Protopseudohypericin

Interspecific variation in localization of hypericins and phloroglucinols in the genus Hypericum as revealed by desorption electrospray ionization mass spectrometry imaging.[Pubmed:26822391]

Physiol Plant. 2016 May;157(1):2-12.

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.

LC-MS and LC-PDA analysis of Hypericum empetrifolium and Hypericum sinaicum.[Pubmed:19791496]

Z Naturforsch C. 2009 Jul-Aug;64(7-8):476-82.

Within the framework of our continuous efforts to explore Hypericum species from Jordan, we report the analysis of the major active metabolites, naphthodianthrones and phloroglucinols, in the methanolic extracts of two under-explored Hypericum species; H. empetrifolium Willd. and H. sinaicum Hochst. & Steud. ex Boiss., using LC-(+,-)-ESI-MS (TIC and SIM) and LC-UV/Vis spectroscopy. Based on their LC-UV/Vis profiles, retention times and (+,-)-ESI-MS (TIC and SIM) spectral data, hypericin, protohypericin and pseudohypericin were identified in both of the investigated species. In addition adhyperfirin was only detected in H. empetrifolium, while hyperforin and Protopseudohypericin were only detected in H. sinaicum. This is the first report documenting the presence of hypericin, protohypericin, pseudohypericin, Protopseudohypericin, and hyperforin in H. sinaicum, and adhyperfirin in H. empetrifolium.

Study of active naphtodianthrone St John's Wort compounds by electrospray ionization Fourier transform ion cyclotron resonance and multi-stage mass spectrometry in sustained off-resonance irradiation collision-induced dissociation and infrared multiphoton dissociation modes.[Pubmed:19224526]

Rapid Commun Mass Spectrom. 2009 Mar;23(6):885-98.

Five well-known active naphtodianthrone constituents of Hypericum perforatum (St John's Wort) extracts have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) and ESI-FTICRMSn. The studied compounds were hypericin, pseudohypericin, protohypericin, Protopseudohypericin (biosynthetic precursors of the two former compounds, respectively) and isopseudohypericin (alkaline degradation product of pseudohypericin). Dissociation mass spectrometry measurements performed on the [M-H]- ion presented a variable efficiency as a function of the used activation mode. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) only led to a restricted number of fragment ions. In contrast, IRMPD ensured the detection of numerous product ions. Ions detected in ESI-FTICRMS and ESI-FTICRMSn experiments were measured with a very high mass accuracy (typically mass error is lower than 0.5 mDa at m/z close to 500) that allowed unambiguous formulae to be assigned to each signal observed in a mass spectrum. In spite of similar structures, specific fragmentation patterns were observed for the different compounds investigated. This study may be useful in the future to characterize in natural extracts these compounds (or derivatives of these compounds) by liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments by considering the MS/MS transitions highlighted in this paper.

Influence of the habitat altitude on the (proto)hypericin and (proto)pseudohypericin levels of hypericum plants from Crete.[Pubmed:18671196]

Planta Med. 2008 Oct;74(12):1496-503.

Environmental factors are known to influence strongly the accumulation of secondary metabolites in plant tissues. In a previous paper, we studied the contents of (pseudo)hypericin and its immediate precursors in wild populations of various HYPERICUM species on the island of Crete, Greece, in dependence on their developmental stage. In this study, we investigated the effect of the habitat altitude on the total hypericins content of the plants, which is defined as the sum of protohypericin, hypericin, Protopseudohypericin and pseudohypericin. Taking into account our previous finding that the highest accumulation is found during the flowering period in June, we collected the aerial parts of spontaneously growing H. PERFORATUM L. , H. TRIQUENTRIFOLIUM Turra , H. EMPETRIFOLIUM Willd. and H. PERFOLIATUM L. during that time frame at elevations between 100 and 600 m above sea level, however, bearing in mind the time lag in development with increasing altitude. HPLC analysis of the plant material, separated again into a flowers and a leaves/petioles fraction, revealed great differences in the total hypericin content in dependence on the altitude of the habitat. Specifically, a clear trend was revealed, showing an increase of the total hypericin content with increasing altitude. However, no changes could be observed in the ratio of hypericin to protohypericin and in that of pseudohypericin to Protopseudohypericin. The habitats of the employed plants were again randomly distributed all over Crete. It is proposed that higher light intensities accompanied by enhanced UV-B radiation and lower air temperature might be responsible for the increasing levels of total hypericins with increasing altitude

Influence of the developmental stage on the (proto)-hypericin and (proto)pseudohypericin levels of Hypericum plants from Crete.[Pubmed:17893828]

Planta Med. 2007 Oct;73(12):1309-15.

