Phenolic Compounds Composition of Hypericum perforatum L. Wild-Growing Plants from the Republic of Macedonia

The aim of this study was to provide comprehensive understanding of phenolic compounds composition in roots (RO), non-flower shoots (NFS) and flower shoots (FS) of Hypericum perforatum wild-growing plants from the Republic of Macedonia. Identification of phenolic compounds in plant methanolic extracts was performed by HPLC-DAD/ESI-MS analysis. Chlorogenic acid and 3-p-coumaroylquinic acid were identified in NFS and FS, while 3-feruloylquinic acid was detected in RO and FS extracts. From the group of flavan- 3-ols, (epi)catechin and procyanidins were found in all tested samples, whereas catechin and B-type procyanidin dimer were confirmed in NFS and FS. Four flavonol glycosides (hyperoside, rutin, quercitrin and kaempferol 3-O-rutinoside) were identified in aerial parts. Guaijaverin and kaempferol 3-O-glucoside were exclusively found in NFS. Quercetin, amentoflavone and I3-II8 biapigenin as flavonoid aglycones were detected only in FS extracts. The NFS and FS extracts showed a capability for the accumulation of cyanidin 3-O-glycoside and cyanidin 3-O-rhamnoside, as well for hyperforin and adhyperforin. Naphthodianthrones were represented with pseudohypericin, hypericin and  protopseudohypericin  in  FS, while only hypericin was detected in NFS. Six xanthones, γ-mangostin, 5-O-methyl-2- deprenylrheediaxanthone B, garcinone C, 3,6-dihydroxy-1,5,7-trimethoxy-xanthone, cadensin G and cadensin C were exclusively confirmed in RO extracts. Padiaxanthone was detected in NFS, while dimethylmangiferin in FS extracts. The major finding of this study is the identification of novel xanthones in H. perforatum roots that could be potentially used as bioactive compounds in food and pharmaceutical industry

Phenolic Profile of Dark-Grown and Photoperiod-Exposed Hypericum perforatum

Hypericum perforatum L. is a medicinal plant considered as an important natural source of secondary metabolites with a wide range of pharmacological attributes. Hairy roots (HR) were induced from root segments of in vitro grown seedlings from H. perforatum after cocultivation with Agrobacterium rhizogenes A4. Investigations have been made to study the production of phenolic compounds in dark-grown (HR1) and photoperiod-exposed (HR2) cultures. The chromatographic analysis of phenolic acids, flavonols, flavan-3-ols, and xanthones revealed marked differences between HR1 and HR2 cultures. The production of quinic acid, kaempferol, and seven identified xanthones was increased in HR2. Moreover, HR2 showed a capability for de novo biosynthesis of two phenolic acids (3-p-coumaroylquinic acid and 3-feruloylquinic acid), three flavonol glycosides (kaempferol hexoside, hyperoside, and quercetin acetylglycoside), and five xanthones (tetrahydroxy-one-methoxyxanthone, 1,3,5-trihydroxy-6-methoxyxanthone, 1,3,5,6-tetrahydroxy-2-prenylxanthone, paxanthone, and banaxanthone E). On the other side, HR1 cultures were better producers of flavan-3-ols (catechin, epicatechin, and proanthocyanidin dimers) than HR2. This is the first comparative study on phenolic profile of H. perforatum HR cultures grown under dark and photoperiod conditions

Generation of flavor compounds by biotransformation of genetically modified hairy roots of Hypericum perforatum (L.) with basidiomycetes

Altogether, 14 basidiomycetes (12BAD, 95PCH, 9WCOC, 5PSA, 96BCI, 331SHIBD, 4MSC, 74HFA, 220MPS, 115PFLA, 111 ICO C, 16LED, 6TSU, and 61LYP) were grown on solid and in liquid media using hairy roots of genetically modified Hypericum perforatum (L.) as the only source of carbon and nitrogen. After the first screening by GC-MS/MS-O, two fungi (115PFLA and 61LYP) which resulted in the most pleasant complex natural flavor by biotransformation were selected for further analysis. Twenty-four new volatile compounds were produced, from which 21 were identified (ethyl hexanoate, ethyl octanoate, benzaldehyde, 2-undecanone, (E,E)-2,4-decadienal, 1-octen-3-one, (E)-2-nonenal, ethyl nonanoate, 2-heptenal, 1-methoxy-4-methylbenzene, 3-octanone, 1-decen-3-one, (E)-2-octenal, 1-octen-3-ol, β-linalool, ±trans-nerolidol, anisole, methyl benzoate, 2-pentylfuran, 1,3-dichloro-2-methoxybenzene, and 1-dodecanol). Thereof, 15 compounds were perceived at the ODP, from which 13 were identified. Compound identification was performed by comparison of Kovats indices (KI) and mass spectra to those of authentic reference compounds on a polar VF-WAXms column using headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS)

