Hypericum Androsaemum Research Articles

A new catalytic activity from tobacco converting 2‐coumaric acid to salicylic aldehyde

Salicylic acid (SA) mediates plant response to pathogen invasion, resulting in hypersensitive response and in the formation of systemic acquired resistance. It is well known that Nicotiana tabacum and other plants respond to Tobacco Mosaic Virus (TMV) infection by increasing the content of SA but the details of SA biosynthesis are still not fully understood. Generally, SA may originate directly from isochorismate (Arabidopsis thaliana), or its C6–C1 skeleton could be synthesized via the phenylpropanoid pathway by β‐oxidation of trans‐cinnamic acid (N. tabacum), 2‐coumaric acid (OCA) (Gaulteria procumbens, Lycopersicum esculentum) or by retro‐aldol reaction of trans‐cinnamoyl‐CoA (Hypericum androsaemum). We report here a novel putative enzyme activity from tobacco, salicylic aldehyde synthase (SAS), catalysing non‐oxidative formation of salicylic aldehyde (SALD) directly from OCA. This chain‐shortening activity is similar to that of 4‐hydroxybenzaldehyde synthase from Vanilla planifolia, Lithospermum erythrorhizon, Daucus carota, Solanum tuberosum and Polyporus hispidus but the enzyme differs in the kinetics of the reaction, substrate specificity and requirements for reducing cofactors. SAS activity is constitutively expressed in healthy tobacco leaves and doubles as a result of infection with TMV. Moreover, the product of SAS activity—SALD, applied exogenously on tobacco leaves, stimulates peroxidase activity and enhances resistance to consecutive infection with TMV. These observations could suggest a contribution of SAS and SALD to the response of tobacco to TMV infection.

Influence of extracellular calcium on flavonoid accumulation by cell cultures of Hypericum androsaemum

Hypericum androsaemum L., commonly known in Portugal as tutsan, is a plant species used in folk medicine for its diuretic and hepatoprotective properties [1]. The diverse flavonoids and phenolic acids present in the plant are supposed to be responsible for such biological activities.

Fertility and Inheritance of Variegated and Purple Foliage Across a Polyploid Series in Hypericum androsaemum L.

Inheritance of two mutant foliage types, variegated and purple, was investigated for diploid, triploid, and tetraploid tutsan (Hypericum androsaemum). The fertility of progeny was evaluated by pollen viability tests and reciprocal crosses with diploids, triploids, and tetraploids and germinative capacity of seeds from successful crosses. Segregation ratios were determined for diploid crosses in reciprocal di-hybrid F1, F2, BCP1, and BCP2 families and selfed F2s with the parental phenotypes. F2 tetraploids were derived from induced autotetraploid F1s. Triploid segregation ratios were determined for crosses between tetraploid F2s and diploid F1s. Diploid di-hybrid crosses fit the expected 9: 3: 3: 1 ratio for a single, simple recessive gene for both traits, with no evidence of linkage. A novel phenotype representing a combination of parental phenotypes was recovered. Data from backcrosses and selfing support the recessive model. Both traits behaved as expected at the triploid level; however, at the tetraploid level the number of variegated progeny increased, with segregation ratios falling between random chromosome and random chromatid assortment models. We propose the gene symbol var (variegated) and pl (purple leaf) for the variegated and purple genes, respectively. Triploid pollen stained moderately well (41%), but pollen germination was low (6%). Triploid plants were highly infertile, demonstrating extremely low male fertility and no measurable female fertility (no viable seed production). The present research demonstrates the feasibility of breeding simultaneously for ornamental traits and non-invasiveness.

Effect of calcium on enzyme activities and phenolic accumulation in Hypericum androsaemum cell cultures

Effect of calcium on enzyme activities and phenolic accumulation in Hypericum androsaemum cell cultures

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae.

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.

INHERITANCE OF VARIEGATED AND PURPLE FOLIAGE PHENOTYPES IN Hypericum androsaemum L.

Inheritance of two mutant foliage types (purple and mottled variegated) was investigated for diploid, triploid, and tetraploid tutsan (Hypericum androsaemum). Segregation ratios were determined for diploid crosses in reciprocal dihybrid F1 and F2, BC1P1, and BC1P2 families. F2 tetraploids were derived from autotetraploid F1s. Triploid segregation ratios were determined from crosses between autotetraploid F1s and diploid F1s. Diploid di-hybrid crosses fit the expected 9:3:3:1 ratio for a single, simple recessive gene for both traits, with no evidence of linkage between each trait. Data from backcross and triploid crosses generally supported this model. In tetraploid crosses we observed twice as many variegated phenotypes as predicted which was not explained by random chromosome or chromatid assortment. Inheritance of purple foliage did not deviate from random chromosome assortment at the tetraploid level.

The essential oil composition of Hypericum androsaemum L. leaves and flowers from Iran

AbstractThe composition of the essential oils obtained from the dried leaves and flowers of Hypericum androsaemum L. (Hypericaceae) was analysed by GC and GC–MS. The major constituents of the essential oil of the leaves were caryophyllene oxide (35.8%), ishwarane (30.5%) and humulene epoxide II (5.6%). The major constituents of the essential oil of the flowers were α‐guaiene (40.2%) and caryophyllene oxide (28.0%); 26 and 27 components were identified in the essential oils of the leaves and flowers, respectively. Copyright © 2004 John Wiley & Sons, Ltd.

Hypericum androsaemum infusion increases tert-butyl hydroperoxide-induced mice hepatotoxicity in vivo

Increasing evidence regarding free radical generating agents indicates that the sustained production of high levels of reactive oxygen species (ROS) can cause hepatotoxicity. Being a short chain analog of lipid peroxide, tert-butyl hydroperoxide ( t-BHP) is metabolized into free radical intermediates by cytochrome P450 in hepatocytes, which initiate lipid peroxidation, glutathione depletion and cell damage. The aim of the present study was to evaluate the putative protective effect of Hypericum androsaemum lyophilised infusion against t-BHP-induced mice hepatotoxicity in vivo, which has already been shown to be antioxidant in vitro. However, the results showed that the oral pretreatment with Hypericum androsaemum infusion (4, 20 and 100 mg/kg) for 4 days before a single intraperitoneal dose of t-BHP (1.8 mmol/kg) potentiated the t-BHP-induced hepatotoxicity. In fact, it was observed a potentiation in the depletion of total glutathione and reduced glutathione (GSH) contents and increase in oxidised glutathione (GSSG) level. Also the histopathological evaluation of the mice livers revealed that the infusion raised the incidence of liver lesions induced by t-BHP. These data do not corroborate any effect of Hypericum androsaemum infusion as hepatoprotector, but rather as a potentiator of hepatotoxicity in the present experimental conditions.

Variation of the essential oil content and composition in leaves from cultivated plants of Hypericum androsaemum L.

The amount and composition of the essential oil from leaves of Hypericum androsaemum L. cultivated in Arouca (Portugal) were determined in six samples harvested during 1 year at intervals of 2 months. The seasonally dependent essential oil content ranged from 0.7 mg/g biomass dry weight in September to 3.4 mg/g in February. The oil contained more than 80 compounds, 70 of which (constituting 88-93% of the total oil) were identified by GC and GC-MS. An approximation of the absolute quantification of each compound and compound class was performed using a GC method with an internal standard. The relative and the absolute content of each compound and compound class changed during the year. At the end of the winter and in the spring, the essential oil was dominated by sesquiterpene hydrocarbons and accumulated a high number of intermediate to long chain n-alkanes and 1-alkenes. In September, the essential oil contained the lowest levels of sesquiterpene hydrocarbons (43%) and the highest levels of 1-octene and 2-hexenal (38%). In February, the essential oil had the highest level of sesquiterpene hydrocarbons (73%) and the highest diversity of intermediate to long chain n-alkanes and 1-alkenes.

Protective activity of Hypericum androsaemum infusion against tert-butyl hydroperoxide-induced oxidative damage in isolated rat hepatocytes

Hypericum androsaemum L. (Guttiferae) is a medicinal plant with antioxidant activity. Increasing evidence regarding free radical generating agents suggests that hepatotoxic-related disorders may involve reactive oxygen species (ROS). The purpose of this study was to investigate the protective effect of Hypericum androsaemum infusion on isolated rat hepatocytes oxidative injury induced by tert-butyl hydroperoxide ( t-BHP). The results showed that pretreatment of the cells with this infusion (16, 62 and 250 μg/ml) prevented the leakage of lactate dehydrogenase (LDH) and lipid peroxidation caused by a 30-min treatment with t-BHP (1 mM). However, infusion-induced alterations on glutathione homeostasis were noticed, as it was observed by the increase in glutathione oxidised form (GSSG) and depletion in total glutathione levels, which indicates that plant-derived antioxidant extracts may not be considered a generalised way of treating pro-oxidant-related diseases.

