Characterization of Constituents with Potential Anti-Inflammatory Activity in Chinese Lonicera Species by UHPLC-HRMS Based Metabolite Profiling.

This study centered on detecting potentially anti-inflammatory active constituents in ethanolic extracts of Chinese Lonicera species by taking an UHPLC-HRMS-based metabolite profiling approach. Extracts from eight different Lonicera species were subjected to both UHPLC-HRMS analysis and to pharmacological testing in three different cellular inflammation-related assays. Compounds exhibiting high correlations in orthogonal projections to latent structures discriminant analysis (OPLS-DA) of pharmacological and MS data served as potentially activity-related candidates. Of these candidates, 65 were tentatively or unambiguously annotated. 7-Hydroxy-5,3',4',5'-tetramethoxyflavone and three bioflavonoids, as well as three C32- and one C34-acetylated polyhydroxy fatty acid, were isolated from Lonicera hypoglauca leaves for the first time, and their structures were fully or partially elucidated. Of the potentially active candidate compounds, 15 were subsequently subjected to pharmacological testing. Their activities could be experimentally verified in part, emphasizing the relevance of Lonicera species as a source of anti-inflammatory active constituents. However, some compounds also impaired the cell viability. Overall, the approach was found useful to narrow down the number of potentially bioactive constituents in the complex extracts investigated. In the future, the application of more refined concepts, such as extract prefractionation combined with bio-chemometrics, may help to further enhance the reliability of candidate selection.

Allanblackia Oil: Phytochemistry and Use as a Functional Food.

The consumption and commercial exploitation of Allanblackia (Clusiaceae) seed oils is of current interest. The favorable physicochemical characteristics of Allanblackia oil (solid at room temperature; high stearic acid content) lend food products that contain it (i.e., vegetable-based dairy products, ice cream, spreads) health advantages over others that contain higher levels of lauric, myristic, and/or palmitic acids, which can increase blood cholesterol levels. Such considerations are important for individuals prone to cardiovascular disease or with hypercholesterolemia. Domestication projects of several Allanblackia species in tropical Africa are underway, but wildcrafting of fruits to meet the seed demand still occurs. Proper species authentication is important, since only authenticated oil can be deemed safe for human consumption. The chemical constituency of Allanblackia seed oils, and potential roles of these phytochemicals in preventive strategies (e.g., as part of a healthy diet) and as pharmacological agents used to treat chronic disease were examined in this review. The primary and secondary metabolite constituency of the seed oils of nearly all Allanblackia species is still poorly known. The presence, identity, and quantity of potentially bioactive secondary metabolites in the seed oils, and pharmacological testing of isolated compounds were identified as important directions for future research.

Aldose reductase inhibition of a saponin-rich fraction and new furostanol saponin derivatives from Balanites aegyptiaca.

BACKGROUND: Balanites aegyptiaca Del. (Zygophyllaceae) fruits are used to treat hyperglycemia in Egyptian folk medicine and are sold by herbalists in the Egyptian open market for this purpose. Nevertheless, the fruits have not yet been incorporated into pharmaceutical dosage forms. The identity of the bioactive compounds and their possible mechanisms of action were not well understood until now. PURPOSE: Aldose reductase inhibitors are considered vital therapeutic and preventive agents to address complications caused by hyperglycemia. The present study was carried out to identify the primary compounds responsible for the aldose reductase inhibitory activity of Balanites aegyptiaca fruits. STUDY DESIGN: The 70% ethanolic extract of Balanites aegyptiaca fruit mesocarp and its fractions were screened for inhibition of the aldose reductase enzyme. Bio-guided fractionation of the active butanol fraction was performed and the primary compounds present in the saponin-rich fraction (D), which were responsible for the inhibitory activity, were characterized. HPLC chromatographic profiles were established for the different fractions, using the isolated compounds as biomarkers. METHODS: Aldose reductase inhibition was tested in vitro on rat liver homogenate. The butanol fraction of the 70% ethanolic extract was fractionated using vacuum liquid chromatography (VLC, RP-18 column). The most active sub-fraction D, which was eluted with 75% methanol, was subjected to preparative HPLC to isolate the bioactive compounds. RESULTS: The butanol fraction displayed inhibitory activity against the aldose reductase enzyme (IC50 = 55.0 ± 6 µg/ml). Sub-fraction D exhibited the highest inhibitory activity (IC50 = 12.8 ± 1 µg/ml). Five new steroidal saponin derivatives were isolated from this fraction. The isolated compounds were identified as compound 1a/b, a 7:3 mixture of the 25R:25S epimers of 26-O-ß-D-glucopyranosyl-furost-5-ene-3,22,26-triol 3-O-[α-L-rhamnopyranosyl-(1→3)- ß-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside; compound 2, 26-O-ß-D-glucopyranosyl-(25R)-furost-5-ene-3,22,26-triol 3-O-[ ß-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside; compound 3, 26-O-ß-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[α-L-rhamnopyranosyl-(1→3)- ß-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside; compound 4, 26-O-ß-D-glucopyranosyl-(25R)-furost-5,20-diene-3,26-diol 3-O-[ ß-D-glucopyranosyl-(1→2)]- α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranoside; and compound 5, which is the 25S epimer of compound 4, by using various spectroscopic methods [MS,1D and 2D NMR (HSQC, HMBC, DQF-COSY, HSQC-TOCSY)]. Compounds 1a/b, 2, 3, 4, 5 exhibited highly significant aldose reductase inhibitory activities (IC50 values were 1.9 ± 0.2, 1.3 ± 0.5, 5.6 ± 0.2, 5.1 ± 0.4, 5.1 ± 0.6 µM, respectively) as compared to the activity of the reference standard quercetin (IC50 = 6.6 ± 0.3 µM). CONCLUSION: The aldose reductase inhibitory activity of Balanites fruits is due to the steroidal saponins present. HPLC chromatographic profiles of the crude butanol fraction and its 4 sub-fractions showed that the most highly bioactive fraction D contained the highest amount of steroidal saponins (75%) as compared to the 21% present in the original butanol fraction. The isolated furostanol saponins proved to be highly active in an in vitro assay.

