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.

Whole-genome sequence data of Hypericum perforatum and functional characterization of melatonin biosynthesis by N-acetylserotonin O-methyltransferase.

Hypericum perforatum is among the most commonly used herbal remedies and supplements. The aerial plant parts are often used to treat depression. Due to the lack of genomic information of H. perforatum, the gene networks regulating secondary metabolite synthesis remain unclear. Here, we present a high-quality genome for H. perforatum with a 2.3-Mb scaffold N50. The draft assembly covers 91.9% of the predicted genome and represents the fourth sequenced genus in the order Malpighiales. Comparing this sequence with model or related species revealed that Populus trichocarpa and Hevea brasiliensis could be grouped into one branch, while H. perforatum and Linum usitatissimum are grouped in another branch. Combined with transcriptome data, 40 key genes related to melatonin, hyperforin, and hypericin synthesis were screened and analyzed. Five N-acetylserotonin O-methyltransferases (HpASMT1-HpASMT5) were cloned and functionally characterized. Purified HpASMT3 protein converted N-acetylserotonin into melatonin with a Vmax of about 1.35 pkat/mg protein. HpASMT1 and HpASMT3 overexpression in Arabidopsis mutants caused 1.5-2-fold higher melatonin content than in mutant and wild-type plants. The endogenous reactive oxygen species (ROS) in transgenic plants was significantly lower than ROS in mutant and wild-type plants, suggesting higher drought tolerance. The obtained genomic data offer new resources for further study on the evolution of Hypericaceae family, but also provide a basis for further study of melatonin biosynthetic pathways in other plants.

Computational screening of miRNAs and their targets in leaves of Hypericum spp. by transcriptome-mining: a pilot study.

MAIN CONCLUSION: Our work provides a survey of mature miRNAs, their target genes and primary precursors identified by in-silico approach in leaf transcriptomes of five selected Hypericum species. MiRNAs are small non-coding RNA molecules found in animals, terrestrial plants, several algae and molds. As their role lies in the post-transcriptional gene silencing, these tiny molecules regulate many biological processes. Phyto-miRNAs are considered the important regulators of secondary metabolism in medicinal plants. The genus Hypericum comprises many producers of bioactive compounds, mainly unique naphtodianthrones with a great therapeutic potential. The main goal of our work was to identify genetically conserved miRNAs, characterize their primary precursors and target sequences in the leaf transcriptomes of five Hypericum species using in-silico approach. We found 20 sequences of potential Hypericum pri-miRNAs, and predicted and computationally validated their secondary structures. The mature miRNAs were identified by target genes screening analysis. Whereas predicted miRNA profiles differed in less genetically conserved families, the highly conserved miRNAs were found in almost all studied species. Moreover, we detected several novel highly likely miRNA-mRNA interactions, such as mir1171 with predicted regulatory role in the biosynthesis of melatonin in plants. Our work contributes to the knowledge of Hypericum miRNAome and miRNA-mRNA interactions.

Isolation, Characterization and Targeted Metabolic Evaluation of Endophytic Fungi Harbored in 14 Seed-Derived Hypericum Species.

Medicinal plants of the genus Hypericum are rich sources of bioactive naphthodianthrones, which are unique in the plant kingdom, but quite common in fungal endophytes. Cultivable endophytic fungi were isolated from 14 different Hypericum spp. originating from seeds grown under in vitro conditions and further acclimated to outdoor conditions. Among 37 fungal isolates yielded from the aerial and underground plant organs, 25 were identified at the species level by the fungal barcode marker internal transcribed spacer rDNA and protein-coding gene region of tef1α. Ten of them were isolated from Hypericum spp. for the first time. The axenic cultures of the isolated endophytes were screened for the production of extracellular enzymes, as well as bioactive naphthodianthrones and their putative precursors by Bornträger's test and HPLC-HRMS. Traces of naphthodianthrones and their intermediates, emodin, emodin anthrone, skyrin, or pseudohypericin, were detected in the fungal mycelia of Acremonium sclerotigenum and Plectosphaerella cucumerina isolated from Hypericum perforatum and Hypericum maculatum, respectively. Traces of emodin, hypericin, and pseudohypericin were released in the broth by Scedosporium apiospermum, P. cucumerina, and Fusarium oxysporum during submerged fermentation. These endophytes were isolated from several hypericin-producing Hypericum spp. Taken together, our results reveal the biosynthetic potential of cultivable endophytic fungi harbored in Hypericum plants as well as evidence of the existence of remarkable plant-endophyte relationships in selected non-native ecological niches. A possible role of the extracellular enzymes in plant secondary metabolism is discussed.

