloba; Laccase; Nanofibers; Nanotubes, Carbon; Nylons; Plant Extracts; Salicylates

Gastric cancer is a frequently occurring cancer with high mortality each year worldwide. Finding new and effective therapeutic strategy against human gastric cancer is still urgently required. Ginkgolic acid (GA), a botanical drug, is extracted from the seed coat of Ginkgo biloba L. with various bioactive properties, including anti-tumor. Unfortunately, if GA has antitumor effect on human gastric cancer and the underlying molecular mechanisms have yet to be investigated. In the present study, we found that GA markedly reduced the gastric cancer cell viability. Furthermore, GA treatment led to the reduced migration ability of gastric cancer cells, which was associated with the decreased protein expression levels of Rho-associated protein kinase 1 (ROCK1), matrix metalloproteinase-2 (MMP-2), MMP-9 and α-smooth muscle actin (α-SMA). In addition, GA dose-dependently induced apoptosis in gastric cancer cells through activating Caspase-9/-3 and poly(ADP-Ribose) polymerase (PARP), which was along with the reduced Bcl-2 and Bcl-xl expression levels, and the elevated Bax and Bad levels. Consistently, Cyto-c protein expression in cytoplasm was also up-regulated by GA. Moreover, the production of reactive oxygen species (ROS) was significantly induced by GA. The activation of signal transducer and activator of transcription 3/janus kinase 2 (Stat3/JAK2) signaling pathway was inhibited by GA treatment. Intriguingly, blocking Stat3/JAK2 activation could further promote apoptosis and reduce cell viability induced by GA. However, GA-induced cell death was clearly abolished by ROS scavenger NAC, while the activation of Stat3/JAK2 signaling was restored by NAC. In vivo, GA showed effective role in reducing gastric tumor growth. Together, the findings here indicated that GA could be considered as an effective therapeutic candidate against human gastric cancer progression in future.

Topics: Actins; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cisplatin; Epithelial-Mesenchymal Transition; Ginkgo biloba; Humans; Janus Kinase 2; Male; Matrix Metalloproteinases; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Reactive Oxygen Species; rho-Associated Kinases; Salicylates; Signal Transduction; STAT3 Transcription Factor; Stomach Neoplasms

Ginkgolic acids (GA) are alkylphenol constituents of the leaves and fruits of Ginkgo biloba. GA has shown pleiotropic effects in vitro, including: antitumor effects through inhibition of lipogenesis; decreased expression of invasion associated proteins through AMPK activation; and potential rescue of amyloid-β (Aβ) induced synaptic impairment. GA was also reported to have activity against Escherichia coli and Staphylococcus aureus. Several mechanisms for this activity have been suggested including: SUMOylation inhibition; blocking formation of the E1-SUMO intermediate; inhibition of fatty acid synthase; non-specific SIRT inhibition; and activation of protein phosphatase type-2C. Here we report that GA inhibits Herpes simplex virus type 1 (HSV-1) by inhibition of both fusion and viral protein synthesis. Additionally, we report that GA inhibits human cytomegalovirus (HCMV) genome replication and Zika virus (ZIKV) infection of normal human astrocytes (NHA). We show a broad spectrum of fusion inhibition by GA of all three classes of fusion proteins including HIV, Ebola virus (EBOV), influenza A virus (IAV) and Epstein Barr virus (EBV). In addition, we show inhibition of a non-enveloped adenovirus. Our experiments suggest that GA inhibits virion entry by blocking the initial fusion event. Data showing inhibition of HSV-1 and CMV replication, when GA is administered post-infection, suggest a possible secondary mechanism targeting protein and DNA synthesis. Thus, in light of the strong effect of GA on viral infection, even after the infection begins, it may potentially be used to treat acute infections (e.g. Coronavirus, EBOV, ZIKV, IAV and measles), and also topically for the successful treatment of active lesions (e.g. HSV-1, HSV-2 and varicella-zoster virus (VZV)).

Topics: Animals; Antiviral Agents; Astrocytes; Chlorocebus aethiops; DNA Replication; DNA Virus Infections; DNA Viruses; DNA, Viral; HEK293 Cells; Humans; RNA Virus Infections; RNA Viruses; Salicylates; Vero Cells; Viral Envelope Proteins; Viral Fusion Proteins; Virion; Virus Internalization; Virus Replication

The alphaviruses Chikungunya (CHIKV), Mayaro (MAYV), Una (UNAV), and the flavivirus Zika (ZIKV) are emerging or re-emerging arboviruses which are responsible for frequent epidemic outbreaks. Despite the large impact of these arboviruses on health systems, there are no approved vaccines or treatments to fight these infections. As a consequence, there is an urgent need to discover new antiviral drugs. Natural products are a rich source of compounds with distinct biological activities, including antiviral properties. Thus, we aimed to explore the potential antiviral activity of Ginkgolic acid against the arboviruses CHIKV, MAYV, UNAV, and ZIKV. Viral progeny production in supernatants from cells treated or not treated with Ginkgolic acid was quantified by plaque-forming assay. Ginkgolic acid's direct virucidal activity against these arboviruses was also determined. Additionally, viral protein expression was assessed using Western blot and immunofluorescence. Our results reveal that Ginkgolic acid promotes a dose-dependent decrease in viral titers in all tested viruses. Moreover, the compound demonstrated strong virucidal activity. Finally, we found that viral protein expression was affected by treatment with this drug. Collectively, these findings suggest that Ginkgolic acid could have broader antiviral activity.

Topics: Alphavirus; Animals; Antiviral Agents; Cells, Cultured; Chikungunya virus; Chlorocebus aethiops; Dose-Response Relationship, Drug; Gene Expression Regulation, Viral; HeLa Cells; Humans; Salicylates; Vero Cells; Viral Plaque Assay; Virus Replication; Zika Virus

Ginkgolic acids (GAs) are found in the leaves, nuts, and testa of

Topics: Autophagy; DNA Fragmentation; Drugs, Chinese Herbal; Ginkgo biloba; HeLa Cells; Humans; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Mitophagy; Organelle Biogenesis; Salicylates

The accumulation of intracytoplasmic inclusion bodies (Lewy bodies) composed of aggregates of the alpha-synuclein (α-syn) protein is the principal pathological characteristic of Parkinson's disease (PD) and may lead to degeneration of dopaminergic neurons. To date there is no medication that can promote the efficient clearance of these pathological aggregates. In this study, the effect on α-syn aggregate clearance of ginkgolic acid (GA), a natural compound extracted from Ginkgo biloba leaves that inhibits SUMOylation amongst other pathways, was assessed in SH-SY5Y neuroblastoma cells and rat primary cortical neurons. Depolarization of SH-SY5Y neuroblastoma cells and rat primary cortical neurons with KCl was used to induce α-syn aggregate formation. Cells pre-treated with either GA or the related compound, anacardic acid, revealed a significant decrease in intracytoplasmic aggregates immunopositive for α-syn and SUMO-1. An increased frequency of autophagosomes was also detected with both compounds. GA post-treatment 24 h after depolarization also significantly diminished α-syn aggregate bearing cells, indicating the clearance of pre-formed aggregates. Autophagy inhibitors blocked GA-dependent clearance of α-syn aggregates, but not increased autophagosome frequency. Western analysis revealed that the reduction in α-syn aggregate frequency obtained with GA pre-treatment was accompanied by little change in the abundance of SUMO conjugates. The current findings show that GA can promote autophagy-dependent clearance of α-syn aggregates and may have potential in disease modifying therapy.

Topics: alpha-Synuclein; Animals; Autophagosomes; Autophagy; Cell Line, Tumor; Cells, Cultured; Humans; Neurons; Neuroprotective Agents; Protein Aggregates; Rats; Rats, Wistar; Salicylates; Sumoylation

Ginkgolic acids (GAs) and Ginkgotoxin (4'-O-methylpyridoxine, MPN) are main toxic compounds in Ginkgo biloba seeds which are widely used in the treatment of coughing in China. To evaluate the pharmacokinetics of GAs, MPN and their metabolites in rat plasma, a highly sensitive method followed by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has been developed and validated. The proposed method is selective, precise and accurate enough of MPN and its metabolites (4-pyridoxic Acid, pyridoxal, and pyridoxine) for the pharmacokinetic study. After oral administration of MPN, the plasma concentrations of MPN and its metabolites were increased rapidly. Meanwhile, an investigation was carried out to compare the interactions of the pharmacokinetic profiles of MPN and GAs. Five GAs and main metabolites of GA (15:1) and GA (17:1) were also analyzed by using our previous method. After coadministration GAs with MPN, T

Topics: Administration, Oral; Animals; Chromatography, High Pressure Liquid; Drug Interactions; Ginkgo biloba; Male; Plant Extracts; Pyridoxine; Rats; Rats, Sprague-Dawley; Salicylates; Seeds; Tandem Mass Spectrometry

Liver cancer is one of the most devastating types of cancer worldwide. Despite years of improvements in treatment, the prognosis of patients with this type of malignancy remains poor due to frequent recurrence and metastasis after surgical resection. Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgo biloba L. that possesses a wide range of bioactive properties. However, to the best of our knowledge, whether GA can inhibit the invasion of liver cancer cells and the underlying mechanisms remains unknown. The aim of the present study was to investigate the effects of GA on the migration and invasion abilities of liver cancer cells and the underlying molecular mechanism. The results revealed that GA suppressed the migration and invasion abilities of HepG2 cells. In addition, GA treatment inhibited the expression of invasion‑related molecules (MMP‑2 and MMP‑9) and prevented the epithelial‑mesenchymal transition (EMT) of HepG2 cells. Further experiments revealed that GA‑reduced hepatocyte growth factor (HGF) production and suppressed c‑Met phosphorylation may be the underlying mechanisms. Exogenous recombinant HGF supplementation improved the cell invasion ability impaired by GA. Moreover, the in vivo experiment revealed that GA inhibited the tumor growth of liver cancer and prevented EMT. Collectively, these data indicated that GA effectively suppressed the invasion and EMT of HepG2 cells via downregulation of HGF/c‑Met signaling, thus GA may serve as a novel chemotherapeutic agent for the treatment of HCC.

Topics: Animals; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Mice; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins c-met; Salicylates; Signal Transduction; Xenograft Model Antitumor Assays

Topics: Cryptosporidiosis; Cryptosporidium; Drugs, Chinese Herbal; Humans; Parasitic Sensitivity Tests; Phytotherapy; Salicylates

In the present investigation, fibrinolytic Ginkgo seeds were produced by solid-state fermentation (SSF) with Bacillus natto strains, and some parameters of the fermentation processes were investigated. Under optimal fermentation conditions, the fibrinolytic activity of Ginkgo seeds reached 3,682 ± 43 IU/g with the fermentation parameters of relative humidity 80%, initial water content 73%, fermentation temperature 38°C, inoculation volume 18%, and fermentation time 38 hr, respectively. The fermented Ginkgo seeds possessed a superior potential for the production of Nattokinase. What's more, the fermented Ginkgo seeds possessed higher total flavonoid and lower ginkgolic acids contents, which could enhance bioactivity and guarantee food safety. Sensory evaluations indicated that Ginkgo seeds produced by SSF could also be consumed as a kind of popular food. PRACTICAL APPLICATIONS: Fermented food is popular in countries. It can not only improve the sensory properties of the products, reduce undesirable constituents, and make nutrients easily absorbable, but also improve the nutritional properties. Ginkgo biloba L is one of the oldest species that has existed on earth for more than 200 million years. However, the application of Ginkgo seeds has been limited because of the ginkgolic acids. In a previous study, immobilized Bacillus natto acted upon Ginkgo seeds to enhance the bioactivity and safety of fermented Ginkgo seeds. However, separating the fermented Ginkgo seeds from the liquid needs a large amount of energy. The solid-state fermentation of Ginkgo seeds is a good choice to produce functional Ginkgo seed products.

Topics: Bacillus subtilis; Fermentation; Fibrinolysis; Flavonoids; Ginkgo biloba; Salicylates; Seeds; Subtilisins

Topics: Apoptosis; Autophagy; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Humans; Reactive Oxygen Species; Salicylates

Sumoylation is a post-translational modification process having an important influence in mesenchymal stem cell (MSC) differentiation. Thus, sumoylation-modulating chemicals might be used to control MSC differentiation for skeletal tissue engineering. In this work, we studied how the differentiation of mouse bone marrow stromal cells (mBMSCs) is affected by ginkgolic acid (GA), a potent sumoylation inhibitor also reported to inhibit histone acetylation transferase (HAT). Our results show that GA promoted the differentiation of mBMSCs into adipocytes when cultured in osteogenic medium. Moreover, mBMSCs pre-treated with GA showed enhanced pre-adipogenic gene expression and were more efficiently differentiated into adipocytes when subsequently cultured in the adipogenic medium. However, when GA was added at a later stage of adipogenesis, adipocyte maturation was markedly inhibited, with a dramatic down-regulation of multiple lipogenesis genes. Moreover, we found that the effects of garcinol, a HAT inhibitor, differed from those of GA in regulating adipocyte commitment and adipocyte maturation of mBMSCs, implying that the GA function in adipogenesis is likely through its activity as a sumoylation inhibitor, not as a HAT inhibitor. Overall, our studies revealed an unprecedented role of GA in MSC differentiation and provide new mechanistic insights into the use of GA in clinical applications.