Effect of plant eztracts and essential oils on root-knot nematode

The nematicidal activity of methanolic extracts (20 µg ml-1) from twenty Jordanian plant species against two species of root-knot nematodes in vitro was evaluated. Whole-plant extract of Hypericum androsaemum showed the highest activity (11% mortality) against Meloidogyne javanica after 24 h of incubation. However, leaf extract of Origanum syriacum also increased M. javanica mortality markedly a day later, reaching 59 and 82% after 48 and 72 h of exposure respectively. Against M. incognita the response of leaf extracts was somewhat different, with leaf extract of Artemisia herba alba the most effective causing 22, 51, 54% mortality after 24, 48 and 72 h of exposure respectively. With a tenfold concentration (200 µg ml-1) of those plant extracts thought to contain volatile oils, the secondstage juveniles (J2) mortality of both nematodes increased after 24 and 72 h of incubation. Nematicidal tests of some volatile oils that are active ingredients of the plants tested revealed that geraniol, thymol, and camphor were the most effective against M. javanica J2s, with 91, 60, 56% mortality respectively after 72 h of exposure. Cineole, menthol, and pinene were not effective against this nematode. Against M. incognita J2s, the most effective oil components were carvacol, thymol, and geraniol, with mortalities of 100, 90, and 74% respectively after 72 h of exposure. Cineole was the least effective against M. incognita.

Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site-directed mutagenesis of two polyketide synthases.

Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.

Essential oils from plants and in vitro shoots of Hypericum androsaemum L.

The essential oil yields obtained by hydrodistillation of the aerial parts of Hypericum androsaemum cultivated plants varied from 0.94 to 4.09 mg/g of biomass dry weight, depending of the harvest time. The respective analyses performed by gas chromatography and gas chromatography-mass spectrometry revealed more than 80 compounds, 72 of which were identified. Most of the compounds were sesquiterpene hydrocarbons, which, depending of the harvest time, corresponded to 43-78% of the total essential oil. The other compounds were distributed as monoterpene hydrocarbons, oxygen-containing sesquiterpenes, n-alkanes, 1-alkenes, and oxygen-containing monoterpenes, these being a minor group. In H. androsaemum in vitro shoots, sesquiterpene hydrocarbons represented >80% of the respective essential oil. Differences in the essential oil composition were found depending on the harvest time and origin, in vivo versus in vitro, of the plant material. The essential oil sampled in November was characterized by the highest levels of sesquiterpene hydrocarbons and a high number of n-alkanes and 1-alkenes, from C(18) to C(28), whereas that sampled in June of the following year showed the highest levels of n-nonane and 1-octene as well as monoterpene hydrocarbons, the second most representative group.

Variability in phenolic composition of hypericum Androsaemum

The methanolic extracts of fifteen Hypericum androsaemum samples, growing spontaneously or cultivated in Portugal, were analysed by HPLC/DAD, allowing the identification of 9 phenolic compounds and the detection of 6 phloroglucinols. Total amounts of phenolics found ranged from 11 to 39 g/kg, and the influence of some factors which may be responsible for this variation is discussed. The individual compounds were also quantified. Four different phenolic profiles were found concerning both qualitative and quantitative composition, indicating the possible existence of chemical polymorphism.

Antioxidant activity of Hypericum androsaemum infusion: scavenging activity against superoxide radical, hydroxyl radical and hypochlorous acid.

Hypericum androsaemum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Since polyphenolic compounds have high antioxidant potential, the ability of H. androsaemum infusion to act as a scavenger of reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid) was investigated. Superoxide radical was generated by the xanthine/xanthine oxidase and phenazine methosulphate/NADH systems. The infusion-mediated prevention of nitroblue tetrazolium reduction by the superoxide radical was used as the measured endpoint. Hydroxyl radical was generated by the Fe3+-EDTA/ascorbate Fenton system, and assayed by evaluating deoxyribose degradation using the thiobarbituric acid method. Hypochlorous acid scavenging activity was tested by measuring the inhibition of hypochlorous acid-induced 5-thio-2-nitrobenzoic acid oxidation to 5,5'-dithiobis(2-nitrobenzoic acid). The tested infusion mainly exhibited a potent scavenging effect on superoxide radicals (although a noncompetitive inhibitory effect on xanthine oxidase was also observed). The infusion also acted as a moderate scavenger of hydroxyl radicals and hypochlorous acid. A phytochemical study of the infusion was also undertaken, and nine phenolic compounds were identified.

Benzoic acid biosynthesis in cell cultures of Hypericum androsaemum.

Biosynthesis of benzoic acid from cinnamic acid has been studied in cell cultures of Hypericum androsaemum L. The mechanism underlying side-chain shortening is CoA-dependent and non-beta-oxidative. The enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone biosynthesis and general phenylpropanoid metabolism, respectively. Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase finally supplies benzoic acid. In cell cultures of H. androsaemum, benzoic acid is a precursor of xanthones, which accumulate during cell culture growth and after methyl jasmonate treatment. Both the constitutive and the induced accumulations of xanthones were preceded by increases in the activities of all benzoic acid biosynthetic enzymes. Similar changes in activity were observed for phenylalanine ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase and benzophenone synthase.