MALDI-HRMS Imaging Maps the Localization of Skyrin, the Precursor of Hypericin, and Pathway Intermediates in Leaves of Hypericum Species.

Hypericum perforatum and related species (Hypericaceae) are a reservoir of pharmacologically important secondary metabolites, including the well-known naphthodianthrone hypericin. However, the exact biosynthetic steps in the hypericin biosynthetic pathway, vis-à-vis the essential precursors and their localization in plants, remain unestablished. Recently, we proposed a novel biosynthetic pathway of hypericin, not through emodin and emodin anthrone, but skyrin. However, the localization of skyrin and its precursors in Hypericum plants, as well as the correlation between their spatial distribution with the hypericin pathway intermediates and the produced naphthodianthrones, are not known. Herein, we report the spatial distribution of skyrin and its precursors in leaves of five in vitro cultivated Hypericum plant species concomitant to hypericin, its analogs, as well as its previously proposed precursors emodin and emodin anthrone, using MALDI-HRMS imaging. Firstly, we employed HPLC-HRMS to confirm the presence of skyrin in all analyzed species, namely H. humifusum, H. bupleuroides, H. annulatum, H. tetrapterum, and H. rumeliacum. Thereafter, MALDI-HRMS imaging of the skyrin-containing leaves revealed a species-specific distribution and localization pattern of skyrin. Skyrin is localized in the dark glands in H. humifusum and H. tetrapterum leaves together with hypericin but remains scattered throughout the leaves in H. annulatum, H. bupleuroides, and H. rumeliacum. The distribution and localization of related compounds were also mapped and are discussed concomitant to the incidence of skyrin. Taken together, our study establishes and correlates for the first time, the high spatial distribution of skyrin and its precursors, as well as of hypericin, its analogs, and previously proposed precursors emodin and emodin anthrone in the leaves of Hypericum plants.

Phenotyping the genus Hypericum by secondary metabolite profiling: emodin vs. skyrin, two possible key intermediates in hypericin biosynthesis.

A wide range of compounds that occur in the genus Hypericum are listed as effective drugs of natural origin. The main biological activities of several Hypericum representatives are due to the presence of naphthodianthrones, phloroglucinols, and other diverse groups of secondary metabolites that synergistically contribute to their therapeutic effects. The regulation of biosynthesis of hypericin as the key bioactive naphthodianthrone remains uncertain. Here, we present liquid chromatography mass spectrometry-based phenotyping of 17 Hypericum species, the results of which suggest an important role for skyrin and its derivatives in the polyketide pathway that leads to hypericin formation. Moreover, we report for the first time the presence of new metabolites in the genus Hypericum that are related to classes of anthraquinones, their derivatives, and phloroglucinols. As skyrin and other species of anthraquinones are rarely found in higher plants but frequently occur in fungal microorganisms, the obtained results suggest that further research on the synthesis pathways of hypericin and the role of anthraquinone derivatives in plant metabolism should be carried out. The fact that these compounds are commonly synthesized in endophytic fungi and perhaps there is some similarity in the metabolic pathways between these organisms should also be investigated.

Interspecific variation in localization of hypericins and phloroglucinols in the genus Hypericum as revealed by desorption electrospray ionization mass spectrometry imaging.

Plants of the genus Hypericum are widely known for their therapeutic properties. The most biologically active compounds of this genus are naphtodianthrones and phloroglucinols. Indirect desorption electrospray ionization mass spectrometry (DESI-MS) imaging allows visualization and localization of secondary metabolites in different plant tissues. This study is focused on localization of major secondary compounds in the leaves of 17 different in vitro cultured Hypericum species classified in 11 sections. Generally, all identified naphtodianthrones, protohypericin, hypericin, protopseudohypericin and pseudohypericin were co-localized in the dark glands of eight hypericin producing species at the site of their accumulation. The known phloroglucinols, hyperforin, adhyperforin, hyperfirin and some new phloroglucinols with m/z [M - H](-) 495 and 569 were localized in the translucent and pale cavities within the leaf in the majority of studied species. The comparison of different Hypericum species revealed an interspecific variation in the distribution of the dark and translucent glands corresponding with the localization of hypericins and phloroglucinols. Moreover, similarities in the localization and composition of the phloroglucinols were observed in the species belonging to the same section. Adding to various quantitative studies focused on the detection of secondary metabolites, this work using indirect DESI-MSI offers additional valuable information about localization of the above-mentioned compounds.