Topics: Adipogenesis; Animals; Bone Marrow Cells; Cells, Cultured; Histone Acetyltransferases; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Salicylates; Sumoylation; Terpenes

Protein modification by small ubiquitin-like modifier (SUMO) plays a critical role in the pathogenesis of heart diseases. The present study was designed to determine whether ginkgolic acid (GA) as a SUMO-1 inhibitor exerts an inhibitory effect on cardiac fibrosis induced by myocardial infarction (MI).. GA was delivered by osmotic pumps in MI mice. Masson staining, electron microscopy (EM) and echocardiography were used to assess cardiac fibrosis, ultrastructure and function. Expression of SUMO-1, PML, TGF-β1 and Pin1 was measured with Western blot or Real-time PCR. Collagen content, cell viability and myofibroblast transformation were measured in neonatal mouse cardiac fibroblasts (NMCFs). Promyelocytic leukemia (PML) protein was over-expressed by plasmid transfection.. GA improved cardiac fibrosis and dysfunction, and decreased SUMO-1 expression in MI mice. GA (>20 μM) inhibited NMCF viability in a dose-dependent manner. Nontoxic GA (10 μM) restrained angiotensin II (Ang II)-induced myofibroblast transformation and collagen production. GA also inhibited expression of TGF-β1 mRNA and protein in vitro and in vivo. GA suppressed PML SUMOylation and PML nuclear body (PML-NB) organization, and disrupted expression and recruitment of Pin1 (a positive regulator of TGF-β1 mRNA), whereas over-expression of PML reversed that.. Inhibition of SUMO-1 by GA alleviated MI-induced heart dysfunction and fibrosis, and the SUMOylated PML/Pin1/TGF-β1 pathway is crucial for GA-inhibited cardiac fibrosis.

Topics: Animals; Animals, Newborn; Cell Survival; Dose-Response Relationship, Drug; Fibrosis; Male; Mice; Myocardial Infarction; Salicylates; Stroke Volume; SUMO-1 Protein

The toxic ginkgolic acids are the main safety concern for the application of Ginkgo biloba. In this study, the degradation ability of salicylic acid decarboxylase (SDC) for ginkgolic acids was examined using ginkgolic acid C15:1 as a substrate. The results indicated that the content of ginkgolic acid C15:1 in Ginkgo biloba seeds was significantly decreased after 5 h treatment with SDC at 40 °Cand pH 5.5. In order to explore the structure of SDC and the interaction between SDC and substrates, homology modeling, molecular docking and molecular dynamics were performed. The results showed that SDC might also have a catalytic active center containing a Zn

Topics: Biocatalysis; Carboxy-Lyases; Models, Molecular; Salicylates; Salicylic Acid

This present investigation examined the mitigating impact of Ginkgolic acid in the organization on oxidized low-density lipoproteinox-LDL (ox- LDL) animated in HUVECs, and to clear up its fundamental molecular components. The levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess examine and catalyst connected immunosorbent test. The declarations of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-initiated protein kinases (MAPKs), and Akt were measured utilizing Western smearing. ox-LDL-instigated was utilized as the HUVECs cell model of inflammation. Ginkgolic acid significantly inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the expression of iNOS and COX-2 in ox-LDL-stimulated HUVECs cells. Ginkgolic acid strongly suppressed NF-κB by preventing degradation of inhibitor of κB-α as well as by inhibiting phosphorylation of Akt and MAPKs. Ginkgolic acid reduced LDL-stimulated inflammation in endothelial cells. These outcomes suggest that the anti-inflammatory properties of Ginkgolic acid are related to a down-control of iNOS, COX-2, and master provocative cytokines through the restraint of NF-κB pathway in ox- LDL-animated endothelial cells.

Topics: Anti-Inflammatory Agents; Cyclooxygenase 2; Cytokines; Dinoprostone; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Lipoproteins, LDL; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Salicylates

Several protein tyrosine phosphatases (PTPs) that disrupt the insulin-signaling pathway were investigated by siRNAs to identify potential antidiabetic targets. Individual knockdown of PTPN9 and DUSP9 in 3T3-L1 preadipocytes increased AMPK phosphorylation, respectively, and furthermore, concurrent knockdown of both PTPN9 and DUSP9 synergistically increased AMPK phosphorylation. Next, 658 natural products were screened to identify dual inhibitors of both PTPN9 and DUSP9. Based on the selectivity and inhibition potency of the compounds, ginkgolic acid (GA) was selected for further study as a potential antidiabetic drug candidate. GA inhibited the enzymatic activity of PTPN9 (K

Topics: AMP-Activated Protein Kinases; Animals; Cell Line; Dual-Specificity Phosphatases; Enzyme Inhibitors; Gene Knockdown Techniques; Glucose; Hypoglycemic Agents; Mice; Mitogen-Activated Protein Kinase Phosphatases; Phosphorylation; Protein Tyrosine Phosphatases, Non-Receptor; Salicylates

A synthetic strategy for phenolic lipids such as anacardic acid and ginkgolic acid derivatives using an efficient and selective redox-relay Heck reaction followed by a stereoselective olefination is reported. This approach controls both the alkene position and stereochemistry, allowing the synthesis of natural and unnatural unsaturated lipids as single isomers. By this strategy, the activities of different anacardic acid and ginkgolic acid derivatives have been examined in a matrix metalloproteinase inhibition assay.

Topics: Alkenes; Anacardic Acids; Lipids; Matrix Metalloproteinase Inhibitors; Molecular Structure; Oxidation-Reduction; Palladium; Phenols; Salicylates; Stereoisomerism; Structure-Activity Relationship

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.

Topics: Countercurrent Distribution; Ginkgo biloba; Molecular Structure; Plant Extracts; Salicylates

Ginkgolic acid obtained as a sphingomyelin synthase inhibitor from a plant extract library inspired the concept of sphingolipid mimics. Ginkgolic acid-derived N-acyl anilines and ginkgolic acid 2-phosphate (GA2P) respectively mimic ceramide and sphingosine 1-phosphate (S1P) in structure and function. The GA2P-induced phosphorylation of ERK and internalization of S1P receptor 1 (S1P1) indicated potent agonist activity. Docking studies revealed that GA2P adopts a similar binding conformation to the bound ligand ML5, which is a strong antagonist of S1P1.

Topics: Animals; Biological Products; CHO Cells; Cricetulus; Dose-Response Relationship, Drug; Drug Design; Enzyme Inhibitors; Humans; Molecular Docking Simulation; Molecular Structure; Receptors, Lysosphingolipid; Salicylates; Sphingolipids; Structure-Activity Relationship; Transferases (Other Substituted Phosphate Groups)

Sumoylation is a downstream effector of aging/oxidative stress; excess oxidative stress leads to dysregulation of a specificity protein1 (Sp1) and its target genes, such as Peroxiredoxin 6 (Prdx6), resulting in cellular damage. To cope with oxidative stress, cells rely on a signaling pathway involving redox-sensitive genes. Herein, we examined the therapeutic efficacy of the small molecule Ginkgolic acid (GA), a Sumoylation antagonist, to disrupt aberrant Sumoylation signaling in human and mouse lens epithelial cells (LECs) facing oxidative stress or aberrantly expressing Sumo1 (small ubiquitin-like modifier). We found that GA globally reduced aberrant Sumoylation of proteins. In contrast, Betulinic acid (BA), a Sumoylation agonist, augmented the process. GA increased Sp1 and Prdx6 expression by disrupting the Sumoylation signaling, while BA repressed the expression of both molecules. In vitro DNA binding, transactivation, Sumoylation and expression assays revealed that GA enhanced Sp1 binding to GC-boxes in the Prdx6 promoter and upregulated its transcription. Cell viability and intracellular redox status assays showed that LECs pretreated with GA gained resistance against oxidative stress-driven aberrant Sumoylation signaling. Overall, our study revealed an unprecedented role for GA in LECs and provided new mechanistic insights into the use of GA in rescuing LECs from aging/oxidative stress-evoked dysregulation of Sp1/Prdx6 protective molecules.

Topics: Animals; Betulinic Acid; DNA-Binding Proteins; Epithelial Cells; Gene Expression Regulation; Humans; Lens, Crystalline; Mice; Oxidative Stress; Pentacyclic Triterpenes; Peroxiredoxin VI; Promoter Regions, Genetic; Reactive Oxygen Species; Salicylates; Signal Transduction; Sp1 Transcription Factor; Sumoylation; Triterpenes

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Topics: Animals; Cells, Cultured; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP3A; Ginkgo biloba; Glucuronosyltransferase; Hepatocytes; Humans; Kinetics; Liver; Microsomes, Liver; Plant Extracts; Rats; Rats, Sprague-Dawley; Salicylates; UDP-Glucuronosyltransferase 1A9

Ginkgolic acids (GA), a group of alkyl phenols found in crude extracts of Ginkgo biloba leaves, are known to have anticancer activity, but their mode of action is not well understood. Our aim in this study was to investigate the anti-migratory activity of seven GA against breast cancer cells and to determine the molecular mechanism behind this activity. All seven GA and their mixture inhibited wound healing in MCF-7 and MDA-MB 231 breast cancer cells. None of the compounds nor the mixture showed cytotoxicity towards the two cell lines, if tested by the resazurin assay. C13:0 inhibited NF-κB activity in the HEK Blue Null 1 reporter cell line. Furthermore, C13:0 inhibited degradation of nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor α (IκBα). Sumoylation assay revealed that GA inhibited sumoylation of NF-κB essential modulator (NEMO). Molecular docking on SUMO-activating enzyme E1 showed that the seven GA bound to the active adenylation site with high calculated affinities ranging from -10.28 to -12.27 kcal/mol. Quantitative RT-PCR using C15:0, C13:0 and the mixture showed a significant down-regulation of urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinase 9 (MMP-9). We conclude that GA revealed considerable anti-migratory activity at non-cytotoxic concentrations, indicating anti-metastatic activity with low toxicity. This effect can be explained by the inhibition of NEMO sumoylation leading to inhibition of IκBα degradation and consequently a reduction of NF-κB activity, leading to the down-regulation of metastasis related genes including uPA, PAI-1, CXCR4, and MMP-9.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Matrix Metalloproteinase 9; MCF-7 Cells; Models, Molecular; Molecular Docking Simulation; NF-kappa B; Plasminogen Activator Inhibitor 1; Receptors, CXCR4; Salicylates; Signal Transduction; Sumoylation; Urokinase-Type Plasminogen Activator

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Survival; Dogs; Ginkgo biloba; Lysosomes; Madin Darby Canine Kidney Cells; Mitochondria; Necrosis; Plant Extracts; Salicylates

Ginkgolic acids (GAs) are thought to be the potentially hazardous constituents corresponding to the toxic side effects of Ginkgo products. In this study, toxicological and metabolomics studies of GAs were carried out by ultra-performance liquid chromatography-high-definition mass spectrometry (UPLC-HDMS). Significant changes in serum clinical chemistry were observed in the both low (100 mg/kg) and high (900 mg/kg) doses. Especially the serum enzyme of ALT, AST, LDH, and CK decreased in treated groups. The histopathological observation demonstrated hepatic steatosis in liver and tubular vacuolar degeneration in kidney. These results demonstrated the hepatotoxicity and nephrotoxicity of GAs. Functional disorders are more likely to be toxic induced by GAs. Metabolic profiling within seven days revealed the change of the body status after oral administration. The results indicated the body function was significantly influenced at the 3rd day and could recover in seven days. Metabolomic analysis showed alterations in 14 metabolites from plasma such as LysoPC(18:0), LysoPC(18:2) and other lipids. The results suggested that exposure to GAs could cause disturbances in liver and kidney function associated with the metabolisms of lipids, glucose and the enzyme activity.

Topics: Administration, Oral; Animals; Chromatography, High Pressure Liquid; Kidney; Liver; Male; Mass Spectrometry; Metabolomics; Rats; Rats, Sprague-Dawley; Salicylates

Topics: Dietary Supplements; Ginkgo biloba; Mass Spectrometry; Plant Leaves; Salicylates

α-Synuclein accumulation is a pathological hallmark of Parkinson's disease (PD). Ubiquitinated α-synuclein is targeted to proteasomal or lysosomal degradation. Here, we identify SUMOylation as a major mechanism that counteracts ubiquitination by different E3 ubiquitin ligases and regulates α-synuclein degradation. We report that PIAS2 promotes SUMOylation of α-synuclein, leading to a decrease in α-synuclein ubiquitination by SIAH and Nedd4 ubiquitin ligases, and causing its accumulation and aggregation into inclusions. This was associated with an increase in α-synuclein release from the cells. A SUMO E1 inhibitor, ginkgolic acid, decreases α-synuclein levels by relieving the inhibition exerted on α-synuclein proteasomal degradation. α-Synuclein disease mutants are more SUMOylated compared with the wild-type protein, and this is associated with increased aggregation and inclusion formation. We detected a marked increase in PIAS2 expression along with SUMOylated α-synuclein in PD brains, providing a causal mechanism underlying the up-regulation of α-synuclein SUMOylation in the disease. We also found a significant proportion of Lewy bodies in nigral neurons containing SUMO1 and PIAS2. Our observations suggest that SUMOylation of α-synuclein by PIAS2 promotes α-synuclein aggregation by two mutually reinforcing mechanisms. First, it has a direct proaggregatory effect on α-synuclein. Second, SUMOylation facilitates α-synuclein aggregation by blocking its ubiquitin-dependent degradation pathways and promoting its accumulation. Therefore, inhibitors of α-synuclein SUMOylation provide a strategy to reduce α-synuclein levels and possibly aggregation in PD.