Cinnamic acid is a precursor of benzoic acids in cell cultures of Hypericum androsaemum L. but not in cell cultures of Centaurium erythraea RAFN.

Benzoic acids are precursors of xanthone biosynthesis which has been studied in cell cultures of Hypericum androsaemum (Hypericaceae) and Centaurium erythraea (Gentianaceae). In both cell cultures, methyl jasmonate induces the intracellular accumulation of a new xanthone. Under these inductive conditions, feeding experiments were performed with [U-14C]L-phenylalanine, [7-14C]benzoic acid and [7-14C]3-hydroxybenzoic acid. All three precursors were efficiently incorporated into the elicited xanthone in H. androsaemum, whereas 3-hydroxybenzoic acid was the only precursor to be incorporated into xanthones in C. erythraea. In addition, an appreciable increase in phenylalanine ammonia-lyase activity occurred only in methyl-jasmonate-treated cell cultures of H. androsaemum. Benzoic acids thus appear to be formed by different pathways in the two cell cultures studied. In H. androsaemum, benzoic acid is derived from cinnamic acid by side-chain degradation. In C. erythraea 3-hydroxybenzoic acid appears to originate directly from the shikimate pathway.

Xanthone 6-hydroxylase from cell cultures of Centaurium erythraea RAFN and Hypericum androsaemum L.

Xanthone 6-hydroxylase activity was detected in the microsomal fractions from two plant cell cultures. The enzyme from cultured cells of Centaurium erythraea (Gentianaceae) exhibited absolute specificity for 1,3,5-trihydroxyxanthone as substrate, whereas xanthone 6-hydroxylase from cell cultures of Hypericum androsaemum (Hypericaceae) preferred the isomeric 1,3,7-trihydroxyxanthone but used 1,3,5-trihydroxyxanthone also to a small extent. Both xanthones were regioselectively hydroxylated in position 6. The xanthone 6-hydroxylases are cytochrome P450 monooxygenases, as shown by their dependence on NADPH and molecular oxygen and their inhibition by carbon monoxide and typical P450 inhibitors. In both cell cultures, xanthone accumulation was preceded by an increase in xanthone 6-hydroxylase activity.

Xanthone biosynthesis and accumulation in calli and suspended cells of Hypericum androsaemum

Calli and suspended cells of Hypericum androsaemum accumulated high levels of 1,3,5,6 and 1,3,6,7 oxygenated xanthones. The major compounds include simple oxygenated xanthones or derivatives with prenyl, pyran or methoxyl groups, four of them being new natural compounds. A hypothetical biosynthetic scheme is proposed based on the isolated compounds and statistical analysis. Xanthone accumulation was influenced greatly by medium factors, namely hormone supplementation. Calli grown with 4.5 μM α-naphtaleneacetic acid (NAA)+2.3 μM kinetin (KIN) had the highest specific xanthone production (1.3% biomass dry weight (DW)), whereas suspended cells grown in similar medium accumulated a lower amount (0.87% DW). Calli displayed a negative linear relationship between total xanthone accumulation and NAA concentration, in the range of 4.5–22.5 μM. However, in this range the xanthone 1,3,6,7/1,3,5,6 ratio and the biomass production showed a positive linear relationship with NAA concentration. Substitution of 4.5 μM of NAA by the same molar amount of 2,4-dichlorophenoxyacetic acid (2,4-D), in the presence of 2.3 μM of KIN, caused a decrease in xanthone accumulation in calli. The use of N 6-benzyladenine (BA) instead of KIN reduced xanthone production, independently of the auxin used. This effect was attenuated when both hormones were present.

THE DEVELOPMENT AND EVALUATION OF AN HPLC-DAD METHOD FOR THE ANALYSIS OF THE PHENOLIC FRACTIONS FROM IN VIVO AND IN VITRO BIOMASS OF HYPERICUM SPECIES

A new HPLC-DAD method for separation and identification of the main phenolics present in in vivo and in vitro biomass of Hypericum perforatum and Hypericum androsaemum has been developed. This method accomplished the direct identification of 22 compounds including flavonoids, hypericins, phloroglucinols, and phenolic acids. The HPLC profiles obtained in the course of this work clearly evidenced a distinct phenolic production between in vivo and in vitro biomass. For example calli and suspended cells produced mainly xanthones while in in vivo plants of both species these compounds were not detected. Some luteolin flavone type compounds were identified in calli and suspended cells of H. perforatum while quercetin was found in suspended cells of H. androsaemum. Flavonoids, namely those related to quercetin, were the major metabolites in methanolic extracts from in vivo plants. Hypericins were detected in in vivo plants and in in vitro shoots of H. perforatum but not in calli or suspended cells of the same species. Aurones, produced by in vitro cultures were not detected in in vivo plants of H. perforatum.