Effect of cryoprotectant solution and of cooling rate on crystallization temperature in cryopreserved Hypericum perforatum cell suspension cultures.

BACKGROUND: The increasing demand for hypericins and hyperforins, the unique pharmaceuticals found in the Hypericum genus, requires the development of effective tools for long-term storage of cells and tissues with unique biochemical profiles. OBJECTIVE: To determine the temperature of crystallization (T(C)) and of ice formation of 14 cryoprotectant mixtures (CMs) for their use in cryoprotection of H. perforatum L. cell suspensions and to evaluate the impact of the lowest Tc on post-cryogenic recovery. MATERIALS AND METHODS: T(C) was determined by real-time microscopy of ice formation during slow cooling to -196° C and heating to 20° C. RESULTS: Exposure of cells to CMs CM2 (PVS3) containing sucrose and glycerol or CM12 and CM13 containing sucrose, glycerol, dimethylsulfoxide and ethylene glycol decreased T(C) below -60° C, prevented intracellular crystallization and considerably reduced both the size of crystals and the rate of extracellular ice propagation. CONCLUSION: The selected CMs proved suitable for cryopreservation of H. perforatum cell suspensions with the maximum of 58 % post-thaw recovery.

Spatial chemo-profiling of hypericin and related phytochemicals in Hypericum species using MALDI-HRMS imaging.

Advanced analytical imaging techniques, including matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) imaging, can be used to visualize the distribution, localization, and dynamics of target compounds and their precursors with limited sample preparation. Herein we report an application of MALDI-HRMS imaging to map, in high spatial resolution, the accumulation of the medicinally important naphthodianthrone hypericin, its structural analogues and proposed precursors, and other crucial phytochemical constituents in the leaves of two hypericin-containing species, Hypericum perforatum and Hypericum olympicum. We also investigated Hypericum patulum, which does not contain hypericin or its protoforms. We focused on both the secretory (dark glands, translucent glands, secretory canals, laminar glands, and ventral glands) and the surrounding non-secretory tissues to clarify the site of biosynthesis and localization of hypericin, its possible precursors, and patterns of localization of other related compounds concomitant to the presence or absence of hypericin. Hypericin, pseudohypericin, and protohypericin accumulate in the dark glands. However, the precursor emodin not only accumulates in the dark glands but is also present outside the glands in both hypericin-containing species. In hypericin-lacking H. patulum, however, emodin typically accumulates only in the glands, thereby providing evidence that hypericin is possibly biosynthesized outside the dark glands and thereafter stored in them. The distribution and localization of related compounds were also evaluated and are discussed concomitant to the occurrence of hypericin. Our study provides the basis for further detailed investigation of hypericin biosynthesis by gene discovery and expression studies.

Xanthones from roots, hairy roots and cell suspension cultures of selected Hypericum species and their antifungal activity against Candida albicans.

KEY MESSAGE: Highest xanthone contents were found in Hypericum pulchrum and H. annulatum untransformed roots. The best anti- Candida activity was obtained for hairy roots extracts of H. tetrapterum clone 2 ATCC 15834. Extracts of root cultures, hairy roots and cell suspensions of selected Hypericum spp. were screened for the presence of xanthones and tested for their antifungal activity against Candida albicans strain ATCC 10231. At least one of the following xanthones, 5-methoxy-2-deprenylrheediaxanthone; 1,3,6,7-tetrahydroxyxanthone; 1,3,5,6-tetrahydroxyxanthone; paxanthone; kielcorin or mangiferin was identified in methanolic extracts of the untransformed root cultures. The highest total xanthone content, with five xanthones, was found in untransformed H. pulchrum and H. annulatum root cultures. Hairy roots and the controls of H. tetrapterum contained 1,7-dihydroxyxanthone, while hairy root cultures and the corresponding controls of H. tomentosum contained toxyloxanthone B, 1,3,6,7- and 1,3,5,6-tetrahydroxyxanthone. Two xanthones, cadensin G and paxanthone, were identified in cell suspension cultures of H. perforatum. Their content increased about two-fold following elicitation with salicylic acid. The anti-Candida activity of the obtained extracts ranged from MIC 64 to >256 µg ml(-1). Among the extracts of Hypericum untransformed roots, the best antifungal activity was obtained for extracts of H. annulatum grown under CD conditions. Extracts of hairy roots clones A4 and 7 ATCC15834 of H. tomentosum and clone 2 ATCC15834 of H. tetrapterum displayed inhibition of 90% of Candida growth with 256 µg ml(-1). Extracts from chitosan-elicitated cells did not show antifungal activity.