In vitro growth inhibition by Hypericum extracts and isolated pure compounds of Paenibacillus larvae, a lethal disease affecting honeybees worldwide.

The in vitro inhibitory potential of 50 extracts from various species of the flowering plant genus Hypericum was investigated using the Kirby-Bauer disk diffusion susceptibility test against Paenibacillus larvae, a spore-forming, Gram-positive bacterial pathogen that causes American foulbrood (AFB), a lethal disease affecting honeybee brood worldwide. Of the tested extracts, 14 were identified as highly active against P. larvae as compared to the activity of the positive control, indicating the presence of highly potent antibacterial compounds in the extracts. Examination of these extracts using TLC and HPLC/MS analyses revealed the presence of acylphloroglucinol and filicinic-acid derivatives. Six pure compounds isolated from these extracts, viz., hyperforin (1), uliginosin B (2), uliginosin A (3), 7-epiclusianone (4), albaspidin AA (5), and drummondin E (6), displayed strong antibacterial activity against the vegetative form of P. larvae (MIC ranging from 0.168-220 µM). Incubation of P. larvae spores with the lipophilic extract of Hypericum perforatum and its main acylphloroglucinol constituent 1 led to the observation of significantly fewer colony forming units as compared to the negative control, indicating that the acylphloroglucinol scaffold represents an interesting lead structure for the development of new AFB control agents.

Reduced Alzheimer's disease pathology by St. John's Wort treatment is independent of hyperforin and facilitated by ABCC1 and microglia activation in mice.

Soluble ß-amyloid peptides (Aß) and small Aß oligomers represent the most toxic peptide moieties recognized in brains affected by Alzheimer's disease (AD). Here we provide the first evidence that specific St. John's wort (SJW) extracts both attenuate Aß-induced histopathology and alleviate memory impairments in APP-transgenic mice. Importantly, these effects are attained independently of hyperforin. Specifically, two extracts characterized by low hyperforin content (i) significantly decrease intracerebral Aß42 levels, (ii) decrease the number and size of amyloid plaques, (iii) rescue neocortical neurons, (iv) restore cognition to normal levels, and (iv) activate microglia in vitro and in vivo. Mechanistically, we reveal that the reduction of soluble Aß42 species is the consequence of a highly increased export activity in the bloodbrain barrier ABCC1transporter, which was found to play a fundamental role in Aß excretion into the bloodstream. These data (i) support the significant beneficial potential of SJW extracts on AD proteopathy, and (ii) demonstrate for the first time that hyperforin concentration does not necessarily correlate with their therapeutic effects. Hence, by activating ABC transporters, specific extracts of SJW may be used to treat AD and other diseases involving peptide accumulation and cognition impairment. We propose that the anti-depressant and anti-dementia effects of these hyperforin-reduced phytoextracts could be combined for treatment of the elderly, with a concomitant reduction in deleterious hyperforin-related side effects.

Flavonoid glycosides and naphthodianthrones in the sawfly Tenthredo zonula and its host-plants, Hypericum perforatum and H. hirsutum.