Topics: alpha-Synuclein; Animals; Cells, Cultured; HEK293 Cells; Humans; Neurons; Parkinson Disease; Protein Inhibitors of Activated STAT; Proteolysis; Rats, Sprague-Dawley; Salicylates; Substantia Nigra; Sumoylation

Epithelial-to-mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N-cadherin, MMP-9, MMP-2, Twist and Snail) and suppressed TGF-β-induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N-cadherin), MMP-9, MMP-2, Twist and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E-cadherin) in both A549 and H1299 cells. TGF-β-induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF-β-induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. J. Cell. Physiol. 232: 346-354, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Cell Line, Tumor; Cell Movement; Cell Survival; Down-Regulation; Enzyme Activation; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Salicylates; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

A highly sensitive method using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has been developed and validated for the simultaneous identification and quantification of ginkgolic acids and semi-quantification of their metabolites in rat plasma. For the five selected ginkgolic acids, the method was found to be with good linearities (r>0.9991), good intra- and inter-day precisions (RSD<15%), and good accuracies (RE, from -10.33% to 4.92%) as well. Extraction recoveries, matrix effects and stabilities for rat plasm samples were within the required limits. The validated method was successfully applied to investigate the pharmacokinetics of the five ginkgolic acids in rat plasma after oral administration of 3 dosage groups (900mg/kg, 300mg/kg and 100mg/kg). Meanwhile, six metabolites of GA (15:1) and GA (17:1) were identified by comparison of MS data with reported values. The results of validation in terms of linear ranges, precisions and stabilities were established for semi-quantification of metabolites. The curves of relative changes of these metabolites during the metabolic process were constructed by plotting the peak area ratios of metabolites to salicylic acid (internal standard, IS), respectively. Double peaks were observed in all 3 dose groups. Different type of metabolites and different dosage of each metabolite both resulted in different T

Topics: Administration, Oral; Animals; Chromatography, High Pressure Liquid; Drug Stability; Limit of Detection; Linear Models; Mass Spectrometry; Rats; Reproducibility of Results; Salicylates

Ginkgolic acid (15:1) is a major toxic component in extracts obtained from Ginkgo biloba (EGb) that has allergic and genotoxic effects. This study is the first to explore the hepatotoxicity of ginkgolic acid (15:1) using a NMR (nuclear magnetic resonance)-based metabolomics approach in combination with biochemistry assays. Mice were orally administered two doses of ginkgolic acid (15:1), and mouse livers and serum were then collected for NMR recordings and biochemical assays. The levels of activity of alanine aminotransferase (ALT) and glutamic aspartate transaminase (AST) observed in the ginkgolic acid (15:1)-treated mice suggested that it had induced severe liver damage. An orthogonal signal correction partial least-squares discriminant analysis (OSC-PLSDA) performed to determine the metabolomic profile of mouse liver tissues indicated that many metabolic disturbances, especially oxidative stress and purine metabolism, were induced by ginkgolic acid (15:1). A correlation network analysis combined with information related to structural similarities further confirmed that purine metabolism was disturbed by ginkgolic acid (15:1). This mechanism might represent the link between the antitumour activity and the liver injury-inducing effect of ginkgolic acid (15:1). A SUS (Shared and Unique Structure) plot suggested that a two-dose treatment of ginkgolic acid (15:1) had generally the same effect on metabolic variations but that its effects were dose-dependent, revealing some of the common features of ginkgolic acid (15:1) dosing. This integrated metabolomics approach helped us to characterise ginkgolic acid (15:1)-induced liver damage in mice.

Topics: Administration, Oral; Animals; Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Liver; Male; Metabolome; Metabolomics; Mice, Inbred ICR; Nuclear Magnetic Resonance, Biomolecular; Salicylates

The present study investigated the antibacterial activity and underlying mechanisms of ginkgolic acid (GA) C15:1 monomer using green fluorescent protein (GFP)-labeled bacteria strains.. GA presented significant antibacterial activity against Gram-positive bacteria but generally did not affect the growth of Gram-negative bacteria. The studies of the antibacterial mechanism indicated that large amounts of GA (C15:1) could penetrate GFP-labeled Bacillus amyloliquefaciens in a short period of time, and as a result, led to the quenching of GFP in bacteria. In vitro results demonstrated that GA (C15:1) could inhibit the activity of multiple proteins including DNA polymerase. In vivo results showed that GA (C15:1) could significantly inhibit the biosynthesis of DNA, RNA and B. amyloliquefaciens proteins.. We speculated that GA (C15:1) achieved its antibacterial effect through inhibiting the protein activity of B. amyloliquefaciens. GA (C15:1) could not penetrate Gram-negative bacteria in large amounts, and the lipid soluble components in the bacterial cell wall could intercept GA (C15:1), which was one of the primary reasons that GA (C15:1) did not have a significant antibacterial effect on Gram-negative bacteria.

Topics: Anti-Bacterial Agents; Bacillus amyloliquefaciens; Bacterial Proteins; Cell Proliferation; Dose-Response Relationship, Drug; Molecular Imaging; Protein Biosynthesis; Salicylates

Ginkgolic acid C 17:1 (GAC 17:1) extracted from

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Ginkgo biloba; Humans; Interleukin-6; Membrane Potential, Mitochondrial; Multiple Myeloma; Phosphorylation; Plant Extracts; Plant Leaves; Protein Binding; Protein Tyrosine Phosphatase, Non-Receptor Type 6; PTEN Phosphohydrolase; Salicylates; STAT3 Transcription Factor

Ginkgo biloba extract is a multicomponent pharmacological agent widely used in neurological disorders therapy. It was shown that ginkgolic acid, a constituent of lipophylic Ginkgo biloba extract, has numerous biological activities. In the present study we have focused on the features of ginkgolic acid action on αl and α2 glycine receptors that make part of the inhibitory system of the brain. Using whole-cell configuration of patch-clamp recording we analysed effects of ginkgolic acid on different subunits of glycine receptors. Experiments were performed on cultured Chinese hamster ovary cells (CHO cells), transfected with αl and α2 glycine receptor subunits. Ionic currents were induced by the fast application of different glycine concentrations. After 20-40 sec of pre-treatment with ginkgolic acid (25μM) currents mediated by al glycine receptors reversibly increased from 364±49 pA, (n=34) to 846±134 pA, (n=34). EC(50) for glycine has changed from 36±6 μM (control) to 17±2 μM. In contrast, the application of ginkgolic acid on glycine receptors formed by α2 subunits did not provoke potentiation. Our results demonstrate that ginkgolic acid is a subunit specific modulator of glycine receptors. The mechanisms of the ginkgolic acid action on glycine receptors require further investigation.

Topics: Animals; CHO Cells; Cricetulus; Gene Expression; Ginkgo biloba; Humans; Membrane Potentials; Patch-Clamp Techniques; Plant Extracts; Protein Subunits; Receptors, Glycine; Salicylates; Transgenes

Analysis of 11 commercial nutraceutical products obtained from ginkgo has been performed using ultra-high performance liquid chromatography coupled to single-stage Orbitrap high resolution mass spectrometry (UHPLC-Orbitrap-MS). The main phytochemicals present in these samples were detected and quantified, utilizing a database containing 65 compounds. Phytochemicals were extracted using a mixture of an aqueous solution of methanol:water (80:20, v/v) in two sequential solid-liquid extractions. Adequate validation parameters were obtained. The validated compounds exhibited suitable linearity with determination coefficients (R(2)) higher than 0.99, and intra and inter-day precision were lower than 17 and 22%, respectively. Limits of detection (LODs) and quantification (LOQs) were calculated, ranging from 2 to 10 μg L(-1), except for myricetin (LOD, 150 μg L(-1) and LOQ, 300 μg L(-1)). Results indicate that the amount of terpenoids greatly varies among samples, ranging from 1133 (C7) to 12706 mg kg(-1) (C11). This emphasizes the importance of improve quality control in ginkgo-based products. Moreover, retrospective analysis allowed the detection of some undesirable substances as ginkgolic acid in the samples evaluated.

Topics: Biological Products; Chromatography, High Pressure Liquid; Dietary Supplements; Ginkgo biloba; Limit of Detection; Mass Spectrometry; Phytochemicals; Quality Control; Retrospective Studies; Salicylates

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.

Topics: Chromatography, Gas; Chromatography, Liquid; Chromatography, Supercritical Fluid; Dietary Supplements; Ginkgo biloba; Lactones; Plant Extracts; Salicylates; Terpenes

An analytical method was developed for the determination of ginkgolic acids in ginkgo biloba extract and its preparations by ultra high performance liquid chromatography-triple quadrupole mass spectrometry. A chromatographic column, Agilent Poroshell 120 EC-C18 (50 mm x 3. 0 mm, 2.7 µm), was used with methanol-l% acetic acid (90 :10, v/v) as the mobile phase. The ginkgo acids were detected by electrospray ionization mass spectrometry in negative mode with multiple reaction monitoring (MRM) mode. Ginkgo acids C13:0, C15:1 and C17:1 possessed good linear correlations in the mass concentration range from 2 to 200 µg/L, with the correlation coefficients more than 0. 999. The mean recoveries at the spiked levels of 5, 20 and 100 µg/L were in the range of 86. 3%-114. 3%, and the RSDs were 0. 5%-13. 6%. The limits of detection and quantification were 0. 003-0. 08 µg/g and 0. 01-0. 19 µg/g, respectively. The method was successfully applied to the analysis of actual samples.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Ginkgo biloba; Plant Extracts; Salicylates; Spectrometry, Mass, Electrospray Ionization

Breast cancer is genetically heterogeneous, and recent studies have underlined a prominent contribution of epigenetics to the development of this disease. To uncover new synthetic lethalities with known breast cancer oncogenes, we screened an epigenome-focused short hairpin RNA library on a panel of engineered breast epithelial cell lines. Here we report a selective interaction between the NOTCH1 signaling pathway and the SUMOylation cascade. Knockdown of the E2-conjugating enzyme UBC9 (UBE2I) as well as inhibition of the E1-activating complex SAE1/UBA2 using ginkgolic acid impairs the growth of NOTCH1-activated breast epithelial cells. We show that upon inhibition of SUMOylation NOTCH1-activated cells proceed slower through the cell cycle and ultimately enter apoptosis. Mechanistically, activation of NOTCH1 signaling depletes the pool of unconjugated small ubiquitin-like modifier 1 (SUMO1) and SUMO2/3 leading to increased sensitivity to perturbation of the SUMOylation cascade. Depletion of unconjugated SUMO correlates with sensitivity to inhibition of SUMOylation also in patient-derived breast cancer cell lines with constitutive NOTCH pathway activation. Our investigation suggests that SUMOylation cascade inhibitors should be further explored as targeted treatment for NOTCH-driven breast cancer.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Microscopy, Fluorescence; Receptor, Notch1; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Salicylates; Signal Transduction; Small Ubiquitin-Related Modifier Proteins; SUMO-1 Protein; Sumoylation; Transcriptional Activation; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitins

Ginkgolic acids (GAs) are alkylphenols which can be found in the fruits and leaves of Ginkgo biloba L. (Ginkgoaceae) used in herbal teas, drugs and food supplements. Standardized leaf extracts of G. biloba are widely used in the therapy of cognitive decline including Alzheimer's diseases. However, GAs are known to have cytotoxic and allergenic potential and are suspected to possess genotoxic properties. Therefore, we examined in this study the cytotoxicity and mutagenicity of three major GAs with different alkyl or alkenyl groups (13:0, 15:1, 17:1). Cytotoxicity was assessed in male Chinese hamster lung fibroblasts (V79 cells) using the resazurin reduction assay. The substances showed concentration dependent cytotoxic effects after 24h of incubation at concentrations of 50μM and higher. Mutagenicity was determined by using the Ames fluctuation assay in different Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) with and without exogenous metabolic activation (S9 mix). Furthermore, we analyzed the mutagenic potency of the three major GAs in V79 cells by performing the hypoxanthine phosphoribosyl transferase (HPRT) assay which detects gene mutations at the HPRT locus. None of the mutagenic assays showed any increase in mutagenicity above background. Therefore, these data provide evidence that the GAs tested have some cytotoxic potency but are not mutagenic. Thus, our findings contribute to the risk assessment of preparations containing plant extracts from G. biloba.

Topics: Animals; Cell Line; Cell Survival; Cricetinae; Cricetulus; Hypoxanthine Phosphoribosyltransferase; Male; Mutagenicity Tests; Mutagens; Salicylates; Toxicity Tests

An HPLC quantification method for ginkgolic acid derivatives in Ginkgo biloba leaf extracts was developed. Using 13 : 0 ginkgolic acid as a marker compound, the relative correlation factors of the four other ginkgolic acid derivatives - namely, 15 : 0 ginkgolic acid, 15 : 1 ginkgolic acid, 17 : 1 ginkgolic acid, and 17 : 2 ginkgolic acid - to 13 : 0 ginkgolic acid were determined by HPLC and subsequently used for calculating their contents in ten hydro-ethanolic refined extract samples. In other words, the content of 13 : 0 ginkgolic acid in the extracts was determined using the isolated compound as an external standard. Subsequently the now known concentration of this compound functioned as an internal standard for the quantification of the other four ginkgolic acid derivatives via the described correlation factors. This HPLC method was validated by two independent control measurements, one with an external standard for every individual compound and one based on the present method with the single marker compound alone. The results did not differ significantly in any of the 10 tested extract samples. The protocol presented here thus not only uses the same reference substance for G. biloba extracts as the current Chinese Pharmacopoeia method but also incorporates the advantages of the current European Pharmacopoeia approach. It is simple, reproducible, and can be used to determine the total contents of ginkgolic acid derivatives in G. biloba leaf extracts.

Topics: Biomarkers; Chromatography, High Pressure Liquid; Ginkgo biloba; Limit of Detection; Plant Extracts; Plant Leaves; Salicylates

SUMOylation is a form of post-translational modification where small ubiquitin-like modifiers (SUMO) are covalently attached to target proteins to regulate their properties. SUMOylation has been demonstrated during cell stress and implicated in cellular stress response. However, it is largely unclear if SUMOylation contributes to the pathogenesis of kidney diseases, such as acute kidney injury (AKI). Here we have demonstrated a dynamic change of protein SUMOylation in ischemic and cisplatin nephrotoxic AKI in mice. In rat kidney proximal tubular cells (RPTC), cisplatin-induced SUMOylation was diminished by two antioxidants (N-acetylcysteine and dimethylurea), supporting a role of oxidative stress in the activation of SUMOylation. In addition, SUMOylation by SUMO-2/3, but not SUMO-1, was partially suppressed by pifithrin-alpha (a pharmacological inhibitor of p53), supporting a role of p53 in SUMOylation by SUMO-2/3. We further examined the role of SUMOylation during cisplatin treatment of RPTC by using ginkgolic acid (GA), a pharmacological inhibitor of SUMOylation. Pretreatment with GA suppressed SUMOylation and importantly, GA enhanced apoptosis during cisplatin incubation. Taken together, the results demonstrate the first evidence of SUMOylation in AKI and suggest that SUMOylation may play a cytoprotective role in kidney tubular cells.

Topics: Acetylcysteine; Acute Kidney Injury; Animals; Antineoplastic Agents; Apoptosis; Cisplatin; Free Radical Scavengers; HEK293 Cells; Humans; Kidney Tubules, Proximal; Mice; Oxidative Stress; Rats; Salicylates; SUMO-1 Protein; Sumoylation; Tumor Suppressor Protein p53

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.

Topics: Chloroplasts; Electrophoresis, Gel, Two-Dimensional; Enzymes; Flavonoids; Ginkgo biloba; Plant Leaves; Plant Proteins; Plants, Medicinal; Proteome; Salicylates; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Ultraviolet Rays

Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgo biloba L. with a wide range of bioactive properties, including anti-tumor effect. However, whether GA has antitumor effect on pancreatic cancer cells and the underlying mechanisms have yet to be investigated. In this study, we show that GA suppressed the viability of cancer cells but has little toxicity on normal cells, e.g, HUVEC cells. Furthermore, treatment of GA resulted in impaired colony formation, migration, and invasion ability and increased apoptosis of cancer cells. In addition, GA inhibited the de novo lipogenesis of cancer cells through inducing activation of AMP-activated protein kinase (AMPK) signaling and downregulated the expression of key enzymes (e.g. acetyl-CoA carboxylase [ACC], fatty acid synthase [FASN]) involved in lipogenesis. Moreover, the in vivo experiment showed that GA reduced the expression of the key enzymes involved in lipogenesis and restrained the tumor growth. Taken together, our results suggest that GA may serve as a new candidate against tumor growth of pancreatic cancer partially through targeting pathway driving lipogenesis.

Topics: Acetyl-CoA Carboxylase; AMP-Activated Protein Kinases; Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Fatty Acid Synthases; Ginkgo biloba; Hep G2 Cells; Human Umbilical Vein Endothelial Cells; Humans; Lipogenesis; Male; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasms; Pancreatic Neoplasms; RNA Interference; Salicylates; Signal Transduction

To develop a LC-MS/MS method for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials, the column was Agilent ZORBAX Eclipse plus C18 (3.0 mm x 50 mm, 1.8 µm), and the mobile phase consisted of methanol-water (containing 0.2% formic acid) (95:5) at a flow rate of 0.5 mL · min(-1). The multiple reaction ion monitoring (MRM) with an ESI interface in the negative ion mode was selected. The results showed that the linear ranges of five kinds of ginkgolic acids were in the range of 0.2-36.0 µg · L(-1) (r ≥ 0.999 5). The lowest limit of quantification (LOQ) of ginkgo acid C13: 0, C15:1, C17:2, C15:0 and C17:1 were 0.18, 0.18, 0.21, 0.10 and 0.20 µg · L(-1), respectively. The average recovery was between 73.28% and 87.56%, and the average content of total ginkgolic acids in three batches of samples was in the range of 0.023-0.028 µg · g(-1), which was much lower than 2 µg · g(-1) prescribed in drug registration standards. This method is simple and rapid with high sensitivity, which can be used for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials.

Topics: Chromatography, Liquid; Ginkgolides; Injections; Limit of Detection; Salicylates; Tandem Mass Spectrometry

Infection by enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem, and there is no effective therapy. Biofilm formation is closely related to EHEC infection and is also a mechanism of antimicrobial resistance. Antibiofilm screening of 560 purified phytochemicals against EHEC showed that ginkgolic acids C15:1 and C17:1 at 5μg/ml and Ginkgo biloba extract at 100μg/ml significantly inhibited EHEC biofilm formation on the surfaces of polystyrene and glass, and on nylon membranes. Importantly, at their working concentrations, ginkgolic acids and G. biloba extract did not affect bacterial growth. Transcriptional analyses showed that ginkgolic acid C15:1 repressed curli genes and prophage genes in EHEC, and these findings were in-line with reduced fimbriae production and biofilm reductions. Interestingly, ginkgolic acids and G. biloba extract did not inhibit the biofilm formation of a commensal E. coli K-12 strain. In addition, ginkgolic acids and G. biloba extract inhibited the biofilm formation of three Staphylococcus aureus strains. The findings of this study suggest that plant secondary metabolites represent an important resource for biofilm inhibitors.

Topics: Anti-Bacterial Agents; Biofilms; Escherichia coli K12; Escherichia coli O157; Gene Expression Regulation, Bacterial; Ginkgo biloba; Plant Extracts; Salicylates; Staphylococcus aureus

Ginkgolic acids (GAs; anacardic acids; 6-alkylsalicylic acids) are both unwanted constituents in standardized Ginkgo biloba (Ginkgo) extracts and desirable constituents for pharmacological assays. Thus, for the quality control of Ginkgo extracts, the availability of pure GAs is important. In this investigation, inexpensive and easily prepared Fe3O4 magnetic nanoparticles (MNPs) in methanol were used to selectively adsorb GAs from crude petroleum ether extracts of Ginkgo leaves in the presence of various lipids including other alkylphenols (cardanols and cardols). The adsorption capacity of the MNPs is high, at 4-5% (w/w). The moiety responsible for the adsorption is the salicylic acid group, which binds strongly to Fe(III). Desorption with acidified methanol gave an extract with a GA content of 73%. This could be further separated by preparative HPLC on a C8 column. In total, eight different GAs were captured by MNPs. The MNP adsorption step can replace more traditional column chromatography and liquid-liquid extraction steps and is superior in terms of solvent consumption, selectivity, labor, and energy consumption. MNPs might become an efficient separation technique for selected high-value phytochemicals that contain a salicylic acid moiety.

Topics: Adsorption; Chromatography, High Pressure Liquid; Ferrosoferric Oxide; Ginkgo biloba; Magnetite Nanoparticles; Molecular Structure; Plant Leaves; Salicylates

Podocyte effacement and the reformation of foot processes and slit diaphragms can be induced within minutes experimentally. Therefore, it seems likely that the slit diaphragm proteins underlie orchestrated recycling mechanisms under the control of posttranslational modifiers. One of these modifiers, SUMO (small ubiquitin-like modifier), is an ubiquitin-like protein with a 20% corresponding identity to ubiquitin. Modification by SUMOs to proteins on lysine residues can block the ubiquitination of the same site leading to the stabilization of the target protein. Here we found in vitro and in vivo that nephrin is a substrate modified by SUMO proteins thereby increasing its steady-state level and expression at the plasma membrane. A conversion of lysines to arginines at positions 1114 and 1224 of the intracellular tail of murine nephrin led to decreased stability of nephrin, decreased expression at the plasma membrane, and decreased PI3K/AKT signaling. Furthermore, treatment of podocytes with the SUMOylation inhibitor ginkgolic acid led to reduced membrane expression of nephrin. Similarly, the conversion of lysine to arginine at position 1100 of human nephrin caused decreased stability and expression at the plasma membrane. As SUMOylation is a reversible process, our results suggest that SUMOylation participates in the tight orchestration of nephrin turnover at the slit diaphragm.

Topics: Animals; Arginine; Cell Membrane; HEK293 Cells; Humans; Kidney Glomerulus; Lysine; Male; Membrane Proteins; Mice; Podocytes; Proteinuria; Salicylates; Signal Transduction; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Transfection; Ubiquitin-Conjugating Enzymes

Ginkgolic acids (GAs) in natural product Ginkgobiloba L. are the pharmacological active but also toxic components. Two compounds, GA (C15:1) and GA (C17:1) are the most abundant GAs. In this study, several in vitro and in vivo models were applied to investigate transport mechanism of GAs. A rapid and sensitive LC-MS/MS method for the simultaneous determination of GA (C15:1) and GA (C17:1) was applied to analyze the biological specimens. The Papp(AP→BL) values of GA (C15:1) and GA (C17:1) were 1.66-2.13×10(-)(6)cm/s and 1.34-1.85×10(-)(6)cm/s determined using MDCK and MDCK-MDR1 cell monolayers, respectively. The Papp(BL→AP) were remarkably greater in the MDCK-MDR1 cell line, which were 6.77-11.2×10(-)(6)cm/s for GA (C15:1) and 4.73-5.15×10(-)(6)cm/s for GA (C17:1). Similar results were obtained in LLC-PK1 and LLC-PK1-BCRP cell monolayers. The net efflux ratio of GA (C15:1) and GA (C17:1) in both cell models was greater than 2 and markedly reduced by the presence of Cyclosporin A (CsA) or GF120918, inhibitors of P-gp and BCRP, suggesting that GAs are P-gp and BCRP substrates. The results from a rat bioavailability study also showed that co-administrating CsA intravenously (20mg/kg) could significantly increase GA (C15:1) and GA (C17:1) AUC0-t by 1.46-fold and 1.53-fold and brain concentration levels of 1.43-fold and 1.51-fold, respectively, due to the inhibition of P-gp and BCRP efflux transporters by CsA.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Availability; Biological Transport; Brain; Cell Culture Techniques; Cell Survival; Cyclosporine; Dogs; LLC-PK1 Cells; Madin Darby Canine Kidney Cells; Male; Neoplasm Proteins; Rats, Sprague-Dawley; Salicylates; Substrate Specificity; Swine; Tissue Distribution; Transfection

Ginkgolic acids and urushiols are natural alkylphenols known for their mutagenic, carcinogenic and genotoxic potential. However, the mechanism of toxicity of these compounds has not been thoroughly elucidated so far. Considering that the SIRT inhibitory potential of anacardic acids has been hypothesized by in silico techniques, we herein demonstrated through both in vitro and computational methods that structurally related compounds such as ginkgolic acids and urushiols are able to modulate SIRT activity. Moreover, their SIRT inhibitory profile and cytotoxicity were comparable to sirtinol, a non-specific SIRT inhibitor (SIRT1 and SIRT2), and different from EX-527, a SIRT1 specific inhibitor. This is the first report on the SIRT inhibition of ginkgolic acids and urushiols. The results reported here are in line with previously observed effects on the induction of apoptosis by this class of compounds, and the non-specific SIRT inhibition is suggested as a new mechanism for their in vitro cytotoxicity.

Topics: Catalytic Domain; Catechols; HEK293 Cells; HeLa Cells; Humans; Models, Molecular; Molecular Docking Simulation; Protein Conformation; Salicylates; Sirtuin 1; Sirtuin 2

Ginkgolic acids are alkylsalicylic acid derivatives with a thermolabile carboxylic group from Ginkgo biloba L., and they exhibit anticancer activity. Their anticancer effects are closely associated with their thermal stability. In this study, the thermal decomposition of ginkgolic acids was analyzed at temperatures of 30, 50, 70 and 250°C. The results clearly showed that an obvious slow decarboxylation of the ginkgolic acids was detected at 70°C. When the temperature increased to 250°C, the decarboxylation reaction was rapidly completed. The ginkgolic acids were decarboxylated to yield ginkgols. The ginkgols C13:0, C15:1 and C17:1 were separated and definitively identified by IR, NMR and GC-MS. The cytotoxic effects of ginkgols C13:0, C15:1 and C17:1 were tested and compared with those of the corresponding ginkgolic acids. An MTT assay showed that ginkgol C17:1 (48-h IC50=8.5 μg·ml(-1)) has the strongest inhibition on SMMC-7721 cells in a dose- and time-dependent manner. The anticancer action may occur via the induction of apoptosis by the activation of caspases-3, the upregulation of Bax expression, and the inhibition migration of SMMC7721 cells. The results indicated that ginkgol C17:1 might be useful for the further development of a hepatocellular carcinoma preventive agent.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Drug; Drug Stability; Ginkgo biloba; Hot Temperature; Humans; Salicylates

Intracellular accumulation of the abnormally modified tau is hallmark pathology of Alzheimer's disease (AD), but the mechanism leading to tau aggregation is not fully characterized. Here, we studied the effects of tau SUMOylation on its phosphorylation, ubiquitination, and degradation. We show that tau SUMOylation induces tau hyperphosphorylation at multiple AD-associated sites, whereas site-specific mutagenesis of tau at K340R (the SUMOylation site) or simultaneous inhibition of tau SUMOylation by ginkgolic acid abolishes the effect of small ubiquitin-like modifier protein 1 (SUMO-1). Conversely, tau hyperphosphorylation promotes its SUMOylation; the latter in turn inhibits tau degradation with reduction of solubility and ubiquitination of tau proteins. Furthermore, the enhanced SUMO-immunoreactivity, costained with the hyperphosphorylated tau, is detected in cerebral cortex of the AD brains, and β-amyloid exposure of rat primary hippocampal neurons induces a dose-dependent SUMOylation of the hyperphosphorylated tau. Our findings suggest that tau SUMOylation reciprocally stimulates its phosphorylation and inhibits the ubiquitination-mediated tau degradation, which provides a new insight into the AD-like tau accumulation.

Topics: Alzheimer Disease; Amino Acid Substitution; Amyloid beta-Peptides; Androstadienes; Animals; Cerebral Cortex; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Hippocampus; Humans; Indoles; Male; Maleimides; Middle Aged; Mutagenesis, Site-Directed; Mutation, Missense; Nerve Tissue Proteins; Peptide Fragments; Phosphorylation; Point Mutation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Proteolysis; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Salicylates; Solubility; SUMO-1 Protein; Sumoylation; tau Proteins; Ubiquitination; Wortmannin

A high-resolution gas chromatography/mass spectrometry (GC/MS) with selected ion monitor method focusing on the characterization and quantitative analysis of ginkgolic acids (GAs) in Ginkgo biloba L. plant materials, extracts, and commercial products was developed and validated. The method involved sample extraction with (1:1) methanol and 10% formic acid, liquid-liquid extraction with n-hexane, and derivatization with trimethylsulfonium hydroxide (TMSH). Separation of two saturated (C13:0 and C15:0) and six unsaturated ginkgolic acid methyl esters with different positional double bonds (C15:1 Δ8 and Δ10, C17:1 Δ8, Δ10, and Δ12, and C17:2) was achieved on a very polar (88% cyanopropyl) aryl-polysiloxane HP-88 capillary GC column. The double bond positions in the GAs were determined by ozonolysis. The developed GC/MS method was validated according to ICH guidelines, and the quantitation results were verified by comparison with a standard high-performance liquid chromatography method. Nineteen G. biloba authenticated and commercial plant samples and 21 dietary supplements purported to contain G. biloba leaf extracts were analyzed. Finally, the presence of the marker compounds, terpene trilactones and flavonol glycosides for Ginkgo biloba in the dietary supplements was determined by UHPLC/MS and used to confirm the presence of G. biloba leaf extracts in all of the botanical dietary supplements.

Topics: Dietary Supplements; Gas Chromatography-Mass Spectrometry; Ginkgo biloba; Molecular Structure; Plant Extracts; Salicylates

Dummy molecularly imprinted polymers (DMIPs) for simultaneously selective removal and enrichment of ginkgolic acids (GAs) during the processing of Ginkgo biloba leaves have been prepared. Two dummy template molecule with similar structural skeleton to GAs, 6-methoxysalicylic acid (MOSA, DT-1) and 6-hexadecyloxysalicylic acid (HOSA, DT-2), have been designed and synthesized. The performance of the DMIPs and NIPs were evaluated including selective recognition capacity, adsorption isotherm, and adsorption kinetics. The selective recognition capacity of the three GAs with four analogues on the sorbents illustrated that the DMIPs sorbents have high specificity for GAs. An efficient method based on DMIP-HOSA coupled with solid-phase extraction (SPE) was developed for simultaneously selective removal and enrichment of ginkgolic acids (GAs) during the processing of Ginkgo biloba leaves. The method showed excellent recoveries (82.5-88.7%) and precision (RSD 0.5-2.6%, n=5) for licorice extracts, Gastrodia elata extracts and pepper extracts spiked at three concentration levels each (50, 100, 200 μg mL(-1)). The results indicated that GAs and standardized Ginkgo biloba leaves extracts could be obtained simultaneously through the DMIP-SPE.

Topics: Adsorption; Ginkgo biloba; Kinetics; Molecular Imprinting; Plant Extracts; Plant Leaves; Polymers; Salicylates; Solid Phase Extraction

In vitro α-glucosidase inhibitory activity of Ginkgo biloba leaves was investigated. The inhibitory activity of methanol extracts from yellow and green leaves was 13.8 and 40.1 μg mL(-1), respectively. Each methanol extract was separated into its respective fraction by solvent-solvent extraction with n-hexane, chloroform, ethyl acetate and n-butanol. The n-hexane fractions (in both methanol extracts from green and yellow leaves) exhibited high α-glucosidase inhibitory activity with IC50 values of 13.6 and 13.4 μg mL(-1), respectively. Further fractionation of the n-hexane fractions by silica gel column chromatography gave the most active fraction which was identified as ginkgolic acid (C13:0) and a mixture (C13:0, C15:0, C15: 1, C17:1 and C17:2). Ginkgolic acid (C13:0) exhibited the highest α-glucosidase inhibitory activity. This is the first study to successfully isolate ginkgolic acids as α-glucosidase inhibitors.

Topics: 1-Butanol; Acetates; Chemical Fractionation; Chloroform; Gas Chromatography-Mass Spectrometry; Ginkgo biloba; Glycoside Hydrolase Inhibitors; Hexanes; Methanol; Molecular Structure; Phytotherapy; Plant Extracts; Plant Leaves; Plants, Medicinal; Salicylates; Solvents

On the basis of liquid chromatography coupled with triple quadrupole mass spectrometry working in multiple reaction monitoring mode, an analytical method has been established to simultaneously determine flavonol glycosides, terpene lactones, biflavones, proanthocyanidins, and ginkgolic acids in Ginkgo biloba leaves. Chromatographic separation was carried out on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7  μ m) with gradient elution of acetonitrile and 0.10% formic acid (v/v) at a flow rate of 0.4 mL/min, and column temperature 30°C. The developed method was validated in terms of linearity, accuracy, precision, stability, and sensitivity. The optimized method was successfully applied to analyze twenty-two G. biloba leaf samples of fruit cultivars collected from different places in China. Furthermore, hierarchical clustering analysis (HCA) was performed to evaluate and classify the samples according to the contents of the twenty-four chemical constituents. All of the results demonstrated that the developed method was useful for the overall evaluation of the quality of G. biloba leaves, and this study was also helpful for the comprehensive utilization and development of G. biloba resources.

Topics: China; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Flavonols; Fruit; Ginkgo biloba; Humans; Lactones; Mass Spectrometry; Plant Leaves; Proanthocyanidins; Salicylates; Terpenes

Imprints of potato sprout (Solanum tuberosum L.), gingko leaves (Gingko biloba L.) and strawberries (Fragaria x ananassa Duch.) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

Topics: Acetonitriles; Alkaloids; Carbohydrates; Flavonoids; Fragaria; Ginkgo biloba; Hot Temperature; Methanol; Molecular Imaging; Plant Leaves; Salicylates; Solanum tuberosum; Solvents; Spectrometry, Mass, Electrospray Ionization

A novel efficient synthesis of ginkgolic acid (13:0) from abundant 2,6-dihydroxybenzoic acid was successfully developed through a state-of-the-art palladium-catalyzed cross-coupling reaction and catalytic hydrogenation with an overall yield of 34% in five steps. The identity of the synthesized ginkgolic acid (13:0) was confirmed by nuclear magnetic resonance, mass spectrometry, infrared, and high-performance liquid chromatography. The reaction sequence of this method can be readily extended to the synthesis of other ginkgolic acids. The synthesized ginkgolic acid (13:0) exhibited promising anti-tyrosinase activity (IC₅₀ = 2.8 mg/mL) that was not correlated to antioxidant activity as probed by 1,1-diphenyl-2-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ferric reducing ability of plasma, and oxygen radical absorbance capacity assays. The synthetic strategy developed in this work will significantly facilitate biological studies of ginkgolic acids that have great potential applications in food and pharmaceuticals.

Topics: Agaricales; Antifungal Agents; Enzyme Inhibitors; Food Preservatives; Fungal Proteins; Molecular Structure; Monophenol Monooxygenase; Salicylates

Fourier transform infrared microspectroscopy is a powerful tool to obtain knowledge about the spatial and/or temporal distributions of the chemical compositions of plants for better understanding of their biological properties. However, the chemical morphologies of plant leaves in the plane of the blade are barely studied, because sections in this plane for mid-infrared transmission measurements are difficult to obtain. Besides, native compositions may be changed by chemical reagents used when plant samples are microtomed. To improve methods for direct infrared microspectroscopic imaging of plant leaves in the plane of the blade, the bulk and surface chemical morphologies of nonmicrotomed Ginkgo biloba leaves were characterized by near-infrared transmission and mid-infrared attenuated total reflection microspectroscopic imaging. A new self-modeling curve resolution procedure was proposed to extract the spectral and concentration information of pure compounds. Primary and secondary metabolites of secretory cavities, veins, and mesophylls of Ginkgo biloba leaf blades were analyzed, and the distributions of cuticle, protein, calcium oxalate, cellulose, and ginkgolic acids on the adaxial surface were determined. By the integration of multiple infrared microspectroscopic imaging and chemometrics methods, it is possible to analyze nonmicrotomed leaves and other plant samples directly to understand their native chemical morphologies in detail.

Topics: Calcium Oxalate; Ginkgo biloba; Plant Leaves; Plant Proteins; Salicylates; Spectroscopy, Fourier Transform Infrared; Spectroscopy, Near-Infrared

Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors. Unlike known SUMOylation inhibitors such as ginkgolic acid, spectomycin B1 directly binds to E2 (Ubc9) and selectively blocks the formation of the E2-SUMO intermediate; that is, Ubc9 is the direct target of spectomycin B1. Importantly, either spectomycin B1 treatment or Ubc9 knockdown inhibited estrogen-dependent proliferation of MCF7 human breast-cancer cells. Our findings suggest that Ubc9 inhibitors such as spectomycin B1 have potential as therapeutic agents against hormone-dependent breast cancers.

Topics: Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; High-Throughput Screening Assays; Humans; Kinetics; Protein Binding; Protein Processing, Post-Translational; Salicylates; Signal Transduction; Spectinomycin; Sumoylation; Ubiquitin-Conjugating Enzymes

Fatty acid synthase (FAS) has been proposed to be a new drug target for the development of anticancer agents because of the significant difference in expression of FAS between normal and tumour cells. Since a n-hexane-soluble extract from Ginkgo biloba was demonstrated to inhibit FAS activity in our preliminary test, we isolated active compounds from the n-hexane-soluble extract and evaluated their cytotoxic activity in human cancer cells. Three ginkgolic acids 1-3 isolated from the n-hexane-soluble extract inhibited the enzyme with IC(50) values 17.1, 9.2 and 10.5 µM, respectively, and they showed cytotoxic activity against MCF-7 (human breast adenocarcinoma), A549 (human lung adenocarcinoma) and HL-60 (human leukaemia) cells. Our findings suggest that alkylphenol derivatives might be a new type of FAS inhibitor with cytotoxic activity.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fatty Acid Synthases; Female; Ginkgo biloba; Hexanes; HL-60 Cells; Humans; Inhibitory Concentration 50; Lung Neoplasms; Molecular Structure; Plant Extracts; Plant Leaves; Salicylates

1. In this article, metabolites of ginkgolic acid (GA) (15:1) in rats plasma, bile, urine and faeces after oral administration have been investigated for the first time by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) with the aid of on-line hydrogen/deuterium (H/D) exchange technique and β-glucuronidase hydrolysis experiments. 2. After oral administration of GA (15:1, M0) to rats at a dose of 10 mg/kg, it was found that metabolites M1-M5 together with parent compound (M0) existed in rat plasma; parent compound (M0) and metabolites M2-M5 were observed in rat bile, and parent compound (M0) with metabolites M1 and M2 were discovered in rat faeces, and there was no parent compound and metabolite detectable in rat urine. 3. Two oxidative metabolites of GA (15:1, M0) were identified as 2-hydroxy-6-(pentadec-8-enyl-10-hydroxy) benzoic acid (M1) and 2-hydroxy-6-(pentadec-8-enyl-11-hydroxy-13-carbonyl) benzoic acid (M2), respectively. Metabolites M3, M4 and M5 were identified as the mono-glucuronic acid conjugates of parent compound (M0), M1 and M2, respectively. 4. The results indicated that M1 and M2 with parent compound (M0) were mainly eliminated in faeces and three glucuronide metabolites (M3, M4 and M5) excreted in bile as the predominant forms after oral administration of GA (15:1) to rats.

Topics: Administration, Oral; Animals; Bile; Chromatography, High Pressure Liquid; Feces; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Salicylates; Tandem Mass Spectrometry

Several HIV protease mutations, which are resistant to clinical HIV protease inhibitors (PIs), have been identified. There is a great need for second-generation PIs with different chemical structures and/or with an alternative mode of inhibition. Ginkgolic acid is a natural herbal substance and a major component of the lipid fraction in the nutshells of the Ginkgo biloba tree. The objective of this study was to determine whether ginkgolic acid could inhibit HIV protease activity in a cell free system and HIV infection in human cells.. Purified ginkgolic acid and recombinant HIV-1 HXB2 KIIA protease were used for the HIV protease activity assay. Human peripheral blood mononuclear cells (PBMCs) were used for HIV infection (HIV-1SF162 virus), determined by a p24gag ELISA. Cytotoxicity was also determined.. Ginkgolic acid (31.2 µg/ml) inhibited HIV protease activity by 60%, compared with the negative control, and the effect was concentration-dependent. In addition, ginkgolic acid treatment (50 and 100 µg/ml) effectively inhibited the HIV infection at day 7 in a concentration-dependent manner. Ginkgolic acid at a concentration of up to 150 µg/ml demonstrated very limited cytotoxicity.. Ginkgolic acid effectively inhibits HIV protease activity in a cell free system and HIV infection in PBMCs without significant cytotoxicity. Ginkgolic acid may inhibit HIV protease through different mechanisms than current FDA-approved HIV PI drugs. These properties of ginkgolic acid make it a promising therapy for HIV infection, especially as the clinical problem of viral resistance to HIV PIs continues to grow.

Topics: Cell Death; Cell-Free System; Dose-Response Relationship, Drug; Ginkgolides; HIV Infections; HIV Protease; HIV Protease Inhibitors; Humans; Jurkat Cells; Lactones; Salicylates

Cryptosporidium is a worldwide waterborne parasite and the treatment is a severe problem in immunocompromised patients. In this study, we used the in vitro culture system to evaluate the anti-Cryptosporidium activity of ginkgolic acids (GAs), nitazoxanide (NTZ), garlicin (GAR), and artemether (ART). The growth of Cryptosporidium andersoni in HCT-8 cells was determined by real-time PCR assay. When exposed to 5.00 μg/ml GAs or 10.00 μg/ml NTZ for 48 h, the number of C. andersoni in cultures was on a very low lever, but the number of parasites did not significantly decrease when exposed to GAR and ART. Our results indicate that GAs could be a potential drug for the treatment of cryptosporidiosis.

Topics: Allyl Compounds; Antiprotozoal Agents; Artemether; Artemisinins; Cell Line; Cell Survival; Cryptosporidium; Disulfides; Humans; Nitro Compounds; Parasitic Sensitivity Tests; Salicylates; Thiazoles

Codling moth, Cydia pomonella (L.), is a cosmopolitan pest of apple, potentially causing severe damage to the fruit. Currently used methods of combating this insect do not warrant full success or are harmful to the environment. The use of plant-derived semiochemicals for manipulation with fruit-infesting behavior is one of the new avenues for controlling this pest. Here, we explore the potential of Ginkgo biloba and its synthetic metabolites for preventing apple feeding and infestation by neonate larvae of C. pomonella. Experiments with crude extracts indicated that deterrent constituents of ginkgo are present among alkylphenols, terpene trilactones, and flavonol glycosides. Further experiments with ginkgo synthetic metabolites of medical importance, ginkgolic acids, kaempferol, quercetin, isorhamnetin, ginkgolides, and bilobalide, indicated that three out of these chemicals have feeding deterrent properties. Ginkgolic acid 15:0 prevented fruit infestation at concentrations as low as 1 mg/mL, bilobalide had deterrent effects at 0.1 mg/mL and higher concentrations, and ginkgolide B at 10 mg/mL. On the other hand, kaempferol and quercetin promoted fruit infestation by codling moth neonates. Ginkgolic acids 13:0, 15:1, and 17:1, isorhamnetin, and ginkgolides A and C had no effects on fruit infestation-related behavior. Our research is the first report showing that ginkgo constituents influence fruit infestation behavior and have potential applications in fruit protection.

Topics: Animals; Cyclopentanes; Fruit; Furans; Ginkgo biloba; Ginkgolides; Insecticides; Lactones; Larva; Malus; Moths; Plant Extracts; Salicylates

A highly sensitive HPLC-ESI-MS method has been developed and validated for the quantification of ginkgolic acid (15:1) in a small quantity of rat plasma (50μL) using its homologous compound ginkgolic acid (17:1) as an internal standard. GA (15:1) and GA (17:1) were extracted from biological matrix by direct protein precipitation with 5-fold volume of methanol and separated on an Elite hypersil BDS C(18) column (2.1×100mm, 3μm), eluted with acetonitrile:water (92:8, v/v, containing 0.3% glacial acetic acid). Linear range was 8-1000ng/mL with the square regression coefficient (r(2)) of 0.996. The lowest concentration (8ng/mL) in the calibration curve was estimated as LLOQ with both deviation of accuracy and RSD of precision <20% (n=6). The intra- and inter-day precision ranged from 3.6% to 9.9%, and the intra- and inter-day accuracy was between 89.9% and 101.3%. This method was successfully applied to study pharmacokinetics of GA (15:1) in rats after oral administration at a dose of 10mg/kg. GA (15:1) pharmacokinetic parameters C(max), T(max), t(1/2), AUC(0-12h) are 1552.9±241.0ng/mL, 0.9±0.7h, 5.5±2.6h, 3356.0±795.3ngh/mL, respectively.

Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Male; Rats; Rats, Sprague-Dawley; Regression Analysis; Reproducibility of Results; Salicylates; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

Ginkgolic acids (GAs), extracted from the seed coat of Ginkgo biloba L. Our previous study has shown that GA monomer could inhibit the growth of Hep-2 significantly and induce the fragmentation of the chromosomal DNA. To further assess the antitumor potential and turn it into a candidate new antitumor drug, the antitumor mechanism of GA was investigated.. The cytotoxicity and antitumor effect of GA monomer were assayed by MTT colorimetric assay with nontumorogenic MC-3T3-E1 as well as tumorogenic Hep-2 and Tac8113 cell lines. The effect of GA monomer on the proliferation of tumor cell lines was analyzed with MTT colorimetric and CFSE labeled assay. Cell cycle distribution and measurement of the percentage of apoptotic cells were performed by flow cytometry following stained with propidium iodide, annexin V-FITC. The expression of apoptotic proteins Bcl-2, Bax and caspase-3 was analyzed with Western blot.. GA only inhibited the growth of tumorogenic cell lines in a both dose- and time-dependent manner. Tumor cells were treated with GA for 72 h, 70.53 ± 4.54% Hep-2 and 63.5 ± 7.2% Tca8113 cells were retarded at GO/G1 phase, and the percentage of apoptosis was 40.4 ± 1.58 and 38.4 ± 1.7%, respectively. GA-treated activated caspase-3 downregulated the expression of anti-apoptotic Bcl-2 protein and upregulated the expression of pro-apoptotic Bax protein, eventually leading to a decrease in the Bcl-2/Bax ratio in tumor cells.. The antitumor action of GA was due to inhibiting the proliferation in a manner of inhibiting division, retarding the progress of cell cycle and inducing apoptosis, making GA a candidate as new antitumor drug.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Colorimetry; Flow Cytometry; G1 Phase; Humans; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Resting Phase, Cell Cycle; Salicylates

Carboplatin (CBP)-resistant cell line (Tca8113/CBP) and pingyangmycin (PYM)-resistant cell line (Tca8113/PYM) were established in vitro. Ginkgolic acids' influence over multidrug resistance (MDR) of drug-resistant cells was discussed by ginkgolic acids coupled with chemotherapy drugs.. The expression of P-glycoprotein (P-gp) was detected by immunohistochemistry. MTT assay was applied to ascertain the resistance index of drug-resistant cells. The effect of different concentrations of ginkgolic acids on the proliferation of drug-resistant cells and parental cell was measured by MTT assay. Making sure the non-toxic concentration of ginkgolic acids and observing the reversal effect of ginkgolic acids on drug-resistant cells. Resistance index was redetermined by MTT assay after ginkgolic acids coupled with chemotherapy drugs induced the cell lines for some time.. Immunohistochemistry showed that P-gp positive expression rate of drug-resistant cells was significantly higher than parental cells. The non-toxic concentration of ginkgolic acids which was determined by MTT assay was 10 microg x mL(-1). The reversal folds of Tca8113/CBP cell line to CBP and Tca8113/PYM cell line to PYM were 2.94 and 2.43 respectively. Before coupled with ginkgolic acids, the resistance indices of Tca8113/CBP and Tca8113/PYM cell lines were 3.24 and 11.9 respectively. When ginkgolic acids was added with chemotherapy drugs for some time, the resistance indices of Tca8113/CBP and Tca8113/PYM cell lines were 2.18 and 4.43 respectively.. This experiment successfully induced the drug-resistant cell lines of Tca8113/CBP and Tca8113/PYM. The method of chemotherapy drugs coupled with ginkgolic acids further confirmed the effect on proliferation of Tca8113/CBP and Tca8113/PYM cell lines was reducing. Non-toxic concentration of ginkgolic acids can partially reverse the drug resistance of Tca8113/ CBP and Tca8113/PYM cell lines. Furthermore, MDR level of drug-resistant cells decreased somewhat when they were induced by ginkgolic acids coupled with chemotherapy drugs for some time.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bleomycin; Carcinoma, Squamous Cell; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Mouth Neoplasms; Salicylates

Protein modification by small ubiquitin-related modifier proteins (SUMOs) controls diverse cellular functions. Dysregulation of SUMOylation or deSUMOylation processes has been implicated in the development of cancer and neurodegenerative diseases. However, no small-molecule inhibiting protein SUMOylation has been reported so far. Here, we report inhibition of SUMOylation by ginkgolic acid and its analog, anacardic acid. Ginkgolic acid and anacardic acid inhibit protein SUMOylation both in vitro and in vivo without affecting in vivo ubiquitination. Binding assays with a fluorescently labeled probe showed that ginkgolic acid directly binds E1 and inhibits the formation of the E1-SUMO intermediate. These studies will provide not only a useful tool for investigating the roles of SUMO conjugations in a variety of pathways in cells, but also a basis for the development of drugs targeted against diseases involving aberrant SUMOylation.

Topics: Anacardic Acids; Ginkgo biloba; Plant Extracts; Plant Leaves; Protein Binding; Salicylates; Small Molecule Libraries; Small Ubiquitin-Related Modifier Proteins; Structure-Activity Relationship; Ubiquitination

Ginkgolic acids and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardous compounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties. To determine whether the phase I metabolism could contribute to their cytotoxicity, we investigated the cytotoxicity of one model compound, ginkgolic acid (15:1), using in vitro bioassay systems. In the first step, cytochrome P450 enzymes involved in ginkgolic acid metabolism were investigated in rat liver microsomes; then, two in vitro cell-based assay systems, primary rat hepatocytes and HepG2 cells, were used to study and the measurement of MTT reduction was used to assess cell viability. Results indicated that the cytotoxicity of ginkgolic acid in primary rat hepatocytes was lower than in HepG2 cells. Ginkgolic acid was demonstrated less cytotoxicity in four-day-cultured primary rat hepatocytes than in 20-h cultured ones. Co-incubation with selective CYP inhibitors, alpha-naphthoflavone and ketoconazole, could decrease the cytotoxicity of ginkgolic acid in primary rat hepatocytes. In agreement, pretreatment with selective CYP inducers, beta-naphthoflavone and rifampin, could increase the cytotoxicity of ginkgolic acid in HepG2 cells. These findings suggest that HepG2 cells were more sensitive to the cytotoxicity of ginkgolic acid than primary rat hepatocytes, and CYP1A and CYP3A could metabolize ginkgolic acid to more toxic compounds.

Topics: Animals; Benzoflavones; beta-Naphthoflavone; Cell Line, Tumor; Cell Survival; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Fomepizole; Hepatocytes; Humans; Isoenzymes; Ketoconazole; Liver; Male; Microsomes, Liver; Pyrazoles; Quinine; Rats; Rats, Sprague-Dawley; Salicylates; Sulfaphenazole

Ginkgolic acids have been shown to possess allergenic as well as genotoxic and cytotoxic properties. The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the in vitro metabolism of ginkgolic acid (15:1, GA), one component of ginkgo acids, was investigated as a model compound in Sprague-Dawley rat liver microsomes. The metabolites were analyzed by ultra-performance liquid chromatography coupled with photodiode array detector/negative-ion electrospray ionization tandem mass spectrometry (UPLC-PDA/ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids.

Topics: Animals; Chromatography, High Pressure Liquid; Deuterium Exchange Measurement; Ginkgo biloba; Male; Microsomes, Liver; Rats; Rats, Sprague-Dawley; Salicylates; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

To study dynamic change of total ginkgolic acids in Ginkgo biloba leaves of the different aged trees and different collecting seasons.. The content of total ginkgolic acids in G. biloba leaves was determined by HPLC. A Alltima C18 (4.6 mm x 250 mm, 5 microm) and the mobile phase of methanol and 1% acetic acid (90:10) were used, the flow rate was 1.0 mL x min(-1), and the wavelength was 310 nm. The content were calculated with external standard method.. The content of total ginkgolic acids in G. biloba leaves was in the range of 0.48% to 2.51% in different collecting seasons. The content reached maximum at the end of May and the beginning of June, and then declined gradually. In different aged trees, the content in the older ages was lower than that in the younger ages.. The results provide scientific basis for the collecting season of G. biloba leaves.

Topics: Chromatography, High Pressure Liquid; Ginkgo biloba; Linear Models; Plant Leaves; Reproducibility of Results; Salicylates; Seasons; Sensitivity and Specificity; Trees

To investigate the anti-cancer effects and mechanism of natural plant ginkgolic acids (GAs) on Human Hep-2 cancer cells.. Hep-2 cancer cells were treated with different concentration of GAs for different times. The proliferation of Hep-2 cancer cells were detected by MTT colorimetric assay. Biochemistry characteristics of cell apoptosis was analyzed by DNA agarose electrophoresis and FCM.. The proliferation of Hep-2 cancer cells were inhibited significantly by GAs in a dose and time-dependent manner. Typical DNA ladder bands of Hep-2 cell DNA treated with GAs were observed on agarose electrophoresis gels. Increased apoptosis of Hep-2 cells treated with GAs was also noticed with FCM analysis.. Ginkgolic acids show an anti-tumor activity through inhibition the growth and inducing apoptosis of tumor cell in vitro.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Laryngeal Neoplasms; Salicylates

The sarcotesta of Ginkgo biloba is a Chinese herbal medicine used for treating toxoplasmosis, a serious disease requiring treatment with antibiotics that can have serious side effects.. To investigate the anti-Toxoplasmagondii activity of ginkgolic acids (GAs) isolated from the Ginkgo biloba sarcotesta in Toxoplasmagondii-infected human foreskin fibroblast (HFF) cells in vitro.. The safe concentration of GAs for HFF cells was determined by methyl thiazolyl tetrazolium (MTT) cell proliferation assay. The presence of Toxoplasmagondii was measured by [3H]-thymine deoxyriboside ([3H]-TdR) and [3H]-leucine ([3H]-Leu) incorporation, as well as Giemsa staining. The positive control was the commonly used and highly effective antibiotic azithromycin.. Light microscopy revealed that most HFF cells were infected after 4h of exposure to Toxoplasmagondii. After 48 h of exposure to either GAs or azithromycin, Toxoplasmagondii DNA and protein synthesis were minimal, there were no visible parasites in HFF cells, and the HFF cells had no significant morphological changes.. These results demonstrate that GAs have significant anti-Toxoplasma activity with low toxicity to HFF cells, suggesting that GAs could be an alternative treatment for toxoplasmosis.

Topics: Animals; Antiprotozoal Agents; Azithromycin; Cells, Cultured; Humans; Salicylates; Toxoplasma

A method to determine ginkgolic acids in Ginkgo biloba leaves was developed on the basis of pyrolytic methylation-gas chromatography. Pyrolytic methylation of G. biloba leaves in the injector at 300 degrees C in the presence of an organic alkali, tetramethylammonium hydroxide (TMAH, 25% methanol), allowed quantitative analysis of ginkgolic acids as their corresponding methyl derivatives. On the basis of the relative areas of these peaks, chemical composition of the ginkgolic acids in G. biloba leaves was determined with the relative standard deviations (RSDs) less than 3.4% (n = 3). The calibration curves of 5 main ginkgolic acids showed good linearity with the correlation coefficients greater than 0.996 6. The detection limits were between 0.8 - 2.8 mg/kg for the 5 compounds (S/N = 3). It is a simple, accurate and highly sensitive method for the chemical analysis and quality control of ginkgolic acids in G. biloba leaves and extracts.

Topics: Calibration; Chromatography, Gas; Ginkgo biloba; Hot Temperature; Methylation; Plant Leaves; Quaternary Ammonium Compounds; Salicylates

To investigate the molluscicidal activities of the ginkgolic acid(GA) monomers isolated and purified from GAs.. Five monomers of GAs from the sarcotesta of Ginkgo biloba. were extracted by petrol ether, separated by silica gel column chromatography, purified by semi-prepared reversed-phased HPLC, and identified by LC-MS analysis. The molluscicidal activities of GAs and their monomers against Oncomelania hupensis were determined as referring to the WHO guidelines for laboratory molluscicidal test.. The five purified ginkgolic acid monomers were GA(13:0), GA(15:0), GA(15:1), GA(17:1) and GA(17:2), with a side chain of 13, 15, 17 alkyl or ethylenic radicals res pectively on their benzene loop. The five monomer proportions to the total GAs were 17.6%, 3.2%, 52.3%, 23.3% and 3.6% respectively. The order of molluscicidal activities for the five monomers was as follows: GA(13:0)>GA(15:1)>GA(15:0)>GA(17:1)>GA(17:2), and their LC50 for snails was 20.79 mg/L, 22.28 mg/L, 33.76 mg/L, 51.89 mg/L, and 59.10 mg/L respectively after immersion for 24 hours. Two monomers, GA(13:0), and GA(15:1) inhibited the snails' climbing up significantly.. The molluscicidal activities of GAs may be dependent on the monomer's structure with different number of carbon molecules and double-bonds on the side carbon-chain. The two monomers, GA(13:0) and GA(15:1), are mainly responsible for the molluscicidal activities of GAs and both effectively inhibit snails' climbing up as well. GA(15:0) also shows certain molluscicidal activity.

Topics: Animals; Ginkgo biloba; Molluscacides; Plant Preparations; Salicylates; Snails; Toxicity Tests

We report here a negative ionization nanoelectrospray ionization mass spectrometry (nanoESI-MS) technique that simultaneously detects active components, terpenes and intact flavonol glycosides, and toxic ginkgolic acids in ginkgo products. Unlike the conventional methods that hydrolyze flavonol glycosides to flavonoids for analysis, this technique directly detects intact flavonol glycosides, enabling differentiation of these natural glycosides from the synthetic flavonoids. Thus, it allows the detection of fortification of ginkgo products, alleviating a common problem encountered by the conventional methods. Analysis of 14 commercial ginkgo products using this technique demonstrates large variations and deviation from the well-accepted standardized ginkgo extract. Four products showed evidence of fortification with synthetic surrogates. Two products were found to have toxic ginkgolic acids that exceed the 5 microg g(-1) limit by as much as 60000 fold. These results emphasize the importance of appropriate monitoring of ginkgo product quality.

Topics: Drug Contamination; Ginkgo biloba; Humans; Phytotherapy; Plant Extracts; Salicylates; Spectrometry, Mass, Electrospray Ionization

A competitive enzyme-linked immunosorbent assay (ELISA) for ginkgolic acids (GAs) was developed using monoclonal antibody (MAb) 9F raised against 6-(13-formylheptyl) salicylic acid covalently coupled to bovine serum albumin (BSA). ELISA, at an effective measuring range of 300 ng/ml-1 microgram/ml of GA15:1, was successful in detecting GAs content in ginkgo leaves and standardized extracts due to the lack of cross-reactivity against various related compounds. The sensitive and simple immunoassay developed in this study was validated to be specific for the quantitative determination of total GAs content in ginkgo crude drugs with no interference from the sample matrix. The analytical recovery of spiked GA15:1 was 103% in a concentration range between 10 and 40 mg/g dry weight of ginkgo leaves.

Topics: Animals; Antibodies, Monoclonal; Enzyme-Linked Immunosorbent Assay; Ginkgo biloba; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred BALB C; Phytotherapy; Plant Extracts; Plant Leaves; Salicylates

To study the influence of ginkgolic acids on human tumor cells and normal cells.. Ginkgolic acids (total concentration 90%) was prepared from ginkgo sarcotestas. The inhibitive effect of ginkgolic acids on human tumor cells and normal cells lines was examined by MTT assay.. When the concentration was 5.0 micrograms/ml, ginkgolic acids obviously inhibited the growth of tumor cells and didn't influence the normal cells. Inhibitive rate of ginkgolic acids on LTEP-a-2 was 59.1%. High-concentration ginkgolic acids had inhibitive effect on the growth of tumor cells and normal cells.. Ginkgolic acids had a obvious inhibitive effect on tumor cells in vitro. When the concentration was under 5.0 micrograms/ml, ginkgolic acids didn't influence the growth of normal cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Division; Chromatography, High Pressure Liquid; Ginkgo biloba; Humans; Lung Neoplasms; Mice; Plant Bark; Plant Extracts; Plants, Medicinal; Salicylates; Seeds; Tumor Cells, Cultured

Ginkgolic acids and their three monomers were separated from ginkgo sarcotestas. The anti-bacterium activity of ginkgolic acids were tested. The relation between the anti-bacterium activity and side chain of ginkgolic acid were studied.. The MIC of ginkgolic acids and their three monomers and salicylic acid were tested.. Ginkgolic acid has strong inhibitive effect on G+-bacterium. Salicylic acid has no side chain, so no anti-bacterial activity. When the length of gingkolic acid side chain is C13:0, it has the strongest anti-bacterial activity in three monomers.. The side chain of ginkgolic acid is the key functional group that possessed anti-bacterial activity. The length of Ginkgolic acid was the main effective factor of anti-bacterial activity.

Topics: Anti-Bacterial Agents; Drugs, Chinese Herbal; Escherichia coli; Ginkgo biloba; Plant Bark; Plant Leaves; Plants, Medicinal; Propionibacterium acnes; Salicylates; Seeds; Staphylococcus aureus

The quality control method of "Lü Kang Yin Xing Ye Pian" (LKYXYP) was studied and established.. The ginkgolides in LKYXYP were identified by TLC. The compounds of fatty acids in Perillae oil were determined by CC/MS. Flavones, ginkgolides and ginkgolic acids were determined by HPLC.. There were good linears relationship between the peak area and concentration, r = 0.999, RSD = 1.2% - 2.5% (n = 5), and recoveries of each composition was above 95%, the contation was stable in 24h.. This method is proved to be accurate and able to be applied for the quality control of this preparation.

Topics: Bilobalides; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Drugs, Chinese Herbal; Flavones; Gas Chromatography-Mass Spectrometry; Ginkgo biloba; Plant Leaves; Plants, Medicinal; Quality Control; Salicylates; Tablets

Extracts from the leaves of Ginkgo biloba are among the most widely used phytotherapeutics. Some alkylphenols (ginkgolic acids, cardanols and cardols) have been described as potentially hazardous constituents in Ginkgo extracts. Accordingly, a requirement for a maximum concentration of ginkgolic acids has been proposed in the UE and US pharmacopoeias Ginkgo monographs establishing a limit value of 5 ppm. A novel HPLC-UV method, developed by the use of HPLC-APCI-MS HPLC-DAD techniques and allowing the identification of ginkgolic acids and related phenols, is described. The new analytical method, not requiring enrichment procedures, can be used for the quantification of ginkgolic acids in Ginkgo extracts.

Topics: Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Ginkgo biloba; Humans; Phenols; Phytotherapy; Plant Extracts; Reproducibility of Results; Salicylates; Spectrophotometry, Ultraviolet; Structure-Activity Relationship

The technology of supercritical CO2 fluid extraction for ginkgolic acids in the epicarp of Ginkgo biloba L. was studied. The effect of pressure, temperature and extraction time on the yield of the ginkgolic acids was explored. The optimum conditions for supercritical CO2 fluid extraction was 30 MPa, 45 degrees C, extraction time 6 h and the flow rate of CO2 2L/min. The ginkgolic acids were determined by HPLC. Supercritical CO2 fluid extraction exceled the traditional solvent extraction in high yield, high purification and easy operation.

Topics: Carbon Dioxide; Chromatography, Supercritical Fluid; Ginkgo biloba; Plants, Medicinal; Pressure; Salicylates; Seeds; Temperature; Time Factors

To isolate ginkgolic acids (GA) from exopleura of Ginkgo biloba and to analyse GA.. GA were ultrasonic-extracted from exopleura of Ginkgo biloba with petroleum ether, and purified by silica gel column chromatography. The content of GA in sample was determined by HPLC with methanol-3% acetic acid (90:10) as mobile phase at a flow rate of 1 ml/min. UV detection wavelength was set at 310 nm. A HiQ sil C18 column was used at 40 degrees C in analysis.. A good linearity was obtained in the range of 1.144-5.720 micrograms (r = 0.9978) for GA. The average recovery of GA was 97.50% with RSD of 1.70%.. The method may be employed to prepare GA from exopleura of Ginkgo biloba. The analysis method for GA is accurate and reliable, and can be used for determination of GA in sample.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Fruit; Ginkgo biloba; Plants, Medicinal; Salicylates

To establish a high performance liquid chromatographic method for determination of ginkgolic acids in Ginkgo biloba extract and its preparations.. Ginkgo biloba extract and its preparations were extracted with petroleum ether in Soxhlet apparatus, and then concentrated under vacuum. The ginkgolic acids were determined directly by HPLC, and identified by LC/DAD/ESI/MS. The chromatographic column was Inertsil ODS-2; the mobile phase was methanol-3% aqueous acetic acid(92:8); the flow rate was 1.0 mL.min-1; the column temperature was 40 degrees C; the detection wavelength was 310 nm.. There were six kinds of ginkgolic acid (C13:0, C15:1, C17:2, C15:0, C17:1 and an unknown compound C17:3 tentatively) in the Ginkgo biloba extract. The relative percentage content of ginkgolic acids C13:0, C15:1 and C17:1 was above 94%. The content of ginkgolic acids in Ginkgo biloba extract containing high content ginkgolic acids was 1.12%, and RSD was 2.4% (n = 5). The content of ginkgolic acids in one kind of EGb preparations (tablet) was 49.2 micrograms.g-1, and RSD was 4.3% (n = 5). The average recovery was 98.2%, RSD was 2.6% (n = 5).. The method is accurate, fast, simple, and can be used for determination of ginkgolic acids in Ginkgo biloba extract and its preparations.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Ginkgo biloba; Plants, Medicinal; Salicylates; Spectrometry, Mass, Electrospray Ionization

Extracts from the leaves of Ginkgo biloba L. belong to the most widely used phytopharmaceuticals. In crude Ginkgo extracts, ginkgolic acids (GA) and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardous compounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties. To further assess the cytotoxic potential of GA, their effect on the human keratinocyte cell line HaCaT and the rhesus monkey kidney tubular epithelial cell line LLC-MK(2) was investigated. The action of a defined mixture of GA on cell growth, viability and integrity was evaluated by the neutral red uptake assay as well as the release of lactate dehydrogenase (LDH) and acid phosphatase (ACP). Cell morphology was examined by electron microscopy. For comparison, the effect of the standardized Ginkgo extract EGb 761, which contains less than 5 ppm GA, was also investigated. Following incubation of cells with EGb 761, neutral red uptake was half-maximally inhibited at concentrations of 900 mg/l (HaCaT) and 1480 mg/ml (LLC-MK(2)). The corresponding IC(50)-values for the mixture of GA ranged between 22 mg/l (HaCaT) and 4.6 mg/l (LLC-MK(2)), respectively. In parallel to the inhibition of neutral red uptake, a concentration dependent release of LDH was observed when cells were incubated in the presence of GA (1-100 mg/l). In contrast, even at a concentration of 1800 mg/l EGb 761 did not cause release of LDH above controls. Since GA interacted with the assay for ACP, no index of lysosomal damage could be established by this method. Incubation of HaCaT cells with GA for 18 h increased the proportion of apoptotic cells from about 6% (control) to nearly 80% at concentrations of >or=30 mg/l. Electron microscopic analysis of HaCaT cells revealed a drug induced formation of myelinosomes possibly due to the inhibition of lysosomal enzymes, while morphological evaluation of LLC-MK(2) cells indicated that the cytotoxic activity of GA in these cells is primarily mediated by transformation of mitochondria, which is probably induced by uncoupling of oxidative phosphorylation.

Topics: Animals; Apoptosis; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Ginkgo biloba; Humans; Keratinocytes; Kidney Tubules; L-Lactate Dehydrogenase; Macaca mulatta; Oxidative Phosphorylation; Plant Extracts; Salicylates

In this article we studied the anti-bacterial activity of the extract from testa of Ginkgo biloba and ginkgolic acids. They can inhibit the growth of Staphylocococus aureus, Escherichia coli, Bacillus subtilis, B. cereus and MRSA. Ginkgolic acids combined with peniciline can enhance their inhibitory activity to MRSA.

Topics: Anti-Bacterial Agents; Bacillus subtilis; Drug Therapy, Combination; Escherichia coli; Ginkgo biloba; Penicillins; Plant Leaves; Salicylates; Seeds; Staphylococcus aureus

To establish a simple pre-treated method and high performance liquid chromatographic method for separation and determination of ginkgolic acids in Ginkgo biloba leaves.. The ginkgolic acids in the German standard sample were identified by LC/DAD/ESI/MS. The methods for pre-treatment and high performance liquid chromatography determination of ginkgolic acids were studied. Ginkgo biloba leaves were extracted with n-hexane in Soxhlet apparatus, then concentrated under vacuum. The ginkgolic acids can be determined directly by HPLC after one-pre-purified-step by silica gel column chromatography. The eluant was petroleum ether-diethyl ether-formic acid (89:11:1). The chromatographic column was Inertsil ODS-2; the mobile phase was methanol-3% acetic acid (92:8); the flow rate was 1.0 mL.min-1; the column temperature was 40 degrees C; the detection wavelength was at 310 nm.. There were five kinds of ginkgolic acid (C13:0, C15:1, C17:2, C15:0 and C17:1) in ginkgo biloba leaves. The relative percentage content of ginkgolic acids C15:1 and C17:1 was about 85%. Ginkgolic acid C17:2 had not been reported in China. The HPLC indicates that there was nearly no impurities except ginkgolic acids after treated by column chromatography. The results showed that the content of ginkgolic acids in the leaves of Ginkgo biloba collected in April, May and June was 1.48%, 1.19% and 1.11% respectively. The average recovery of Ginkgo biloba leaves collected in June was 97.0%, RSD was 1.7% (n = 6).. The method is accurate, simple and reliable, and can be used for determination of ginkgolic acids in Ginkgo biloba leaves.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Ginkgo biloba; Molecular Structure; Plant Leaves; Plants, Medicinal; Salicylates

A reversed-phase argentation high-performance liquid chromatographic method has been achieved for the determination of ginkgolic acids. Liquid chromatography coupled with electrospray ionization (ESI) mass spectrometry in the negative ion mode is applied to identify ginkgolic acids from ginkgo leaves. The leaves are extracted with ethanol and then cleaned-up by extraction of analytes with hexane after addition of an acidified saturated solution of sodium sulfate and siliceous earth to the matrix solution. Ginkgolic acids are determined within 30 min on a C18 column with methanol-5% aqueous acetic acid (90:10) containing 0.03 mol l(-1) silver nitrate as eluent and with ultraviolet detection at 310 nm. Addition of silver ions as complexation agent into the mobile phase decreases retention time of ginkgolic acids with an unsaturated side chain. Four ginkgolic acids are successfully separated from each other and from other interfering components by the high selectivity of reversed-phase argentation HPLC, which is confirmed by the spectra identification. The average recovery of the method is around 97%. Good reproducibility is obtained with relative standard deviations varying from 2 to 5%.