Influence of cryopreservation on the antioxidative activity of in vitro cultivated Hypericum species.

Antioxidative activity of two in vitro cultivated Hypericum species - H. rumeliacum Boiss. and H. tetrapterum Fr. - was estimated after cryopreservation. Both species were successfully regenerated after a cryopreservation procedure performed by the vitrification method. H. tetrapterum did not manifest any significant oxidative stress-induced changes caused by low-temperature treatment. Conversely, a decrease in green pigments' content of H. rumeliacum was measured, particularly pronounced in chlorophyll b, which was accompanied by an increase of carotenoids in the regenerated plants. A strong increase of malone dialdehyde and H2O2 levels in H. rumeliacum tissues was detected. Superoxide dismutase activity was enhanced by 170%, as well as the catalase activity, which was 220% above the control. The same trend was observed in H. tetrapterum, although less pronounced - 143% increase of superoxide dismutase and 112% of catalase. Cryopreservation did not influence the phenol content in the examined plants, but it led to an increase of flavonoid content, especially in H. tetrapterum, by 237%. Total antioxidant activity in regenerated H. tetrapterum varied around the control level, but it was increased in H. rumeliacum. The free proline content in H. tetrapterum remained almost unaffected after freezing, as opposed to H. rumeliacum, where a strong increase of proline content (208% above the control) occurred. An electrolyte leakage from the cells of H. rumeliacum regenerated after cryopreservation was also registered, albeit not significant.

Recent advances in the identification of biosynthetic genes and gene clusters of the polyketide-derived pathways for anthraquinone biosynthesis and biotechnological applications.

Natural anthraquinones are represented by a large group of compounds. Some of them are widespread across the kingdoms, especially in bacteria, fungi and plants, while the others are restricted to certain groups of organisms. Despite the significant pharmacological potential of several anthraquinones (hypericin, skyrin and emodin), their biosynthetic pathways and candidate genes coding for key enzymes have not been experimentally validated. Understanding the genetic and epigenetic regulation of the anthraquinone biosynthetic gene clusters in fungal endophytes would help not only understand their pathways in plants, which ensure their commercial availability, but also favor them as promising systems for prospective biotechnological production.

Does phenotyping of Hypericum secondary metabolism reveal a tolerance to biotic/abiotic stressors?

In this review we summarize the current knowledge about the changes in Hypericum secondary metabolism induced by biotic/abiotic stressors. It is known that the extreme environmental conditions activate signaling pathways leading to triggering of enzymatic and non-enzymatic defense systems, which stimulate production of secondary metabolites with antioxidant and protective effects. Due to several groups of bioactive compounds including naphthodianthrones, acylphloroglucinols, flavonoids, and phenylpropanes, the world-wide Hypericum perforatum represents a high-value medicinal crop of Hypericum genus, which belongs to the most diverse genera within flowering plants. The summary of the up-to-date knowledge reveals a relationship between the level of defense-related phenolic compounds and interspecific differences in the stress tolerance. The chlorogenic acid, and flavonoids, namely the amentoflavone, quercetin or kaempferol glycosides have been reported as the most defense-related metabolites associated with plant tolerance against stressful environment including temperature, light, and drought, in association with the biotic stimuli resulting from plant-microbe interactions. As an example, the species-specific cold-induced phenolics profiles of 10 Hypericum representatives of different provenances cultured in vitro are illustrated in the case-study. Principal component analysis revealed a relationship between the level of defense-related phenolic compounds and interspecific differences in the stress tolerance indicating a link between the provenance of Hypericum species and inherent mechanisms of cold tolerance. The underlying metabolome alterations along with the changes in the activities of ROS-scavenging enzymes, and non-enzymatic physiological markers are discussed. Given these data it can be anticipated that some Hypericum species native to divergent habitats, with interesting high-value secondary metabolite composition and predicted high tolerance to biotic/abiotic stresses would attract the attention as valuable sources of bioactive compounds for many medicinal purposes.

