Clock protein LHY targets SNAT1 and negatively regulates the biosynthesis of melatonin in Hypericum perforatum.

Hypericum perforatum, also known as "natural fluoxetine," is a commonly used herbal remedy for treating depression. It is unclear whether melatonin in plants regulated by the endogenous circadian clock system is like in vertebrates. In this work, we found that the melatonin signal and melatonin biosynthesis gene, serotonin N-acetyltransferase HpSNAT1, oscillates in a 24-hour cycle in H. perforatum. First, we constructed a yeast complementary DNA library of H. perforatum and found a clock protein HpLHY that can directly bind to the HpSNAT1 promoter. Second, it was confirmed that HpLHY inhibits the expression of HpSNAT1 by targeting the Evening Element. Last, it indicated that HpLHY-overexpressing plants had reduced levels of melatonin in 12-hour light/12-hour dark cycle photoperiod, while loss-of-function mutants exhibited high levels, but this rhythm seems to disappear as well. The results revealed the regulatory role of LHY in melatonin biosynthesis, which may make an important contribution to the field of melatonin synthesis regulation.

Single-cell RNA sequencing facilitates the elucidation of the complete biosynthesis of the antidepressant hyperforin in St. John's wort.

Hyperforin is the compound responsible for the effectiveness of St. John's wort (Hypericum perforatum) as an antidepressant, but its complete biosynthetic pathway remains unknown. Gene discovery based on co-expression analysis of bulk RNA-sequencing data or genome mining failed to discover the missing steps in hyperforin biosynthesis. In this study, we sequenced the 1.54-Gb tetraploid H. perforatum genome assembled into 32 chromosomes with the scaffold N50 value of 42.44 Mb. By single-cell RNA sequencing, we identified a type of cell, "Hyper cells", wherein hyperforin biosynthesis de novo takes place in both the leaves and flowers. Through pathway reconstitution in yeast and tobacco, we identified and characterized four transmembrane prenyltransferases (HpPT1-4) that are localized at the plastid envelope and complete the hyperforin biosynthetic pathway. The hyperforin polycyclic scaffold is created by a reaction cascade involving an irregular isoprenoid coupling and a tandem cyclization. Our findings reveal how and where hyperforin is biosynthesized, enabling synthetic-biology reconstitution of the complete pathway. Thus, this study not only deepens our comprehension of specialized metabolism at the cellular level but also provides strategic guidance for elucidation of the biosynthetic pathways of other specializied metabolites in plants.

Phytochemical Analysis, Biological Activities, and Docking of Phenolics from Shoot Cultures of Hypericum perforatum L. Transformed by Agrobacterium rhizogenes.

Hypericum perforatum transformed shoot lines (TSL) regenerated from corresponding hairy roots and non-transformed shoots (NTS) were comparatively evaluated for their phenolic compound contents and in vitro inhibitory capacity against target enzymes (monoamine oxidase-A, cholinesterases, tyrosinase, α-amylase, α-glucosidase, lipase, and cholesterol esterase). Molecular docking was conducted to assess the contribution of dominant phenolic compounds to the enzyme-inhibitory properties of TSL samples. The TSL extracts represent a rich source of chlorogenic acid, epicatechin and procyanidins, quercetin aglycone and glycosides, anthocyanins, naphthodianthrones, acyl-phloroglucinols, and xanthones. Concerning in vitro bioactivity assays, TSL displayed significantly higher acetylcholinesterase, tyrosinase, α-amylase, pancreatic lipase, and cholesterol esterase inhibitory properties compared to NTS, implying their neuroprotective, antidiabetic, and antiobesity potential. The docking data revealed that pseudohypericin, hyperforin, cadensin G, epicatechin, and chlorogenic acid are superior inhibitors of selected enzymes, exhibiting the lowest binding energy of ligand-receptor complexes. Present data indicate that H. perforatum transformed shoots might be recognized as an excellent biotechnological system for producing phenolic compounds with multiple health benefits.

Differentiated response of Hypericum perforatum to foliar application of selected metabolic modulators: Elicitation potential of chitosan, selenium, and salicylic acid mediated by redox imbalance.