Larvae of the sawfly Tenthredo zonula are specialized on Hypericum. Whether the sawfly is able to sequester plant metabolites was unknown. Aerial materials of Hypericum perforatum and H. hirsutum, as well as dissected larvae and prepupae of T. zonula, were analyzed by HPLC to determine the presence and content of flavonoid glycosides (rutin, hyperoside, isoquercitrin, and quercitrin) and naphthodianthrones (pseudohypericin and hypericin). All flavonoid glycosides were detected in both Hypericum species, with hyperoside as major compound in H. perforatum (ca. 1.7 µmol/g fresh weight, FW) and isoquercitrin in H. hirsutum (0.7 µmol/g FW). Naphthodianthrones were present at low concentrations (0.02 µmol/g FW) in the former, and almost undetected in the latter species. In the body parts (i.e., hemolymph, digestive tract, salivary glands, or miscellaneous organs) of T. zonula, the surveyed compounds were detected more frequently in prepupae than in larvae. The compounds were not present in every sample, and flavonoid glycosides especially occurred in highly variable amounts, with maximal concentrations of 41 µg rutin/prepupa in salivary glands, 8 µg hyperoside/prepupa in hemolymph (= 0.36 µmol/g FW), 32 µg isoquercitrin/prepupa in salivary glands, and 63 µg quercitrin/larva in miscellaneous organs (mainly composed of the integument). We conclude that flavonoid glycosides are sequestered since they were detected in organs other than the digestive tract of larvae, and because prepupae are a non-feeding stage. The naphthodianthrone pseudohypericin, but not hypericin, occurred generally in the digestive tract (up to 0.25 µg/larva). Both naphthodianthrones and related unidentified compounds, but not flavonoid glycosides, were found in the larval excrement. The highly variable distributions of flavonoid glycosides and naphthodianthrones in T. zonula larvae and prepupae make it difficult to determine the ecological significance of these metabolites.

Bioactive xanthones from the roots of Hypericum perforatum (common St John's wort).

BACKGROUND: Extracts of Hypericum perforatum L. (common St John's wort; Hypericaceae) are sold as phytopharmaceuticals and herbal supplements to treat mild to moderate depression and as food additives. Extensively cultivated in Europe, plants can be infected by anthracnose (Colletotrichum gloeosporioides), a virulent fungal pathogen that causes tissue necrosis and dramatically decreases crop value. Such infections triggered the production of new secondary metabolites, specifically xanthones, in cell culture experiments. RESULTS: Bioassay-guided fractionation of H. perforatum root extracts, testing for growth inhibition of plant pathogenic fungi from the genera Colletotrichum, Botrytis, Fusarium and Phomopsis, was performed. In vitro anti-inflammatory activity through inhibition of COX-1, COX-2 and 5-LOX-catalyzed LTB(4) formation was also evaluated. Extracts were analyzed by various chromatographic means and structure elucidation was performed using data from nuclear magnetic resonance and mass spectrometry. CONCLUSION: Researchers have previously described constituents from the aerial parts of this species, but few reports describe secondary metabolites found in underground parts, of particular interest because the lower stem and upper root are often sites of fungal infection. This work resulted in the isolation of three xanthones: 1,6-dihydroxy-5-methoxy-4',5'-dihydro-4',4',5'-trimethylfurano-(2',3':3,4)-xanthone; 4,6-dihydroxy-2,3-dimethoxyxanthone; and cis-kielcorin, one of which possessed novel bioactivity against species of Phomopsis and inhibited 5-LOX-mediated LTB(4) formation.

Taxonomy and Chemotaxonomy of the Genus Hypericum.

The genus Hypericum L. (St. John's Wort, Hypericaceae) includes, at the most recent count, 469 species that are either naturally occurring on, or which have been introduced to, every continent in the world, except Antarctica. These species occur as herbs, shrubs, and infrequently trees, and are found in a variety of habitats in temperate regions and in high mountains in the tropics, avoiding only zones of extreme aridity, temperature and/or salinity. Monographic work on the genus has resulted in the recognition and description of 36 taxonomic sections, delineated by specific combinations of morphological characteristics and biogeographic distribution ranges. Hypericum perforatum L. (Common St. John's wort, section Hypericum), one of the best-known members of the genus, is an important medicinal herb of which extracts are taken for their reported activity against mild to moderate depression. Many other species have been incorporated in traditional medicine systems in countries around the world, or are sold as ornamentals. Several classes of interesting bioactive secondary metabolites, including naphthodianthrones (e.g. hypericin and pseudohypericin), flavonol glycosides (e.g. isoquercitrin and hyperoside), biflavonoids (e.g. amentoflavone), phloroglucinol derivatives (e.g. hyperforin and adhyperforin) and xanthones have been identified from members of the genus. A general overview of the taxonomy of the genus and the distribution of relevant secondary metabolites is presented.

Essential oil and volatile components of the genus Hypericum (Hypericaceae).