Phytochemical profiling of several Hypericum species identified using genetic markers.

In the present study, we performed phytochemical profiling of several under-exploited Hypericum representatives taxonomically belonging to the sections Ascyreia, Androsaemum, Inodora, Hypericum, Coridium, Myriandra, and Adenosepalum. The authenticity of the starting plant material was confirmed using the nuclear ribosomal internal transcribed spacer as a molecular marker, DNA content and chromosome number. Phenolic constituents were analyzed using high-performance liquid chromatography to complement species-specific metabolic profiles. In several Hypericum representatives, the pharmacologically important compounds, including naphthodianthrones; phloroglucinol derivatives; chlorogenic acid; and some classes of flavonoids, particularly the flavonols rutin and hyperoside, flavanol catechin, and flavanones naringenin and naringin, were reported for the first time. Comparative multivariate analysis of chemometric data for seedlings cultured in vitro and acclimated to the outdoor conditions revealed a strong genetically predetermined interspecific variability in phenolic compound content. In addition to hypericins, which are the most abundant chemomarkers for the genus Hypericum, rarely employed phenolic metabolites, including phloroglucinol derivatives, chlorogenic acid, catechin, naringenin, naringin, and kaempferol-3-O-glucoside, were shown to be useful for discriminating between closely related species. Given the increasing interest in natural products of the genus Hypericum, knowledge of the spectrum of phenolic compounds in shoot cultures is a prerequisite for future biotechnological applications. In addition, phytochemical profiling should be considered as an additional part of the integrated plant authentication system, which predominantly relies upon genetic markers.

Genotoxicity and antigenotoxicity evaluation of non-photoactivated hypericin.

The potential genotoxicity and antigenotoxicity of non-photoactivated hypericin was investigated in five experimental models. Hypericin was non-mutagenic in the Ames assay, with and without metabolic activation. It did not exert a protective effect against mutagenicity induced by 9-aminoacridine. In a yeast (Saccharomyces cerevisiae) assay, hypericin did not increase the frequency of mitotic crossovers or total aberrants at the ade(2) locus, the number of convertants at the trp5 locus, or the number of revertants at the ilv1 locus. In combined application with 4-nitroquinoline-1-oxide, it significantly enhanced the number of revertants at the ilv1 locus at the highest concentration used. Hypericin was not mutagenic in the alga Chlamydomonas reinhardtii. However, in combined application with methyl methane sulfonate, toxicity and mutagenicity were slightly reduced. In a chromosome aberration assay using three mammalian cell lines, hypericin did not alter the frequency of structural chromosome aberrations, and in the DPPH radical scavenging assay, it did not exert any antioxidant effects.

Effect of the number of rol genes integrations on phenotypic variation in hairy root-derived Hypericum perforatum L. plants.

The extent of phenotypic variation of St. John's wort (Hypericum perforatum L.) plants transformed with wild agropine ATCC 15834 Agrobacterium rhizogenes plasmid was evaluated with respect to the number of rol genes integrations. The transfer of T(L)-DNA to plant explants during each transformation event was incomplete with different rolA, rolB, and rolC copy numbers. Along with typical features representing the hairy root syndrome, an altered size, number and density of dark and translucent glands, changes in ability to synthesize secondary metabolites, and reduced fertility were observed. The highest copy number of transferred rol genes resulted in weak expression of transgenic character and comparable quantitative parameters with the controls. Only 1 out of 11 transgenic clones was able to produce seed progeny and not more than 4 out of its 35 offsprings were positive for rolC gene integration. Sterility of the clones was due to retarded development of both gametophytes.

Physiological, biochemical and molecular characteristics of cryopreserved Hypericum perforatum L. shoot tips.

Hypericum perforatum L. in vitro cultured shoot tips were characterised at the physiological, biochemical and molecular levels following recovery from cryogenic treatment using the plant vitrification solutions PVS2 and PVS3. This comparative study revealed an increase in recovery and regrowth of explants cryoprotected with PVS3. Among the physiological markers only lipid peroxidation in the regenerants treated with PVS2 significantly increased indicating membrane damage. Genotype-specific interactions were found in most characteristics studied, with some variation detected within control and cryopreserved samples. Analyses of metabolite biosynthesis and genetic stability showed no significant differences in hypericin content, RAPD and minisatellite amplification profiles between PVS2- and PVS3-treated explants. This study demonstrates and discusses the criteria selective for PVS3 to improve the cryopreservation of H. perforatum L.