Topics: Chromatography, High Pressure Liquid; Ginkgo biloba; Plant Leaves; Reproducibility of Results; Salicylates; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet

Ginkgo biloba-containing brands are one of the top sellers within the growing market for herbal remedies in many European countries as well as in the USA. In the consumers' interest, these brands should feature a certain quality and should be transparent in quality claims. In this investigation, a variety of products on the USA market was studied with respect to pharmaceutical quality, such as quantity of constituents and in-vitro dissolution. In terms of the content of active substances, flavone glycosides ranged from 24% to 36% and terpene lactones from 4% to 11%. With ginkgolic acids, there was a very large range, from < 500 ppm to about 90000 ppm. Comparing the dissolution rates of terpene lactones and flavone glycosides within the single products, most were approximately the same. Thus, terpene lactones and flavone glycosides were released from these products and dissolved at the same rate in most cases. Furthermore, most of the products investigated released more than the required 75% of the content of both components within 30 min. However, several products showed clear and relevant differences in dissolution rates to the rest (e.g. < 75% within 30 min or even less than 25% after 60 min in one case, indicating much poorer pharmaceutical quality). Beside the comparability respectively standardisation of the extracts used, the in-vitro dissolution of the relevant constituents should be similar to other drugs to guarantee comparable in-vivo performance of herbal products. An important step in standardising pharmaceutical quality is the pharmacopoeial monograph for Ginkgo biloba extract in Germany, standardising the content of pharmacologically relevant substances (flavone glycosides 22-27% and terpenlactones 5-7%, 2.8-3.4% ginkgolides A, B, C and 2.6-3.2% bilobalide thereof). Many of the investigated products, which refer to the German Commission E (of the Federal Institute for Drugs and Medicinal Devices) monograph, are not in accordance with this specification. Thus, they can not be considered to be pharmaceutically equivalent.

Topics: Chromatography, High Pressure Liquid; Flavonoids; Ginkgo biloba; Glycosides; Plant Extracts; Quality Control; Salicylates; Solubility; Terpenes

An analytical method has been firstly achieved for the quantification of ginkgolic acids from Ginkgo biloba leaves by reversed-phase argentation high performance liquid chromatography. Analytical sample was cleaned-up after addition of acidic salt solution and adsorbent to the matrix solution by counter-extraction of analytes with hexane. Ginkgolic acids were determined by HPLC with methanol and 5% aqueous acetic acid (90:10, V/V) containing 0.03 mol.L-1 silver ion as mobile phase and UV detection at 310 nm. Results showed ginkgolic acids were separated successfully from each other and from other interfering components, which were confirmed by spectra analysis and purity assay. The linearity of the calibration curve was good in the range of 0.084 microgram-10.56 micrograms (r = 0.9998). The average recovery was 97.3% and RSD was 1.6%. The detection limit was 0.026 microgram (S/N = 3). The convenient method can be used as a reliable tool for the quantitative analysis of ginkgolic acids.

Topics: Chromatography, High Pressure Liquid; Ginkgo biloba; Plant Leaves; Salicylates

To develop a reverse phase argentation high performance liquid chromatographic (RP-AHPLC) method for the separation and determination of ginkgolic acids.. Liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) was applied to identify ginkgolic acids from Ginkgo biloba leaves and four ginkgolic acids of the samples were separated and quantified by RP-AHPLC. Leaves were extracted with ethanol and analytes were extracted with hexane after addition of acid/salt solution and adsorbent to matrix solution. Ginkgolic acids were separated and determined within 30 minutes by RP-AHPLC under optimum chromatographic conditions. Methanol and 5% aqueous acetic acid (90:10) containing 0.03 mol.L-1 silver ion was used as mobile phase, column temperature was selected at 30 degrees C, flow rate was 1.0 mL.min-1, UV detection wavelength was at 310 nm. The spectra analysis and purity identification of chromatographic peaks of ginkgolic acids were further confirmed by means of diode array detection.. Four ginkgolic acids were baseline separated from each other and from other interfering components. The average recovery and relative standard deviation of the method were 97.3% and 1.6%, respectively.. RP-AHPLC was an excellent method for separation of homologous with different carbon atom numbers and double bond. The method is useful for the quality control of extract of Ginkgo biloba leaves.

Topics: Chromatography, High Pressure Liquid; Ginkgo biloba; Plant Leaves; Plants, Medicinal; Salicylates; Spectrometry, Mass, Electrospray Ionization

Extracts from the leaves of the Gingko tree (Ginkgo biloba L.) are therapeutically used for the treatment of peripheral and cerebral vascular disorders as well as multi-infarct or Alzheimer-type dementia. As constituents with potential contact allergenic and toxic properties in crude Ginkgo extracts a group of alkylphenols (e.g., ginkgolic acids, ginkgol, bilobol) has been described. Thus, for reasons of drug safety a maximal concentration (< or = 5 ppm) of ginkgolic acids is requested by the Monograph of the Commission E of the former German Federal Health Agency (Bundesgesundheitsamt, BGA). During production of the standardized Ginkgo extract EGb 761, alkylphenols are largely eliminated as water insoluble compounds (decanter sludge) from the primary acetone extract. To further assess the adverse properties of alkylphenols, different fractions derived from the decanter sludge were evaluated for their embryotoxic effects in the hen's egg test (HET). A fraction enriched for ginkgolic acids (16%) and biflavones (6.7%) was found to induce death of 50% of the chick embryos (LD50) at a dose of 1.8 mg/egg (approximately/= 33 ppm). A similar strong lethal effect (LD50: 3.5 mg/egg; 64 ppm) was oberserved for a fraction which contained 58% ginkgolic acids but less than 0.02% biflavones. In contrast, an extreme low toxic potential (LD50: 250 mg/egg or 4540 ppm) was established for a fraction containing 16% biflavones and 1% ginkgolic acids. Thus, the present investigations confirm the high toxic potential of ginkgolic acids, although it can not be excluded that biflavones or some other constituents in the different fractions may amplify the adverse effect of these substances. Since no contribution of alkylphenols to the therapeutic efficacy of Ginkgo extracts has been confirmed and their elimination during the manufacturing process does not cause technical problems, these results further support the requirement for the completest possible removal of these compounds under toxicological considerations.

Topics: Animals; Anti-Anxiety Agents; Carcinogens; Chickens; Dose-Response Relationship, Drug; Flavonoids; Ginkgo biloba; Ovum; Phenols; Plant Extracts; Plant Leaves; Plants, Medicinal; Resorcinols; Salicylates; Survival Rate; Vasodilator Agents

A chromatographic procedure for the preparative isolation of six different 6-alkylsalicylic acids (syn. ginkgolic acids) with as alkyl substituents C13:0, C15:0, C15:1, C17:1, C17:2 and, tentatively C17:3 from Ginkgo biloba leaves was developed. The procedure consisted of a combination of normal-phase, reversed-phase and argentation chromatography. The compounds were characterised by means of UV, 1H-NMR and 13C-NMR spectroscopy, and mass spectrometry after silylation. A 15 cm C18 RP-HPLC column connected in series with a 20 cm silver(I) loaded cation exchanger HPLC column in combination with the solvent methanol-water (93:7) acidified with 0.1% formic acid was capable of separating the ginkgolic acids C13:0, C15:1, C17:2, C15:0 and C17:1 within 21 min on an analytical scale. The separation is based on a combination of reversed-phase mechanisms and double bond complexation. Detection took place by UV at 311 nm. The separation is a good starting point for the development of a quantitative procedure for the five major ginkgolic acids in Ginkgo leaves and standardised extracts.

Topics: Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Ginkgo biloba; Salicylates; Spectrum Analysis

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

Topics: Animals; Anti-Anxiety Agents; Apoptosis; Brain; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Chick Embryo; Dose-Response Relationship, Drug; Enzyme Activation; Ginkgo biloba; In Situ Nick-End Labeling; Isoenzymes; Neurons; Phosphoprotein Phosphatases; Plant Extracts; Protein Phosphatase 2; Protein Phosphatase 2C; Saccharomyces cerevisiae Proteins; Salicylates; Staining and Labeling; Time Factors; Trypan Blue

A method is developed for qualitative analysis of ginkgolic acids in the leaves and fruits of Ginkgo biloba by high-performance liquid chromatography (HPLC)-electrospray ionization-mass spectrometry technique. Negative ionization mode is successful in obtaining a very abundant deprotonated molecule [M - H]-. The mass detection sensitivity is higher than ultraviolet detection but relies heavily on the concentration of acetic acid in the HPLC eluent, which consists of acetonitrile-water-acetic acid. The method is also very specific for the analysis of ginkgolic acid with no interferences from the sample matrix.

Topics: Acetic Acid; Chromatography, High Pressure Liquid; Fruit; Ginkgo biloba; Mass Spectrometry; Plant Extracts; Plant Leaves; Plants, Medicinal; Salicylates; Sensitivity and Specificity

Ginkgolic acids (GAs) are toxic phenolic compounds present in the fruits and leaves of Ginkgo biloba L. (Ginkgoacae). Their maximum level in phytopharmaceuticals containing ginkgo extracts has been recently restricted to 5 microg/g by the Commission E of the former Federal German Health Authority. In order to detect ginkgolic acids at these low levels, a sensitive and selective analytical method, based on liquid chromatography-electrospray mass spectrometry (LC-ES-MS) has been developed. The three main phenolic acids (1-3) of the chloroform fruit extract were isolated and used as standards for quantification. In the LC-ES-MS negative ion mode, calibration curves with good linearities (r=0.9973, n=6) were obtained in the range of 0.5-10 microg/g for compounds 1, 2 and between 0.1 and 7.5 microg/g (r=0.9949, n=6) for ginkgolic acid 3. The detection limits at a SIN ratio of 3 were 0.1 (3) and 0.25 microg/g (1, 2). Recoveries were around 101% at 5 microg/g for the substances detected in the leaf extracts. Good precision was achieved with relative standard deviations of less than 4% (n=6). The optimised method was applied to verify whether the amount of gingkolic acids was below 5 microg/g in a standardised leaf extract which is a constituent of a phytopreparation.

Topics: Calibration; Chromatography, High Pressure Liquid; Ginkgo biloba; Mass Spectrometry; Phytotherapy; Plant Extracts; Plant Leaves; Plants, Medicinal; Reference Standards; Reproducibility of Results; Salicylates; Sensitivity and Specificity

Topics: Animals; Anti-Anxiety Agents; Coloring Agents; DNA Damage; DNA Repair; Electrophoresis, Polyacrylamide Gel; Ginkgo biloba; Hepatocytes; Male; Neutral Red; Plants, Medicinal; Rats; Rats, Wistar; Salicylates

Topics: Animals; Cell Line; Cell Survival; Flavonoids; Ginkgo biloba; Humans; Macaca mulatta; Phenols; Plant Extracts; Plants, Medicinal; Salicylates; Swine

Ginkgolic acid conjugates (GAC) (6-alkylsalicylates, namely n-tridecyl-, n-pentadecyl-, n-heptadecyl-, n-pentadecenyl- and n-heptadecenylsalicylates) isolated from the leaves of Indian Ginkgo biloba Linn., (IGb) were tested for their putative role in anxiety in rats. Elevated plus maze, open-field behaviour, novelty-induced feeding latency and social interaction were the rodent behavioural models used in this study. GAC (0.3 and 0.6 mg/kg, each, p.o.) on single acute administration, showed dose-related changes in the behaviour. GAC (0.6 mg/kg) and DZ augmented open arm entries, the open arm/closed arm entries ratio and increased time spent in the open arm on the elevated plus maze. In the open field, GAC (0.6 mg/kg) and DZ significantly increased ambulation and reduced the immobility time. EGb 761 showed a similar profile. GAC (0.6 mg/kg) and DZ significantly attenuated the increased latency to feed in novel environment. By contrast, EGb 761 and Ginkocer further augmented feeding latency. None of the drugs tested showed any significant effect in the social interaction test. GAC showed consistent and significant anxiolytic activity in all the variables investigated. By contrast, EGb 761 and Ginkocer, which are devoid of GAC, did not evoke significant activity. However, increased rearing and decreased immobility time only in open field behaviour shown by EGb 761 may be due to some antianxiety activity of a lesser degree. Our observations suggest that GAC may be the active constituents of Ginkgo biloba responsible for the anxiolytic activity.

Topics: Animals; Anti-Anxiety Agents; Behavior, Animal; Diazepam; Female; Flavonoids; Ginkgo biloba; India; Male; Plant Extracts; Plants, Medicinal; Rats; Salicylates

Ginkgo biloba possesses fruits that have caused numerous cases of allergic contact dermatitis. Low amounts of the ginkgolic acids occur in the leaves as well.. Leaf extracts are used to treat cerebrovascular and peripheral vascular disorders. The question arises whether skin hypersensitivity reactions may be adverse effects because the pharmaceutical preparations contain low amounts of ginkgolic acids.. Guinea pigs were sensitized experimentally with pure ginkgolic acids as well as with leaf extracts containing approximately 1,000 ppm of ginkgolic acids.. The guinea pigs could be sensitized successfully with the pure ginkgolic acids. The animals could not be sensitized with the leaf extract.. Leaf extracts of Ginkgo biloba taken orally or given by infusion to treat diffuse cerebral disturbances can be considered safe, even when they might contain up to 1,000 ppm of the sensitizing ginkgolic acids.

Topics: Administration, Oral; Allergens; Animals; Cerebrovascular Disorders; Dermatitis, Allergic Contact; Dermatitis, Irritant; Disease Models, Animal; Female; Ginkgo biloba; Guinea Pigs; Immunization; Infusions, Intravenous; Peripheral Vascular Diseases; Phytotherapy; Plant Extracts; Plant Leaves; Plants, Medicinal; Salicylates