The aim of this study was to evaluate the non-enzymatic and enzymatic antioxidant response of fifteen Hypericum perforatum L. dark-grown (HR1 A-HR1 O) and photoperiod-exposed (HR2 A-HR2 O) hairy root ...clones. Dark-grown HR1 clones were characterized with high biomass accumulation and up-regulated phenylpropanoid metabolism through phenylalanine ammonia lyase (PAL)-mediated production of flavonoids, flavan-3-ols and anthocyanins. These groups of phenolics showed superior hydrogen-donating capability and significantly contributed to the antioxidant capacity of dark-grown HR1 clones. Photoperiod-exposed HR2 clones showed green coloration with shoot regenerative potential and reduced biomass accumulation. Photoperiod exposition improved the production of hypericins, as well as ferrous chelating properties and lipid peroxidation inhibition activity in HR2 cultures. Furthermore, HR2 clones were represented with enhanced activity of antioxidant enzymes (guaiacol peroxidase, catalase and superoxide dismutase) that resulted in suppression of oxidative stress markers (hydrogen peroxide, superoxide anion and malondialdehyde). These observations revealed the involvement of an efficient antioxidant defense system in the adaptive response of HR to photooxidative stress. Altogether, photoperiod-exposed H. perforatum HR2 clones were considered as a promising alternative for further scale-up production of naphthodianthrones that could be used in the pharmaceutical industry.
Investigations have been made to study the production of phenolic compounds (total phenolics, flavonoids and phenylpropanoids) and total antioxidant capacity in 27 Macedonian traditional medicinal ...plants to improve its potential as a source of natural antioxidants. Antioxidant potential of plant extracts was analyzed by five different assays: cupric reducing antioxidant capacity (CUPRAC), phosphomolybdenum method (PM), reducing power (RP), 2,2-diphenyl-1-picrylhydrazyl (DPPH·) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS·⁺) radical scavenging activity. Origanum vulgare extract consistently exhibited the highest content of phenolic compounds and the strongest antioxidant capacity based on the tests performed, and can be proposed as a promising source of natural antioxidants. Melissa officinalis and Salvia ringens were also identified as valuable sources of antioxidant compounds. A positive linear correlation between antioxidant activity and total phenolics, flavonoids and phenylpropanoids indicates that these compounds are likely to be the main antioxidants contributing to the observed activities of evaluated plants. These findings suggest that the medicinal plants studied in this paper are good sources of bioactive compounds for the food and pharmaceutical industries.
Hypericum perforatum L. cell suspensions were evaluated for their growth, antioxidant activity, phenolic compound productions and xanthone profile after elicitation with Agrobacterium tumefaciens and ...Agrobacterium rhizogenes. Secondary metabolite production in H. perforatum elicited cells was enhanced without loss of biomass. A. tumefaciens elicited cells showed largely increased amounts of total phenolics and flavonoids, whereas total flavanol contents were slightly enhanced. The production of phenolic compounds was clearly less marked in A. rhizogenes treated cells. Antioxidant activity of elicited cells was remarkably elevated throughout the post-elicitation period. A significant correlation between antioxidant activity and phenolic production in elicited cells was found. Xanthone profile of H. perforatum cells was notably changed after bacterial elicitation. H. perforatum elicited cells yielded about 17-fold higher levels of xanthones compared to control cells. Among the twenty-one detected xanthones, five of them identified as 1,3,5,6-tetrahydroxyxanthone C-prenyl isomer, toxyloxanthone, 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone, 1,3,6-trihydroxy-7-methoxy-8-prenyl xanthone and 1,3,6,7-tetrahydroxyxanthone 2-prenyl xanthone were de novo synthesized in elicited cells. Altogether, these results indicated that H. perforatum cells elicited with Agrobacterium represent promising experimental system for enhanced production of xanthones.
Hypericum perforatum
hairy root clones (HR A–HR O) transformed with
Agrobacterium rhizogenes
A4 were evaluated for growth characteristics, phenolic compounds production, antioxidant/radical ...scavenging activity, antioxidant enzymes, and oxidative stress markers. The screening of growth characteristics revealed that HR clones were characterized with higher biomass accumulation, root elongation, and lateral branching in comparison to non-transformed roots. Significant increase of phenolics production in HR clones was related to phenylalanine ammonia lyase activity indicating an up-regulation of phenylpropanoid/flavonoid metabolism. Positive correlation of phenolics, flavonoids, and tannins with antioxidant assays indicated that these secondary metabolites significantly contributed to the antioxidant/radical scavenging properties of HR cultures. Regarding the enzymatic antioxidant state, an enhancement of superoxide dismutase activity in HR lines coincided with decrease in O
2
•−
production rate, while ascorbate peroxidase and catalase greatly contributed to the reduction of H
2
O
2
levels. The substantial accumulation of malondialdehyde in HR clones indicated an efficiency of antioxidant enzymes to reduce O
2
•−
and H
2
O
2
at levels that are not deleterious for membrane lipids. These results confirmed the involvement of an efficient antioxidant defense system in the response adjustment of
H. perforatum
HR cultures to transformation process. Two superior clones denoted as HR B and HR F were selected as fast-growing and high biomass yielding lines with up-regulated phenolic compounds’ production, antioxidant, and radical scavenging activity. Altogether, the best performing
H. perforatum
HR clones could be proposed as a prospective biotechnological system for scale-up production of antioxidant phenolic compounds.
