Effect of Methyl Jasmonate on the Terpene Trilactones, Flavonoids, and Phenolic Acids in Ginkgo biloba L. Leaves: Relevance to Leaf Senescence

The present study compared the effects of natural senescence and methyl jasmonate (JA-Me) treatment on the levels of terpene trilactones (TTLs; ginkgolides and bilobalide), phenolic acids, and flavonoids in the primary organs of Ginkgo biloba leaves, leaf blades, and petioles. Levels of the major TTLs, ginkgolides B and C, were significantly higher in the leaf blades of naturally senesced yellow leaves harvested on 20 October compared with green leaves harvested on 9 September. In petioles, a similar effect was found, although the levels of these compounds were almost half as high. These facts indicate the importance of the senescence process on TTL accumulation. Some flavonoids and phenolic acids also showed changes in content related to maturation or senescence. Generally, the application of JA-Me slightly but substantially increased the levels of TTLs in leaf blades irrespective of the difference in its application side on the leaves. Of the flavonoids analyzed, levels of quercetin, rutin, quercetin-4-glucoside, apigenin, and luteolin were dependent on the JA-Me application site, whereas levels of (+) catechin and (−) epicatechin were not. Application of JA-Me increased ferulic acid and p-coumaric acid esters in the petiole but decreased the levels of these compounds in the leaf blade. The content of p-coumaric acid glycosides and caffeic acid esters was only slightly modified by JA-Me. In general, JA-Me application affected leaf senescence by modifying the accumulation of ginkogolides, flavonoids, and phenolic acids. These effects were also found to be different in leaf blades and petioles. Based on JA-Me- and aging-related metabolic changes in endogenous levels of the secondary metabolites in G. biloba leaves, we discussed the results of study in the context of basic research and possible practical application.

Protective Potential of Ginkgo biloba Against an ADHD-like Condition

Background: Attention deficit hyperactivity disorder (ADHD) is a psychiatric disorder commonly found in children, which is recognized by hyperactivity and aggressive behavior. It is known that the pathophysiology of ADHD is associated with neurobiological dysfunction. Although psychostimulants are recognized as the therapeutic drugs of choice for ADHD patients, the side effects might be of great concern. Ginkgo biloba is a promising herbal complementary supplement that may modulate the neuronal system in an ADHD-like condition. The beneficial effect of Ginkgo biloba on ADHD-like symptoms may be related to the modulation of the system by novel molecular mechanisms. Ginkgo biloba is known to modulate dopamine, serotonin, and norepinephrine signaling. Flavonoid glycosides and terpene trilactones are the two major phytochemical components present in the Ginkgo biloba preparations, which can exhibit antioxidant and neuroprotective activities. The pharmacological mechanisms of the phytochemical components may also contribute to the neuroprotective activity of Ginkgo biloba. Conclusion: In this review, we have summarized recent findings on the potential of various Ginkgo biloba preparations to treat ADHD-like symptoms. In addition, we have discussed the pharmacological mechanisms mediated by Ginkgo biloba against an ADHD-like condition.

Quantitative GC-FID and UHPLC-DAD Evaluation of Bioactive Compounds Extracted from Ginkgo biloba

Background: The official compendium of the quantification of ginkgo flavonoids from Ginkgo biloba extract has been proposed using HPLC. The drawbacks of this technique appear to be due to the restricted efficiency in terms of the recovery results and suitability of the system for the quantification of these compounds. This study investigated the potential advantages and limitations of the development of efficient extraction methods for the recovery of flavonol glycosides (quercetin, kaempferol and isorhamnetin) and terpene trilactones (bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) using extraction, quantification and detection techniques, namely, GC-FID and UHPLC-DAD, which are alternatives to those techniques available in the literature. Methods: Two different extraction methodologies have been developed for the determination of flavonoids (quercetin, kaempferol and isorhamnetin) and terpene trilactones (bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) using ultra-high-pressure liquid chromatography coupled to a diode array detector and gas chromatography coupled to a flame ionization detector. Results: In this study, the Ginkgo biloba extract mass, hydrolysis preparation method (with or without reflux), and volume of the extraction solution seemed to affect the ginkgo flavonoid recovery. The UHPLC-based method exhibited higher extraction efficiency for ginkgo flavonoid quantification compared to the pharmacopoeial method. The developed method exhibited higher extraction efficiency for terpene quantification compared to the previous method that used extractive solution without pH adjustment, with less time of extraction and less amount of the sample and organic solvent aliquots. Conclusion: The UHPLC and GC analysis methods established in this study are both effective and efficient. These methods may improve the quality control procedures for ginkgo extract and commercial products available in today´s natural health product market. The results indicate that redeveloped extraction methods can be a viable alternative to traditional extraction methods.