Plants plastically alter their metabolism in response to environmental stimuli, which induces changes in the accumulation of specialized metabolites. This ability can be utilized to manipulate plant phytochemistry in a desired direction. However, the abundance of secondary metabolites in the different plant species, especially medicinal, is enormous; therefore, it is difficult to establish a clear direction for the effects of metabolic modulators on phytochemical composition, especially given the possibility of using different types thereof. In order to gain insight into these changes, we investigated the effects of foliar-applied chitosan (ChL, 100 mg/L), selenium (Se, 10 mg/L), salicylic acid (SA, 150 mg/L), or an equal volume mixture thereof on Hypericum perforatum L. metabolism. Selenium and SA proved to be the more effective than ChL in enhancing the accumulation of phenolic compounds. The greatest increase was found in the concentration of neochlorogenic acid after Se-spraying. The treatment with the elicitors generally increased the concentration of identified flavonoids, but not the level of naphthodianthrone or phloroglucinol metabolites. The most pronounced response was observed on day 10 following the application of the compounds, and is likely the consequence of elevated levels of O2-˙, free proline, and modulated activity of enzymatic antioxidants.

In silico prediction of polyketide biosynthetic gene clusters in the genomes of Hypericum-borne endophytic fungi.

BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized. RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein). CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.

Regiodivergent biosynthesis of bridged bicyclononanes.

Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.

Do nanoparticles delivered to roots affect plant secondary metabolism? A comprehensive analysis in float seedling cultures of Hypericum perforatum L.

Since nanoparticles (NPs) released into the environment from household or industrial wastes and applied directly on plants as agrochemicals can accumulate in the rhizosphere, it is imperative to understand how these NPs affect plant secondary metabolism upon their contact with the roots of intact plants. Here, the effects of Pd, Au, ZnO and Fe2O3 NPs on secondary metabolism were comprehensively investigated in Hypericum perforatum L float seedlings by analyzing 41 major secondary metabolites using ultra-performance liquid chromatography coupled with photodiode array, fluorescence detector and high-resolution mass spectrometry (UPLC-PDA-FLR-HRMS). The results showed that exposure of H. perforatum roots to Pd, Au, ZnO and Fe2O3 NPs rapidly led to fluctuations in the levels of secondary metabolites. Although these fluctuations did not correlate with NP type, concentration and duration of treatment, a total of 22 compounds were significantly altered by the NPs tested. In particular, 1 ppm Au increased the content of quercetin 3-(2″-acetylgalactoside), cadensin G and leutoskyrin by 5.02-, 2.12- and 2.58-fold, respectively after 24 h; 25 ppm Pd NPs led to a 2.1-fold increase in miquelianin content after 6 h; 50 ppm Fe2O3 NPs increased the level of furohyperforin by 3.09-fold and decreased the content of miquelianin 5.22-fold after 24 h and 50 ppm ZnO led to a 2.13-fold increase in hypericin after 48 h. These results emphasise the need to understand the intricate interplay between NPs and plant secondary metabolism in order to enable safer and efficient applications of NPs in agriculture.

Effect of Tacrolimus Formulation (Prolonged-Release vs Immediate-Release) on Its Susceptibility to Drug-Drug Interactions with St. John's Wort.

Tacrolimus is metabolized by cytochrome P450 3A (CYP3A) and is susceptible to interactions with the CYP3A and P-glycoprotein inducer St. John's Wort (SJW). CYP3A isozymes are predominantly expressed in the small intestine and liver. Prolonged-release tacrolimus (PR-Tac) is largely absorbed in distal intestinal segments and is less susceptible to CYP3A inhibition. The effect of induction by SJW is unknown. In this randomized, crossover trial, 18 healthy volunteers received single oral tacrolimus doses (immediate-release [IR]-Tac or PR-Tac, 5 mg each) alone and during induction by SJW. Concentrations were quantified using ultra-high performance liquid chromatography coupled with tandem mass spectrometry and non-compartmental pharmacokinetics were evaluated. SJW decreased IR-Tac exposure (area under the concentration-time curve) to 73% (95% confidence interval 60%-88%) and maximum concentration (Cmax ) to 61% (52%-73%), and PR-Tac exposure to 67% (55%-81%) and Cmax to 69% (58%-82%), with no statistical difference between the 2 formulations. The extent of interaction appeared to be less pronounced in volunteers with higher baseline CYP3A4 activity and in CYP3A5 expressors. In contrast to CYP3A inhibition, CYP3A induction by SJW showed a similar extent of interaction with both tacrolimus formulations. A higher metabolic baseline capacity appeared to attenuate the extent of induction by SJW.