The contents of (pseudo)hypericin and their immediate precursors were studied in wild populations of various Hypericum species on the island of Crete, Greece. Therefore, the aerial parts of wild grown H. perforatum, H. triquentrifolium, H. empetrifolium and H. perfoliatum shoots were collected throughout the island and the quantitative variations in (proto)hypericin and (proto)pseudohypericin examined. The plant material was harvested at different stages of the life cycle of the species and the contents in the above-mentioned compounds analyzed discriminating between flowers/fruits and leaves/petioles. HPLC analysis of hypericin, pseudohypericin and their immediate precursors, protohypericin and Protopseudohypericin, revealed great differences in the contents of the compounds in dependence on the developmental stage of the plants. In all examined species the highest concentrations of hypericin were found during blossoming whereas the lowest concentrations were present during ripening of the fruits. H. perforatum and H. triquentrifolium show much higher hypericin levels in flowers/fruits compared to leaves/petioles, whereas the species H. empetrifolium and H. perfoliatum show similar concentrations of total hypericins in both flowers/fruits and leaves/petioles. In the different species the levels of (proto)hypericin and (proto)pseudohypericin varied, but in almost all samples from flowers/fruits and leaves/petioles the ratio of (proto)hypericin to (proto)pseudohypericin was higher than one. When the total amount of hypericins per entire aerial part of a plant was calculated for all developmental stages, we found that H. perforatum contained the highest amount of hypericin. This in combination with the comparatively high concentration of hypericins in flowers/fruits and in leaves/petioles in this species, as well as the high ratio of (proto)hypericin to (proto)pseudohypericin, especially during the developmental stage of blossoming, encourages us to think about the possibility of cultivating Hypericum perforatum in Crete as a medicinal plant in the future.

Simultaneous determination of total hypericin and hyperforin in St. John's wort extracts by HPLC with electrochemical detection.[Pubmed:17500362]

Phytochem Anal. 2007 May-Jun;18(3):204-8.

An analytical procedure was developed for the simultaneous determination of total hypericin (Protopseudohypericin, pseudohypericin, protohypericin and hypericin) and hyperforin in Hypericum perforatum (St. John's wort) extracts and its preparations. The determination of total hypericin and hyperforin in one step was achieved by exposing the samples to artificial daylight in amber glass vials. This procedure allows both the photoconversion of the protoforms into the appropriate hypericins and the protection of the photosensitive hyperforin. For quantification, an HPLC method with electrochemical detection was applied. As an example of the application of the principle, two preparations containing St. John's wort were assayed.

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS.[Pubmed:17196625]

Phytochemistry. 2007 Feb;68(3):383-93.

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, Protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.

A high-performance liquid chromatography with electrochemical detection for the determination of total hypericin in extracts of St. John's Wort.[Pubmed:16749423]

Phytochem Anal. 2006 May-Jun;17(3):162-7.

An HPLC method for the quantitation of hypericin using a new and sensitive amperometric detection is presented. Hypericin was eluted isocratically using a mobile phase consisting of ammonium acetate, methanol and acetonitrile. The oxidation was carried out with a glassy carbon electrode at a potential of + 1.1 V vs. an Ag-AgCl-KCl reference electrode. Under the conditions described, hypericin was separated at a retention time (Rt) of 12 min. Linearity was obtained over the range 0.035-1.30 microg/mL (r = 0.9994). The limit of detection was determined to be 0.010 ng on-column for hypericin. The method was applied to the determination of total hypericin (hypericin, pseudohypericin, protohypericin and Protopseudohypericin) in extracts of St. John's wort using hypericin as an external standard. The protoforms were converted into hypericin and pseudohypericin by subjecting the sample to artificial light prior to chromatographic analysis. For the evaluation of total hypericin, the peak areas of pseudohypericin (Rt 3.7 min) and hypericin (Rt 12.0 min) were combined. The relative standard deviation in analysing samples containing Hypericum ranged from 2.5 to 5.4%.