The flowering plant genus Hypericum (Hypericaceae) contains the well-known medicinally valuable species Hypericum perforatum (common St. John's wort). Species of Hypericum contain many bioactive constituents, including proanthocyanins, flavonoids, biflavonoids, xanthones, phenylpropanes and naphthodianthrones that are characterized by their relative hydrophilicity, as well as acylphloroglucinols and essential oil components that are more hydrophobic in nature. A concise review of the scientific literature pertaining to constituents of Hypericum essential oils and volatile fractions is presented.

Chemical composition and antifungal activity of Arnica longifolia, Aster hesperius, and Chrysothamnus nauseosus essential oils.

Essential oils from three different Asteraceae obtained by hydrodistillation of aerial parts were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). Main compounds obtained from each taxon were found as follows: Arnica longifolia carvacrol 37.3%, alpha-bisabolol 8.2%; Aster hesperius hexadecanoic acid 29.6%, carvacrol 15.2%; and Chrysothamnus nauseosus var. nauseosus beta-phellandrene 22.8% and beta-pinene 19.8%. Essential oils were also evaluated for their antimalarial and antimicrobial activity against human pathogens, and antifungal activities against plant pathogens. No antimalarial and antimicrobial activities against human pathogens were observed. Direct bioautography demonstrated antifungal activity of the essential oils obtained from three Asteraceae taxa and two pure compounds, carvacrol and beta-bisabolol, to the plant pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides. Subsequent evaluation of antifungal compounds using a 96-well micro-dilution broth assay indicated that alpha-bisabolol showed weak growth inhibition of the plant pathogen Botrytis cinerea after 72 h.

Phytochemical profiling of New and Old World Hypericum (St. John's Wort) species.

Botanical extracts of Hypericum perforatum L. (common St. John's Wort) are used in the USA and in Europe as a treatment for mild to moderate depression, although controversy surrounds the identity of the active constituent(s). RP-HPLC with photodiode array detection was used to separate and quantify nine compounds of pharmacological interest in extracts from 74 taxa of Hypericum native to the Old and New World. Chemical profiles of these constituents may be used to distinguish extracts of H. perforatum from those of other species of Hypericum, and to indicate species that may be of interest for further phytochemical investigation.

Genetic profiling of Hypericum (St. John's Wort) species by nuclear ribosomal ITS sequence analysis.

A PCR-based DNA amplification method was applied to genetically distinguish the popular dietary supplement Hypericum perforatum L. (common St. John's Wort) from other related Hypericum species. Nuclear ribosomal gene sequences of the internal transcribed spacer (ITS) region were analyzed for 50 Hypericum taxa native to the Old and New Worlds, representing 11 of the 36 currently accepted taxonomic sections. This study provides a genetic method for authentication of commercial H. perforatum preparations. In addition, these data allow a preliminary assessment of phylogenetic relationships within the genus, revealing three strongly supported monophyletic clades, plus several secondary monophyletic groupings. Using ITS gene sequences, we were able to distinguish H. perforatum from all other species of Hypericum included in this study.

Steam distillation-solid-phase microextraction for the detection of Ephedra sinica in herbal preparations.

A new method involving concurrent solid-phase microextraction combined with continuous hydrodistillation of essential oil was developed. This new methodology allowed for the detection by GC-MS of very small amounts of a diagnostic peak for the authentication of Ephedra sinica, in a short period of time and using only small sample sizes. This diagnostic peak was identified as 4-vinylanisole, and elucidated from the chromatographic profile allowed for the identification of a sample as E. sinica among other species investigated in this study. To the best of our knowledge this is the first report on using continuous solid-phase microextraction coupled to hydrodistillation for the investigation of essential oil components, and the first report of 4-vinylanisole as a marker compound for E. sinica. A total of 46 collections representing 21 species of Ephedra were studied.

The role of chemical fingerprinting: application to Ephedra.

Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in Traditional Chinese Medicine (TCM). Preparations, namely teas, of E. sinica have been used for over 5000 years as a stimulant and as an antiasthmatic. In the West, extracts of E. sinica, E. intermedia or E. equisetina are most commonly used in dietary supplements as a stimulant and to promote weight loss. More than 50 species of Ephedra are native to both hemispheres, but the detection of ephedrine alkaloids has been limited to species in Eurasia. Currently, methods exist to quantitate the ephedrine alkaloids in extracts of plant material or dietary supplements, but the methods are not able to verify the extract is of an Ephedra species. Reverse phase high performance liquid chromatography with photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two regions of comparison were determined in the chromatograms at 320 nm. The series of peaks between 52 and 64 min confirms an Ephedra species is being analyzed. The aforementioned peaks also could distinguish between Ephedra species from Eurasia, North America and South America. Peaks at ca. 57 and 59 min were isolated and determined to be two new compounds, 4-(2-eicosyloxycarbonyl-vinyl)-benzoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid respectively. Authentication of ground plant material as Ephedra can be achieved by this chemical fingerprinting method.