St. John's Wort Formulations Induce Rat CYP3A23-3A1 Independent of Their Hyperforin Content.

The pregnane X receptor (PXR) is a ligand-activated regulator of cytochrome P450 (CYP)3A enzymes. Among the ligands of human PXR is hyperforin, a constituent of St John's wort (SJW) extracts and potent inducer of human CYP3A4. It was the aim of this study to compare the effect of hyperforin and SJW formulations controlled for its content on CYP3A23-3A1 in rats. Hyperiplant was used as it contains a high hyperforin content and Rebalance because it is controlled for a low hyperforin content. In silico analysis revealed a weak hyperforin-rPXR binding affinity, which was further supported in cell-based reporter gene assays showing no hyperforin-mediated reporter activation in presence of rPXR. However, cellular exposure to Hyperiplant and Rebalance transactivated the CYP3A reporter 3.8-fold and 2.8-fold, respectively, and they induced Cyp3a23-3a1 mRNA expression in rat hepatoma cells compared with control 48-fold and 18-fold, respectively. In Wistar rats treated for 10 days with 400 mg/kg of Hyperiplant, we observed 1.8 times the Cyp3a23-3a1 mRNA expression, a 2.6-fold higher CYP3A23-3A1 protein amount, and a 1.6-fold increase in activity compared with controls. For Rebalance we only observed a 1.8-fold hepatic increase of CYP3A23-3A1 protein compared with control animals. Even though there are differing effects on rCyp3a23-3a1/CYP3A23-3A1 in rat liver reflecting the hyperforin content of the SJW extracts, the modulation is most likely not linked to an interaction of hyperforin with rPXR. SIGNIFICANCE STATEMENT: Treatment with St John's wort (SJW) has been reported to affect CYP3A expression and activity in rats. Our comparative study further supports this finding but shows that the pregnane X receptor-ligand hyperforin is not the driving force for changes in rat CYP3A23-3A1 expression and function in vivo and in vitro. Importantly, CYP3A induction mimics findings in humans, but our results suggest that another so far unknown constituent of SJW is responsible for the expression- and function-modifying effects in rat liver.

Comparative Genomics and Physiological Investigations Supported Multifaceted Plant Growth-Promoting Activities in Two Hypericum perforatum L.-Associated Plant Growth-Promoting Rhizobacteria for Microbe-Assisted Cultivation.