Induction of hypericins in Hypericum perforatum in response to chromium.[Pubmed:16554124]

Fitoterapia. 2006 Apr;77(3):164-70.

Seedlings of Hypericum perforatum were grown with 0.01 and 0.1 mM of chromium added to the nutrient media. A treatment with 0.01 mM Cr(VI) for seven days resulted in an increased production of Protopseudohypericin (+135%), hypericin (+38%) and pseudohypericin (+5%). Treatment with 0.1 mM Cr(VI) for two days also caused an increase of Protopseudohypericin (+167%), hypericin (25%) and pseudohypericin (+5%). The greatest effect of chromium treatment was observed at a concentration of 0.1 mM for seven days: Protopseudohypericin increased +404% and pseudohypericin to +379%. Hypericin was not affected by this treatment.

Simultaneous determination of protopseudohypericin, pseudohypericin, protohypericin, and hypericin without light exposure.[Pubmed:16526439]

J AOAC Int. 2005 Nov-Dec;88(6):1607-12.

St. John's wort products are commonly standardized to total naphthodianthrones and hyperforin. Determination of these marker compounds is complicated because of the photochemistry of the naphthodianthrones pseudohypericin and hypericin and the instability of hyperforin in solution. Protopseudohypericin and protohypericin have been identified as naturally occurring naphthodianthrones and, when exposed to light, they are converted into pseudohypericin and hypericin, respectively. However, exposure to light and the resulting naphthodianthrone free-radical reactions oxidize hyperforin. A mathematical relationship between the response of the proto compound and the resulting naphthodianthrone can be established by comparing the analytical response of the proto compound in a solution protected from light with the increase in the analytical response of naphthodianthrone in the same solution after exposure to light. By mathematically converting the proto compounds to their respective products, exposure to light can be avoided while still including proto compounds in a single assay. The method presented here details the reporting of all significant naphthodianthrones, including Protopseudohypericin and protohypericin, without exposure to light. This approach includes the benefits of improved naphthodianthrone precision and protection of hyperforin from oxidation.

Liquid chromatography-mass spectrometry studies of St. John's wort methanol extraction: active constituents and their transformation.[Pubmed:15708671]

J Pharm Biomed Anal. 2005 Feb 23;37(2):303-12.

The influence of light and solution pH on the stability behavior of phloroglucinols (hyperforin and adhyperforin) and naphthodianthrones (hypericin, pseudohypericin, protohypericin and Protopseudohypericin) extracted with methanol from St. John's wort powder (Hypericum perforatum L.) were studied using liquid chromatography-mass spectrometry (LC-MS). When exposed to light, hyperforin and adhyperforin in this extract solution degraded rapidly, particularly at pH 7, where within 12h complete transformation was observed. Contrastingly, when protected from light, the solutions regardless of pH, underwent minimal transformation after 36h. Under light and neutral pH conditions, phloroglucinols and naphthodianthrones had different stability behaviors, which were attributed to the different oxidation mechanisms. Four experiments performed on naphthodianthrones exhibited serious transformation at acidic pHs. One hyperforin transformation product was studied using LC-MS. The molecular structure was proposed on the basis of ion fragmentation patterns obtained from MS/MS studies.

Fast high-performance liquid chromatographic analysis of naphthodianthrones and phloroglucinols from Hypericum perforatum extracts.[Pubmed:14516003]

Phytochem Anal. 2003 Sep-Oct;14(5):306-9.

Hypericum perforatum L. (St. John's Wort) has been used in modern medicine for treatments of depression and neuralgic disorders. An HPLC method with photodiode array detection for the rapid determination of the major active compounds, naphthodianthrones and phloroglucinols, has been developed. The method permits the determination of hypericin, protohypericin, pseudohypericin, Protopseudohypericin, hyperforin and adhyperforin in an extract in less than 5 min. Good linearity over the range 0.5-200 microg/mL for hyperforin and 0.02-100 microg/mL for hypericin was observed. Intra-assay accuracy and precision varied from 0.1 to 17% within these ranges. Lower levels of quantitative determination were 2 microg/mL for hyperforin and 0.5 microg/mL for hypericin, while detection limits were 0.1 and 0.02 microg/mL, respectively.