Light-independent metabolomics of endophytic Thielavia subthermophila provides insight into microbial hypericin biosynthesis.

The possible microbial mechanism of hypericin (1) and emodin (2) biosynthesis was studied in axenic submerged culture conditions in the endophytic fungus Thielavia subthermophila, isolated from Hypericum perforatum. The growth and secondary metabolite production of the endophyte remained independent of the illumination conditions. This production remained unaltered on spiking the medium with 3 or 5 mM 2, although the biomass accumulation was reduced. Neither emodin anthrone (3) nor protohypericin (4) could be detected at any stage of fermentation, irrespective of either spiking or illumination conditions. The endophytic metabolites exhibited photodynamic cytotoxicity against the human acute monocytic leukemia cell line (THP-1), at 92.7 vs 4.9%, and 91.1 vs 1.0% viability by resazurin and ATPlite assays, in light and in the dark, respectively. In trying to ascertain the presence/expression of the candidate hyp-1 gene in the endophyte, it was revealed that the hyp-1 gene was absent in T. subthermophila, indicating that the biosynthetic pathway in the endophytic fungus might be different and/or governed by a different molecular mechanism than the host plant or host cell suspension cultures. We have discussed the biosynthetic principles and evolutionary implications relating to endophytic T. subthermophila based on the results obtained.

Targeted metabolomic profiling reveals interspecific variation in the genus Hypericum in response to biotic elicitors.

Shoot cultures of eight Hypericum species belonging to the sections Hypericum, Oligostema, Ascyreia and Webbia were evaluated for their phytochemical profiles by high-performance liquid chromatography. In total, 17 secondary metabolites assigned to the groups of anthraquinones, phloroglucinols, hydroxycinnamic acids and flavonoids were detected. Furthermore, the elicitation potential of 18 biotic factors derived from saccharides, endophytic fungi and Agrobacterium rhizogenes was examined and statistically analysed with the paired two-sample t-test and principal component analysis. The production of naphthodianthrones and emodin was predominantly stimulated by elicitors derived from Fusarium oxysporum and Trichoderma crassum, while Piriformospora indica promoted the phloroglucinols production. Among flavonoids, the aglycone amentoflavone was readily increased by several elicitors up to 15.7-fold in H. humifusum treated by potato-dextrose broth. However, the chlorogenic acid proved to be the most susceptible metabolite to elicitation, when 31.7-times increase was detected in H. maculatum shoots upon D-glucose treatment. In spite of several biotic factors have been tested, no metabolite was commonly induced in all Hypericum spp. as a response to elicitor treatments.

Chemometric evaluation of hypericin and related phytochemicals in 17 in vitro cultured Hypericum species, hairy root cultures and hairy root-derived transgenic plants.

OBJECTIVES: The objective of this study was to ascertain the presence and correlations among eight important secondary metabolites viz. hypericin, pseudohypericin, emodin, hyperforin, rutin, hyperoside, quercetin and quercitrin in different organs of 17 in vitro cultured Hypericum species, along with H. tomentosum and H. tetrapterum hairy root cultures, and hairy root-derived transgenic plants of H. tomentosum. METHODS: Samples were extracted and analysed by LC-MS. The LC-MS data were subjected to chemometric evaluations for metabolite profiling and correlating the phytochemical compositions in different samples. KEY FINDINGS: Hypericin, pseudohypericin and their proposed precursor emodin were detected in various levels in the leaves of eight Hypericum species. The highest content of hypericins and emodin was found in H. tetrapterum, which contains the studied secondary metabolites in all plant organs. A significant positive correlation between hypericins and emodin was observed both by principal component analysis (PCA) and multidimensional scaling (MDS), indicating the role of emodin as a possible precursor in the biosynthetic pathway of hypericins. Flavonoids were found in all tested plant organs except roots of H. pulchrum. The hairy roots lacked hypericin, pseudohypericin, emodin, hyperforin and rutin. However, the hairy root-derived transgenic plants showed a significant increase in flavonoids. CONCLUSIONS: This study broadens knowledge about the phytochemical composition of selected in vitro cultured Hypericum species, compared to that of hairy root cultures and hairy root-derived transgenic plants.