Plants are no longer considered standalone entities; instead, they harbor a diverse community of plant growth-promoting rhizobacteria (PGPR) that aid them in nutrient acquisition and can also deliver resilience. Host plants recognize PGPR in a strain-specific manner; therefore, introducing untargeted PGPR might produce unsatisfactory crop yields. Consequently, to develop a microbe-assisted Hypericum perforatum L. cultivation technique, 31 rhizobacteria were isolated from the plant's high-altitude Indian western Himalayan natural habitat and in vitro characterized for multiple plant growth-promoting attributes. Among 31 rhizobacterial isolates, 26 produced 0.59 to 85.29 µg mL-1 indole-3-acetic acid and solubilized 15.77 to 71.43 µg mL-1 inorganic phosphate; 21 produced 63.12 to 99.92% siderophore units, and 15 exhibited 103.60 to 1,296.42 nmol α-ketobutyrate mg-1 protein h-1 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity. Based on superior plant growth-promoting attributes, eight statistically significant multifarious PGPR were further evaluated for an in planta plant growth-promotion assay under poly greenhouse conditions. Plants treated with Kosakonia cowanii HypNH10 and Rahnella variigena HypNH18 showed, by significant amounts, the highest photosynthetic pigments and performance, eventually leading to the highest biomass accumulation. Comparative genome analysis and comprehensive genome mining unraveled their unique genetic features, such as adaptation to the host plant's immune system and specialized metabolites. Moreover, the strains harbor several functional genes regulating direct and indirect plant growth-promotion mechanisms through nutrient acquisition, phytohormone production, and stress alleviation. In essence, the current study endorsed strains HypNH10 and HypNH18 as cogent candidates for microbe-assisted H. perforatum cultivation by highlighting their exclusive genomic signatures, which suggest their unison, compatibility, and multifaceted beneficial interactions with their host and support the excellent plant growth-promotion performance observed in the greenhouse trial. IMPORTANCE Hypericum perforatum L. (St. John's wort) herbal preparations are among the top-selling products to treat depression worldwide. A significant portion of the overall Hypericum supply is sourced through wild collection, prompting a rapid decline in their natural stands. Crop cultivation seems lucrative, although cultivable land and its existing rhizomicrobiome are well suited for traditional crops, and its sudden introduction can create soil microbiome dysbiosis. Also, the conventional plant domestication procedures with increased reliance on agrochemicals can reduce the diversity of the associated rhizomicrobiome and plants' ability to interact with plant growth-promoting microorganisms, leading to unsatisfactory crop production alongside harmful environmental effects. Cultivating H. perforatum with crop-associated beneficial rhizobacteria can reconcile such concerns. Based on a combinatorial in vitro, in vivo plant growth-promotion assay and in silico prediction of plant growth-promoting traits, here we recommend two H. perforatum-associated PGPR, Kosakonia cowanii HypNH10 and Rahnella variigena HypNH18, to extrapolate as functional bioinoculants for H. perforatum sustainable cultivation.

Biosynthesis of polyprenylated xanthones in Hypericum perforatum roots involves 4-prenyltransferase.

Polyprenylated xanthones are natural products with a multitude of biological and pharmacological activities. However, their biosynthetic pathway is not completely understood. In this study, metabolic profiling revealed the presence of 4-prenylated 1,3,5,6-tetrahydroxyxanthone derivatives in St. John's wort (Hypericum perforatum) root extracts. Transcriptomic data mining led to the detection of 5 variants of xanthone 4-prenyltransferase (HpPT4px) comprising 4 long variants (HpPT4px-v1 to HpPT4px-v4) and 1 short variant (HpPT4px-sh). The full-length sequences of all 5 variants were cloned and heterologously expressed in yeast (Saccharomyces cerevisiae). Microsomes containing HpPT4px-v2, HpPT4px-v4, and HpPT4px-sh catalyzed the addition of a prenyl group at the C-4 position of 1,3,5,6-tetrahydroxyxanthone; 1,3,5-trihydroxyxanthone; and 1,3,7-trihydroxyxanthone, whereas microsomes harboring HpPT4px-v1 and HpPT4px-v3 additionally accepted 1,3,6,7-tetrahydroxyxanthone. HpPT4px-v1 produced in Nicotiana benthamiana displayed the same activity as in yeast, while HpPT4px-sh was inactive. The kinetic parameters of HpPT4px-v1 and HpPT4px-sh chosen as representative variants indicated 1,3,5,6-tetrahydroxyxanthone as the preferred acceptor substrate, rationalizing that HpPT4px catalyzes the first prenylation step in the biosynthesis of polyprenylated xanthones in H. perforatum. Dimethylallyl pyrophosphate was the exclusive prenyl donor. Expression of the HpPT4px transcripts was highest in roots and leaves, raising the question of product translocation. C-terminal yellow fluorescent protein fusion of HpPT4px-v1 localized to the envelope of chloroplasts in N. benthamiana leaves, whereas short, truncated, and masked signal peptides led to the disruption of plastidial localization. These findings pave the way for a better understanding of the prenylation of xanthones in plants and the identification of additional xanthone-specific prenyltransferases.