Use of an on-line, precolumn photochemical reactor in high-performance liquid chromatography of naphthodianthrones in Hypericum perforatum preparations.[Pubmed:12613810]

J Chromatogr A. 2003 Feb 14;987(1-2):181-7.

A method has been developed for the determination of naphthodianthrones in Hypericum perforatum L. extracts and phytopharmaceutical preparations by high-performance liquid chromatography combined with on-line, precolumn photochemical conversion followed by photodiode-array detection. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions. An on-line, precolumn photochemical reactor equipped with a knitted PTFE reaction coil around a visible light source was used in order to transform the light sensitive naphthodianthrones, protohypericin and Protopseudohypericin, very easily into the non-protoforms, hypericin and pseudohypericin, respectively. Two UV chromatograms (photochemical reactor "on" and "off") were compared and were quite useful in characterizing the sample. Validation studies demonstrated that this HPLC method is simple, rapid, reliable and reproducible. The time-consumptive manual irradiation of the samples is omitted by this automated on-line irradiation step. The developed method was successfully applied to the quality control of Hypericum perforatum L. extracts and its phytopharmaceutical preparations.

Improved procedure for the quality control of Hypericum perforatum L.[Pubmed:11793812]

Phytochem Anal. 2001 Nov-Dec;12(6):355-62.

A new, fast and reliable procedure for the quantification of the major compounds of Hypericum perforatum L. has been developed. Four naphthodianthrones (Protopseudohypericin, pseudohypericin, protohypericin, hypericin) and two phloroglucinols (hyperforin, adhyperforin) were assayed by HPLC using a short (17 min) linear gradient, with hypericin and hyperforin as external standards. Extraction of dried plant material with methanol in the dark at room temperature for 2 h led to a complete recovery of phloroglucinols but only a partial recovery of the naphthodianthrone derivatives. Treatment of plant material with water:ethanol (40:60, v/v) in a water bath shaker at 80 degrees C led to the total extraction of hypericins, but a 10% loss of total hyperforins was also observed. The two extraction methods, applied successively to the same sample, allowed the complete extraction of all compounds of interest. A 5 min exposure of the crude extract of H. perforatum to sunlight (1 E/m2) induced a 96% loss of hyperforins, whereas the dry plant material lost only 20% of hyperforins after 2 h exposure to sunlight (24 E/m2).

Separation of hypericins and hyperforins in extracts of Hypericum perforatum L. using non-aqueous capillary electrophoresis with reversed electro-osmotic flow.[Pubmed:11682223]

J Pharm Biomed Anal. 2002 Jan 1;27(1-2):167-76.

The separation of the lipophilic compounds in extracts of Hypericum perforatum L. is demonstrated in a non-aqueous capillary electrophoresis system with reversed electro-osmotic flow. Solvent mixtures of methanol, dimethylsulfoxide and N-methylformamide were used for the electrophoresis media, with addition of ammonium acetate and sodium acetate as electrolytes. The flow was reversed by the addition of the polycation hexadimethrine bromide, and thus negative voltage was applied. The method shows baseline separation between the four hypericins-Protopseudohypericin, pseudohypericin, protohypericin and hypericin-whereas total baseline separation between the two hyperforins-hyperforin and adhyperforin-was not achieved. Using a fused-silica capillary (30 cm x 25 microm ID) and a voltage of -25 kV the analysis time of the hypericins and hyperforins was obtainable within 3 min. Application of the method with a fused-silica capillary of a larger internal diameter (48.5 cm x 50 microm ID) and a voltage of -20 kV resulted in analysis times of 8 min, but also lower limits of detection. The maximal attainable voltage was applied in both cases. Simultaneous separation of the flavonoids-although less efficient-may also be achieved. The technique of non-aqueous capillary electrophoresis with reversed electro-osmotic flow provides a very fast technique to evaluate the composition of hypericins and hyperforins in extracts of Hypericum perforatum L.