Fifteen transgenic shoot clones (TS A-TS O) regenerated from Hypericum perforatum hairy roots were evaluated for growth characteristics, phenylpropanoid, and naphthodianthrone production, as well as ...non-enzymatic and enzymatic antioxidant status. Transgenic shoots displayed higher biomass production and non-altered phenotypes compared to control shoots. Phenylpropanoid production in TS was not related to phenylalanine ammonia lyase activity indicating that alternative enzymes could be involved in the regulation of phenylpropanoid/flavonoid biosynthesis. The positive correlation between phenylpropanoids (phenolic acids, flavonoids, flavan-3-ols, tannins) and non-enzymatic antioxidant assays suggested that these compounds are the main contributors to the redox state and radical scavenging properties of TS clones. More importantly, enhanced naphthodianthrone production (hypericin, pseudohypericin and protopseudohypericin) in TS was associated with the number of leaf dark glands. Concerning the enzymatic antioxidant status of TS, guaiacol peroxidase was shown as the main enzyme that turns down H2O2 levels, while the upregulation of superoxide dismutase coincided with decreased O2•− production. The efficiency of antioxidant enzymes for O2•− and H2O2 neutralization decreased the level of MDA and consequently delayed lipid peroxidation in TS clones. Altogether, H. perforatum transgenic shoots could be proposed as a prospective biotechnological system for scale-up production of naphthodianthrones and phenylpropanoids with antioxidant properties.
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.
Hypericum perforatum callus cultures exhibited qualitatively and quantitatively different phenolic profile compared to wild-growing plants (WGP). This study revealed the presence of phenolic acids, ...flavonoid glycosides and aglycones, flavan-3-ols and naphtodianthrones in callus and WGP extracts. Phloroglucinols were detected only in WGP, while xanthones were exclusively found in callus cultures. Among the caffeoyl-, coumaroyl- and feruloyl-quinic acid derivatives identified in both extracts, 3-feruloylquinic acid was identified only in callus cultures. Flavonoid glycosides were represented with quercetin and kaempferol derivatives in both extracts, whereas calli produced a new flavone of luteolin type. Callus cultures showed superior potential for the production of quercetin 6-C-glucoside, hyperoside and kaempferol 3-O-glucoside compared to WGP. Flavonoid aglycones, quercetin and kaempferol were found in both extracts, while I3,II8-biapigenin was accumulated only in WGP. Catechin and procyanidin dimers were abundant in WGP, while epicatechin was dominant flavan-3-ol in callus cultures. With respect to naphtodianthrones, undifferentiated calli were able to produce only pseudohypericin, but in minor amounts compared to WGP. Among the eighteen detected xanthones, mangiferin, mangiferin C-prenyl hexoside and 1,3,6,7-tetrahydroxyxanthone were found as the major compounds in callus cultures. Four xanthones identified as roeperanone, 1,3,7-trihydroxy-2-(2-hydroxy-3-methyl-3-butenyl)-xanthone, trihydroxy-1-methoxy C-prenyl xanthone and 1,5-dihydroxy-2-methoxyxanthone were shown for the first time in H. perforatum callus extracts. Altogether, these results indicated that H. perforatum callus cultures represent a promising experimental system for the production of novel xanthone derivatives and other bioactive metabolites of commercial importance.
Hypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of ...secondary metabolites in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones.
Hypericum perforatum
cell suspensions were evaluated for their growth, phenylpropanoid and naphtodianthrone productions, and antioxidant activity after treatments with polysaccharide elicitors chitin ...(CHI), pectin (PEC) and dextran (DEX). Polysaccharide elicitation of cell cultures showed a considerable improving effect on phenylpropanoid production (phenolics, flavonoids, flavanols and anthocyanins) during the post-elicitation period. Even that enhanced phenylpropanoid production was related to growth suppression of CHI elicited cells, PEC and DEX were presented as effective elicitors without loss of cell biomass. Phenylpropanoid accumulation in elicited cells was correlated to markedly higher enzyme activities of phenylalanine ammonia lyase and chalcone-flavanone isomerase. Cell cultures were found to respond rapidly towards the applied elicitors through an early stimulation of both naphtodiantrones, hypericin (HYP) and pseudohypericin (PHYP). The CHI was proposed as a much more prominent elicitor in stimulation of HYP and PHYP levels in cell suspensions. All tested polysaccharide elicitors stimulated non-enzymatic antioxidant properties in cell extracts indicating the potential role of phenolic compounds as effective antioxidants. With regards to the antioxidant enzymes, an up-regulation of peroxidase and catalase activities was observed in PEC and DEX elicited cells, whereas CHI did not give any stimulatory effect on enzymatic activities. These findings suggest the involvement of an efficient antioxidant defense system in the adaptive response of cells to polysaccharide elicitation. Altogether, these results indicated that
H. perforatum
cells elicited with polysaccharides represent a promising experimental system for enhanced production of phenylpropanoids and naphtodianthrones.
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