Effects of Aronia melanocarpa and Hypericum perforatum aqueous extracts on hexavalent chromium induced toxicity in rat's thyrocytes.

BACKGROUND: Hexavalent chromium known as oxidizing agent is able to form reactive oxygen species. Aronia melanocarpa and Hypericum perforatum are two plants known for their antioxidant effects. Our study aimed to establish if CrVI induces apoptosis and structural changes in thyrocytes and if its effect can be counteracted by the administration of both extracts. MATERIALS AND METHODS: Wistar rats divided in five groups: C - distilled water (DW), Cr - 75 mg/L CrVI in DW for 3 months, Cr 2 - 75 mg/L CrVI in DW for 3 months followed by 1 month DW, CrA - 3 months 75 mg/L CrVI in DW and 1 month Aronia 2.5% extract, CrH - 3 months 75 mg/L CrVI in DW and 1 month Hypericum 2.5% extract. Histological assessment and qRT-PCR for evaluation of BAX and Bcl2 protein levels performed on thyroid samples. RESULTS: The Cr and Cr2 groups were those with altered cytoarchitecture: increase in the diameter of many thyroid follicles, a decrease in their number, a decrease in the height of the follicular cells. The histological examination of the CrH group revealed almost recovery of structural architecture. The BAX gene levels were higher in the Cr and Cr2 groups indicating the apoptotic activity of chromium. In extract receiving groups the BAX gene expressions were significantly lower, but the lowest level presented the CrH group. Bcl2 gene expression levels indicate antiapoptotic activity being elevated in the Cr group, followed by CrA, Cr2, and CrH groups. The BAX/Bcl2 ratio which significantly increased in the case of the Cr and Cr2 group compared to the groups that were administered the two plant extracts. CONCLUSION: The results obtained in this study confirm that CrVI has toxic effects on thyroid endocrine cells and H. perforatum has stronger antioxidant properties against the action of hexavalent chromium in thyrocytes than A. melanocarpa.

Rapid screening of α-glucosidase inhibitors in Hypericum perforatum L. using bio-affinity chromatography coupled with UPLC/MS.

α-glucosidase inhibitors (AGIs) are widely used for the treatment of type 2 diabetes, but their side effects have made it to develop novel and alternative AGIs immediately. In this study, the extract of Hypericum perforatum L. (HPE) has been confirmed to have α-glucosidase inhibitory activity in vitro and in vivo. Seven active compounds, rutin, hyperoside, isoquercitrin, avicularin, quercitrin, quercetin, and biapigenin, were screened based on a bio-affinity chromatography column with α-glucosidase enzyme-conjugated solid phase and UPLC/MS, which exhibited excellent α-glycosidase inhibitory effects by the determined IC50 values. The mechanism of α-glycosidase inhibitory activity of biapigenin was studied for the first time. The results showed that biapigenin was a high-potential, reversible, and mixed enzyme inhibitor. Analysis by molecular docking further revealed that hydrophobic interactions were generated by interactions between biapigenin and amino acid residues LYS156, PHE303, PHE314, and LEU313. In addition, hydrogen bonding occurred between biapigenin and α-glucosidase amino acid residues ASP307, SER241, and LYS156. This research identified that biapigenin could be a novel AGI and further applied to the development of potential anti-diabetic drugs. Furthermore, our studies established a rapid in vitro screening method for AGIs from plants.

St. John's Wort Exacerbates Acetaminophen-Induced Liver Injury by Activation of PXR and CYP-Mediated Bioactivation.

St. John's wort (SJW) is a medicinal herb remedy for mild depression. However, long-term use of SJW has raised safety concerns in clinical practice because of drug-drug interactions. Excessive use of acetaminophen (APAP) causes severe hepatotoxicity, but whether SJW modulates APAP-induced liver injury remains unclear. In this study, the effect of long-term SJW administration on APAP-induced acute hepatotoxicity and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated that SJW exacerbates APAP-induced toxicity. Moreover, SJW markedly promoted glutathione depletion and increased the levels of the APAP-cysteine and APAP-N-acetylcysteinyl adducts in mice, which enhanced APAP metabolic activation and aggravated APAP-induced liver injury. To further elucidate APAP metabolic activation in liver injury induced by SJW, the activities and expression levels of CYP2E1 and CYP3A were measured. The results showed that the activities and expression levels of CYP2E1 and CYP3A were increased after SJW treatment. Furthermore, the PXR-CYP signaling pathway was activated by SJW, and its downstream target genes were upregulated. Collectively, this study demonstrated that the long-term administration of SJW extract led to the metabolic activation of APAP and significantly exacerbated APAP-induced liver injury, which may suggest caution for the clinical use of SJW and APAP.

Nickel uptake in hydroponics and elemental profile in relation to cultivation reveal variability in three Hypericum species.

The Hypericum species (H. perforatum, H. olympicum, and H. orientale) were cultured in hydroponics with excess nickel (Ni, 1 or 100 µM Ni) to compare the metallic and metabolite content. Identical species were collected outdoor to assess the same parameters (including uranium and lanthanides) with total of 53 elements. The results showed that Ni was less accumulated in shoots in hydroponics (translocation factor of 0.01-0.25) and the highest absolute amount was detected in H. olympicum. Essential elements were typically depleted by Ni excess, but Co and Na increased. Soluble phenols, sum of flavonols and catechin rather increased in response to Ni but quercetin glycosides and free amino acids decreased in the shoots of H. olympicum mainly. Comparison of laboratory and outdoor growing plants showed more phenols in outdoor samples but not in H. olympicum and individual metabolites differed too. Plants cultured in hydroponics contained lower amount of non-essential, toxic and rare earth elements (30-100-fold) and shoot bioaccumulation factor in outdoor samples was low for most elements (<0.01) but not for Cd and Pt. Data reveal that H. olympicum is a potent source of phenolic metabolites whereas H. orientale accumulates many elements (38 out of 53 elements).

Discovery of dearomatized isoprenylated acylphloroglucinols with colon tumor suppressive activities in mice via inhibiting NFκB-FAT1-PDCD4 signaling activation.

Dearomatized isoprenylated acylphloroglucinols (DIAPs) are specific natural products mainly distributed in the plants of genus Hypericum. In this study, guided by HPLC-UV screening, 46 DIAPs (approximately 70% of all DIAPs) including 20 new ones and an unprecedented architecture, were discovered from the roots of Hypericum henryi, which were elucidated by comprehensive spectroscopic, X-ray crystallography, and ECD methods. Compounds 1-7, 39, and 41-42 exhibited remarkable cytotoxicities (IC50 = 0.84-5.63 µM) in human colon cancer HCT116 cells, in which 2 and 6 possessed selective cytotoxicities towards colon cancer cells. The preliminary structure-activity relationships of these tested compounds were discussed. In addition, mechanistic investigations demonstrated that 2 and 6 could significantly suppress the expressions of NFκB, FAT1, and promoted novel tumor suppressor gene PDCD4 in HCT116 cells. Furthermore, in HCT116 colon xenograft-bearing mouse model, treatments with 2 and 6 reduced the growth of xenograft tumors in dose-dependent manner. Expressions of FAT1 in tumors were also decreased in mice treated with 2 and 6, suggesting their anti-tumor effects were via FAT1 signaling pathway. In conclusion, this is the first report on the mechanistic and in vivo studies of DIAP, indicating that these metabolites can be considered as a new type of anti-colon cancer lead agents for further drug development.

Convergent gene clusters underpin hyperforin biosynthesis in St John's wort.

