Metabolite profiling and identification of novel umami compounds in the chaya leaves of two species using multiplatform metabolomics.

Chaya (Cnidoscolus chayamansa and C. aconitifolius) is a fast-growing medicinal plant, and its leaves exhibit a strong umami taste. Here metabolite variation and umami-related compounds in the leaves of two chaya species were determined using a multiplatform untargeted-metabolomics approach, electronic tongue, and in silico screening. Metabolite profiles varied between the leaves of the two species and among leaf maturation stages. Young leaves exhibited the highest umami taste intensity, followed by mature and old leaves. Partial least square regression and computational molecular docking analyses revealed five potent umami substances (quinic acid, trigonelline, alanyl-tyrosine, leucyl-glycyl-proline, and leucyl-aspartyl-glutamine) and three known umami compounds (l-glutamic acid, pyroglutamic acid, and 5'-adenosine monophosphate). The five substances were validated as novel umami compounds using electronic tongue assay; leucyl-glycyl-proline exhibited synergism with monosodium glutamate, thereby enhancing the umami taste. Thus, substances contributing to the taste of chaya leaves were successfully identified.

Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

De novo transcriptome analysis and identification of candidate genes associated with triterpenoid biosynthesis in Trichosanthes cucumerina L.

KEY MESSAGE: De novo transcriptome analysis from callus, leaf, and fruit of Trichosanthes cucumerina L. for the identification of genes associated with triterpenoid biosynthesis, especially bryonolic acid and cucurbitacin B. Trichosanthes cucumerina L. (TC) has been used as a medicinal plant in Thailand with various potential functions. Two major triterpenoids found in this plant, bryonolic acid and cucurbitacin B, are receiving increased attention for their activities. Here, we provide TC transcriptome data to identify genes involved in the triterpenoid biosynthetic pathway through callus, where was previously suggested as a novel source for bryonolic acid production as opposed to leaf and fruit. A de novo assembly of approximately 290-thousand transcripts generated from these tissues led to two putative oxidosqualene cyclases: isomultiflorenol synthase (IMS) and cucurbitadienol synthase (CBS). TcIMS and TcCBS, genes that encode substrates for two characteristic triterpenoids in cucurbitaceous plants, were identified as isomultiflorenol synthase and cucurbitadienol synthase, respectively. These two genes were functionally characterised in mutant yeast Gil77 systems, which led to the productions of isomultiflorenol and cucurbitadienol. Moreover, the callus-specific gene expression profiles were also presented. These obtained information showed candidate cytochrome P450s with predicted full-length sequences, which were most likely associated with triterpenoid biosynthesis, especially bryonolic acid. Our study provides useful information and a valuable reference for the further studies on cucurbitaceous triterpenoids.

Overview of Cannabis including Kampo Medicine and Therapy for Treatment of Dementia: A Review.

Cannabis sativa L. is an annual herb oldest cultivated plants as a source of fiber since about 5000 B.C. On the other hand, the cannabis flower and seed are listed in Shennong's classic Materia Medica approximately 2000 years ago. The formulas prescribed with cannabis in Kampo medicine have been summarized. Cannabidiol (CBD) and tetrahydrocannabinol (THC) are the major neurological and psychiatric cannabinoids, and develop to drugs. It becomes evident that the therapeutic CBD and/or THC are the important candidate of anti-dementia drugs having different mechanism for Alzheimer's patients. Two receptors and endocannabinoids are also discussed for underlying mechanism of action. In order to promote the breeding of cannabis plant containing higher concentration of target cannabinoid the biosynthetic enzymes were isolated, cloning and the tertiary structure of THCA synthase determined by x-ray analysis resulting in the possibility of molecular breeding for cannabinoids.

Enhanced Production of Bryonolic Acid in Trichosanthes cucumerina L. (Thai Cultivar) Cell Cultures by Elicitors and Their Biological Activities.

Bryonolic acid is a triterpenoid compound found in cucurbitaceous roots. Due to its biological activities, this compound gets more attention to improve production. Herein, we carried out efficient ways with high bryonolic acid productions from Trichosanthes cucumerina L., a Thai medicinal plant utilizing plant cell cultures. The results showed that calli (24.65 ± 1.97 mg/g dry weight) and cell suspensions (15.69 ± 0.78 mg/g dry weight) exhibited the highest bryonolic acid productions compared with natural roots (approximately 2 mg/g dry weight). In the presence of three elicitors (methyl jasmonate, yeast extract, and chitosan), cell suspensions treated with 1 mg/mL of chitosan for eight days led to higher bryonolic acid contents (23.56 ± 1.68 mg/g dry weight). Interestingly, cell culture and root extracts with high bryonolic acid contents resulted in significantly higher percent cell viabilities than those observed under control (1% v/v DMSO) treatment in Saos-2 and MCF-7 cells. The present study indicated that T. cucumerina L. cell cultures are alternative and efficient to produce the biologically important secondary metabolite.

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol.

BACKGROUND: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

Camptothecin: therapeutic potential and biotechnology.

Camptothecin (CPT) and its derivatives have been received considerable attention recently. Two semi-synthetic derivatives, topotecan and irinotecan, are currently prescribed as anticancer drugs. Several more are now in clinical trial. CPT is produced in many plants belonging to unrelated orders of angiosperms. At present, CPT supplied for pharmaceutical use is extracted from the plants, Camptotheca acuminata and Nothapodytes foetida. Several efforts have been made to sustain a stable production of CPT by in vitro cell cultures of C. acuminata, N. foetida and Ophiorrhiza pumila. Recent report showed that plants are not the only sources that produce CPT. CPT was reported to be produced from the endophytic fungus isolated from the inner bark of N. foetida. The hairy root cultures of C. acuminata and O. pumila produce and secrete CPT into the medium in large quantities. These reports suggest the possibility to develop large-scale production of CPT. In addition, recent advance in the cloning and characterization of biosynthetic enzymes involved in CPT biosynthetic pathway provides valuable information for developing genetically engineered CPT-producing plants.