Development and optimization of HPLC-MS method for simultaneous determination of four terpene trilactones in Ginkgo biloba leaf extracts based on Quality by Design principles

Background Ginkgo biloba leaf extract (EGBL) is one of the most commonly used and most studied herbal medicines around the world. Taking into account that previously reported HPLC-ELSD methods for terpene trilactones determination in EGBL are time-consuming with complicated sample preparation, it is reasonable and meaningful to developing a simple, sensitive and robust HPLC-MS method based on a novel analytical quality by design (AQbD) approach. Methods Firstly, analytical target profile (ATP) and systematic risk analysis were carried out to identify potential critical method attributes (CMAs) and critical method parameters (CMPs). Secondly, CMPs were identified using a standard partial regression coefficient method. Thirdly, Box-Behnken design (BBD) was employed to establish the quantitative relationship between CMAs and CMPs. Fourthly, the Monte Carlo simulation method was used to build hypercube design space. Then, the verification experiments were performed. Fifthly, the optimized method was validated and utilized. Finally, the paired t test was used to compare the developed method with HPLC-ELSD. Results After the screening experiments, flow rate of mobile phase, the proportion of formic acid in the mobile phase, gas flow rate and gas temperature were identified as CMPs. Models to quantitatively describe the relationship between CMAs and CMPs were built. The operational hypercube design spaces of the HPLC-MS method for terpene trilactones analysis in EGBL were successfully calculated and found to be robust, which led to the analytical control strategy. The verification experiments were successfully performed within the design space and model was found to be accurate. The method had been successfully used for quality analysis of development batches of EGBL and obtained almost identical results to data determinated using HPLC. Conclusions In this work, an analytical control strategy for HPLC-MS method for terpene trilactones analysis in EGBL was developed using AQbD concepts, which is promising for application to other Chinese medicines. The developed HPLC-MS method is an alternative method for quantification of terpene trilactones in commercial EGBL and will be applicable throughout the life cycle of the product.

Transcriptome-based Discovery of AP2/ERF Transcription Factors Related to Terpene Trilactones Synthesis in Ginkgo biloba

Ginkgo biloba is a unique tree in China with medicinally and phylogenetically important characteristics. Terpene trilactones (TTL) is a key active pharmaceutical ingredient in Ginkgo, so the content of TTL in Ginkgo has become one of the important indices for evaluating quality of the medicinal materials. By transcriptome sequencing on samples treated by chlormequat, ultraviolet (UV) and drought, totally 59820 contigs and 37564 unigenes were obtained. Furthermore, 18234 unigenes were annotated through COG, KEGG and GO analysis. There were 78 AP2/ERF transcription factors, 23 factors of up-regulation and 66 factors of down-regulation that were related with synthetic pathway of TTL in Ginkgo. Phylogenetic tree clustering analysis indicated that there were 42 AP2s could be clustered into ERF, DREB and RVA subfamilies. EMSA analysis demonstrated that GbERF13, GbERF25 and GbERF27 could bind with regulatory elements, such as E-box, in the upstream of GbMECPs promoter. Expression analysis showed that the expression level of GbERF25 was the highest in root, and GbERF25 and GbERF27 were expressed in relatively high transcription levels in leaf and other tissues. The results of qRT-PCR indicated that CCC treatment could significantly improve expression levels of ERF25 and ERF27, and UV and drought could induce transcription levels of ERF13 and ERF25, respectively. The results implied that ERF25 and ERF27 might involve in the induction and regulation of CCC treatment on synthesis of bilobalide in G. biloba. ERF13 might participate in the regulation of bilobalide synthesis induced by UV, and EFR25 might involve in the regulation of the synthesis induced by drought. During annual cycle of expression, the transcription levels of ERF13, ERF25 and ERF27 had significantly positive correlation with diterpene level with correlation coefficient 0.975. It implied that these transcription factors mainly acted on the MEP pathway that regulated synthesis of bilobalide. The aim of the research was to indicate the mechanism of environment or cultivation measure regulating target gene of TTL metabolic pathway by AP2/ERF, and establish metabolic network of AP2/ERF regulating TTL synthesis. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********