The meroterpenoid hyperforin is responsible for the antidepressant activity of St John's wort extracts, but the genes controlling its biosynthesis are unknown. Using genome mining and biochemical work, we characterize two biosynthetic gene clusters (BGCs) that encode the first three steps in the biosynthesis of hyperforin precursors. The findings of syntenic and phylogenetic analyses reveal the parallel assembly of the two BGCs. The syntenous BGC in Mesua ferrea indicates that the first cluster was assembled before the divergence of the Hypericaceae and Calophyllaceae families. The assembly of the second cluster is the result of a coalescence of genomic fragments after a major duplication event. The differences between the two BGCs - in terms of gene expression, response to methyl jasmonate, substrate specificity and subcellular localization of key enzymes - suggest that the presence of the two clusters could serve to generate separate pools of precursors. The parallel assembly of two BGCs with similar compositions in a single plant species is uncommon, and our work provides insights into how and when these gene clusters form. Our discovery helps to advance our understanding of the evolution of plant specialized metabolism and its genomic organization. Additionally, our results offer a foundation from which hyperforin biosynthesis can be more fully understood, and which can be used in future metabolic engineering applications.

Herb-drug Interactions in Neuropsychiatric Pharmacotherapy - A Review of Clinically Relevant Findings.

The management of neuropsychiatric disorders relies heavily on pharmacotherapy. The use of herbal products as complimentary medicine, often concomitantly, is common among patients taking prescription neuropsychiatric drugs. Herb-drug interaction, a clinical consequence of this practice, may jeopardize the success of pharmacotherapy in neuropsychiatry. Besides the wellknown ability of phytochemicals to inhibit and/or induce drug-metabolizing enzymes and transport proteins, several phytoconstituents are capable of exerting pharmacological effects on the central nervous system. This study reviewed the relevant literature and identified 13 commonly used herbal products - celery, echinacea, ginkgo, ginseng, hydroxycut, kava, kratom, moringa, piperine, rhodiola, St. John's wort, terminalia/commiphora ayurvedic mixture and valerian - which have shown clinically relevant interactions with prescription drugs used in the management of neuropsychiatric disorders. The consequent pharmacokinetic and pharmacodynamic interactions with orthodox medications often result in deleterious clinical consequences. This underscores the importance of caution in herb-drug co-medication.

Decoupling of Plant Growth and Accumulation of Biologically Active Compounds in Leaves, Roots, and Root Exudates of Hypericum perforatum L. by the Combination of Jasmonate and Far-Red Lighting.

The plant hormone jasmonic acid (JA) fine tunes the growth-defense dilemma by inhibiting plant growth and stimulating the accumulation of secondary compounds. We investigated the interactions between JA and phytochrome B signaling on growth and the accumulation of selected secondary metabolites in Hypericum perforatum L., a medically important plant, by spraying plants with methyl jasmonate (MeJA) and by adding far-red (FR) lighting. MeJA inhibited plant growth, decreased fructose concentration, and enhanced the accumulation of most secondary metabolites. FR enhanced plant growth and starch accumulation and did not decrease the accumulation of most secondary metabolites. MeJA and FR acted mostly independently with no observable interactions on plant growth or secondary metabolite levels. The accumulation of different compounds (e.g., hypericin, flavonols, flavan-3-ols, and phenolic acid) in shoots, roots, and root exudates showed different responses to the two treatments. These findings indicate that the relationship between growth and secondary compound accumulation is specific and depends on the classes of compounds and/or their organ location. The combined application of MeJA and FR enhanced the accumulation of most secondary compounds without compromising plant growth. Thus, the negative correlations between biomass and the content of secondary compounds predicted by the growth-defense dilemma were overcome.

Identification and characterization of glycosyltransferases catalyzing direct xanthone 4-C-glycosylation in Hypericum perforatum.

Xanthones are compounds with a diphenyl ether skeleton mainly found in plants and often glycosylated at carbon atoms. Although many C-glycosyltransferases (CGTs) participating in flavone C-glycosylation have been identified, MiCGT from Mangifera indica, adding sugar to an open-chain benzophenone skeleton, is the only identified xanthone biosynthesis-related CGT. Here, we identified two CGTs from Hypericum perforatum that add sugar to the closed-ring xanthone, but not benzophenone. These CGTs catalyze sugar transfer to the C-4 position of norathyriol (1,3,6,7-tetrahydroxyxanthone) to form isomangiferin (1,3,6,7-tetrahydroxyxanthone 4-C-glucoside), a major xanthone C-glucoside. This is the first study to report CGTs that mediate the direct C-glycosylation of xanthone.