Phenolic Compounds Composition of Hypericum perforatum L. Wild-Growing Plants from the Republic of Macedonia

The aim of this study was to provide comprehensive understanding of phenolic compounds composition in roots (RO), non-flower shoots (NFS) and flower shoots (FS) of Hypericum perforatum wild-growing plants from the Republic of Macedonia. Identification of phenolic compounds in plant methanolic extracts was performed by HPLC-DAD/ESI-MS analysis. Chlorogenic acid and 3-p-coumaroylquinic acid were identified in NFS and FS, while 3-feruloylquinic acid was detected in RO and FS extracts. From the group of flavan- 3-ols, (epi)catechin and procyanidins were found in all tested samples, whereas catechin and B-type procyanidin dimer were confirmed in NFS and FS. Four flavonol glycosides (hyperoside, rutin, quercitrin and kaempferol 3-O-rutinoside) were identified in aerial parts. Guaijaverin and kaempferol 3-O-glucoside were exclusively found in NFS. Quercetin, amentoflavone and I3-II8 biapigenin as flavonoid aglycones were detected only in FS extracts. The NFS and FS extracts showed a capability for the accumulation of cyanidin 3-O-glycoside and cyanidin 3-O-rhamnoside, as well for hyperforin and adhyperforin. Naphthodianthrones were represented with pseudohypericin, hypericin and  protopseudohypericin  in  FS, while only hypericin was detected in NFS. Six xanthones, γ-mangostin, 5-O-methyl-2- deprenylrheediaxanthone B, garcinone C, 3,6-dihydroxy-1,5,7-trimethoxy-xanthone, cadensin G and cadensin C were exclusively confirmed in RO extracts. Padiaxanthone was detected in NFS, while dimethylmangiferin in FS extracts. The major finding of this study is the identification of novel xanthones in H. perforatum roots that could be potentially used as bioactive compounds in food and pharmaceutical industry

Effects of Polysaccharide Elicitors on Secondary Metabolite Production and Antioxidant Response in Hypericum perforatum L. Shoot Cultures

The effects of polysaccharide elicitors such as chitin, pectin, and dextran on the production of phenylpropanoids (phenolics and flavonoids) and naphtodianthrones (hypericin and pseudohypericin) in Hypericum perforatum shoot cultures were studied. Nonenzymatic antioxidant properties (NEAOP) and peroxidase (POD) activity were also observed in shoot extracts. The activities of phenylalanine ammonia lyase (PAL) and chalcone-flavanone isomerase (CHFI) were monitored to estimate channeling in phenylpropanoid/flavonoid pathways of elicited shoot cultures. A significant suppression of the production of total phenolics and flavonoids was observed in elicited shoots from day 14 to day 21 of postelicitation. This inhibition of phenylpropanoid production was probably due to the decrease in CHFI activity in elicited shoots. Pectin and dextran promoted accumulation of naphtodianthrones, particularly pseudohypericin, within 21 days of postelicitation. The enhanced accumulation of naphtodianthrones was positively correlated with an increase of PAL activity in elicited shoots. All tested elicitors induced NEAOP at day 7, while chitin and pectin showed increase in POD activity within the entire period of postelicitation. The POD activity was in significantly positive correlation with flavonoid and hypericin contents, suggesting a strong perturbation of the cell redox system and activation of defense responses in polysaccharide-elicited H. perforatum shoot cultures

Production of phenolic compounds, antioxidant and antimicrobial activities in hairy root and shoot cultures of Hypericum perforatum L.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties