Identification of human intracellular targets of the medicinal Herb St. John's Wort by chemical-genetic profiling in yeast

St. John's wort (SJW; Hypericum perforatum L.) is commonly known for its antidepressant properties and was traditionally used to promote wound healing, but its molecular mechanism of action is not known. Here, chemical-genetic profiling in yeast was used to predict the human intracellular targets of an aqueous extract of SJW. SJW source material was authenticated by TLC, digital microscopy, and HPLC and further characterized by colorimetric methods for antioxidant activity, protein content, and total soluble phenolic content. SJW extract contained 1.76 microg/mL hyperforin, 10.14 microg/mL hypericin, and 46.05 microg/mL pseudohypericin. The effect of SJW extract on approximately 5900 barcoded heterozygous diploid deletion strains of Saccharomyces cerevisiae was investigated using high-density oligonucleotide microarrays. Seventy-eight yeast genes were identified as sensitive to SJW and were primarily associated with vesicle-mediated transport and signal transduction pathways. Potential human intracellular targets were identified using sequence-based comparisons and included proteins associated with neurological disease and angiogenesis-related pathways. Selected human targets were confirmed by cell-based immunocytochemical assays. The comprehensive and systematic nature of chemical-genetic profiling in yeast makes this technique attractive for elucidating the potential molecular mechanisms of action of botanical medicines and other bioactive dietary plants.     

Supercritical fluid extraction and high-performance liquid chromatographic determination of phloroglucinols in St. John's Wort (Hypericum perforatum L.)

A small-scale supercritical fluid extraction (SFE) method was developed for the selective extraction of phloroglucinols from St. John's wort (SJW) leaf/flower mixtures using supercritical carbon dioxide (CO(2)). The extraction efficiency was investigated as influenced by pressure, temperature, time, and modifier. The optimized condition of SFE was carried out at 3.80 x 10(4) kpa (5500 psi) and 50 degrees C. Samples were held in static extraction for 10 min, followed by a dynamic extraction for 90 min at the flow rate of 1 mL/min. A simple and sensitive HPLC method was developed for the analysis of hyperforin and adhyperforin, the major phloroglucinols, in the SFE extract of SJW.     

Hypericin and hyperforin production in St. John's wort in vitro culture: influence of saccharose, polyethylene glycol, methyl jasmonate, and Agrobacterium tumefaciens

Influence of saccharose in the presence or absence of polyethylene glycol (PEG), methyl jasmonate, and an inactivated bacterial culture of Agrobacterium tumefaciens in cultivation medium on morphology of Hypericum perforatum L. and production of hypericin and hyperforin was studied under in vitro conditions. Production of hypericin and hyperforin was influenced by the presence of different concentrations of saccharose (10-30 g L(-1)) in cultivation medium. Addition of PEG (1.25-5 g L(-1)) in the presence of saccharose (10-30 g L(-1)) increased production of hypericin and hyperforin in the H. perforatum in vitro culture. Synthesis of hypericin and hyperforin was unchanged or reduced for most of the experimental plants at higher contents of PEG (10 and 15 g L(-1)). Concentrations of hypericin and hyperforin in the H. perforatum were on the order 100 and 103 microg g(-1) of dry plant material, respectively. Production of hypericin and hyperforin was stimulated either in the presence of a chemical elicitor (methyl jasmonate) or an inactivated bacterial culture of A. tumefaciens. Morphological changes induced by the abovementioned substances were observed and described in detail. The obtained results will be applied in experimental botany and in the technology of H. perforatum cultivation for pharmaceutical applications.     

Analysis of plant complex matrices by use of nuclear magnetic resonance spectroscopy: St. John's wort extract

The efficiency of two-dimensional homonuclear (1)H--(1)H correlated spectroscopy and two-dimensional reverse heteronuclear shift correlation spectroscopy (i.e., heteronuclear multiple quantum correlation) in characterizing and evaluating the relative content of herbal extract constituents is demonstrated. These experiments are able to fully assign the proton and carbon resonances of all three classes of constituents present in dried commercial extract of St. John's wort, that is, flavonols, phloroglucinols, and naphthodianthrones, with particular regard to the very unstable phloroglucinols. In addition, shikimic and chlorogenic acids, sucrose, lipids, polyphenols, and traces of solvents of the extractive process (methanol) were also identified. These experiments can be considered to be a very simple and fast analytical method for determining the quality and stability of the titled commercial extract. They represent a generally applicable technique for a rapid screening and a specific measurement of other commercial phytochemicals or, in selected cases, an alternative to the classical analytical techniques such as high-performance thin-layer chromatography, high-performance liquid chromatography, capillary gas chromatography, and electrophoresis.     

Instability of St. John's wort (Hypericum perforatum L.) and degradation of hyperforin in aqueous solutions and functional beverage

Several bioactive botanicals including St. John's wort (SJW; Hypericum perforatum L.) have been used to formulate functional foods and beverages. This study aimed to investigate the stability of SJW components in aqueous solutions and fruit-flavored drinks. Changes of active marker components (hypericin, pseudohypericin, hyperforin, and adhyperforin) as affected by pH and light exposure were determined by HPLC, and the degradation of hyperforin was analyzed by LC-MS/MS and NMR. SJW components were found to be unstable in acidic aqueous solutions. More changes occurred under light exposure, with hyperforin and adhyperforin decreasing the most. Less severe changes were observed in the drink sample as compared to the pH 2.65 solution. Major degradation products of hyperforin in acidic aqueous solutions were identified as furohyperforin, furohyperforin hydroperoxide, and furohyperforin isomer a. The latter was also found in the drink product containing SJW as an ingredient. Biological activities and potential quality and safety implications of these chemical changes are yet to be evaluated.     

Sotolone production by hairy root cultures of Trigonella foenum-graecum in airlift with mesh bioreactors.

3-Hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone) and 3-amino-4,5-dimethyl-2(5H)-furanone, the postulated precursor of sotolone, were detected in hairy root cultures of Trigonella foenum-graecum (fenugreek) by GC-MS. The hairy root cultures in both conical flasks and airlift with mesh bioreactors were achieved from hypocotyl of seedling by infection with Agrobacterium rhizogenes. In flasks, the mathematical relationship between hairy root growth and conductivity was established and afterward used to evaluate the biomass evolution in bioreactor cultures due to the difficulty of obtaining direct biomass samples from the bioreactor. The GC-MS analyses of ethanolic extracts from hairy roots revealed the presence of two important compounds: sotolone (1.2% of the volatile fraction) and 3-amino-4,5-dimethyl-2(5H)-furanone (17% of the volatile fraction). These results point out that biotechnological production of sotolone in bioreactors is possible. Additionally, these hairy root cultures offer, for the first time, an excellent biological model to study the biosynthetic pathway of sotolone in fenugreek.     

Effect of nitrogen supply on nitrogen and carbohydrate constituent accumulation in rhizomes and storage roots of Curcuma alismatifolia Gagnep.

Abstract

Curcuma (Curcuma alismatifolia cv. Gagnep.), a tropical flowering plant known as “Siam tulip”, were cultivated in a pot with vermiculite and supplied with different levels of nitrogen (N). Rhizomes with storage roots were harvested at 215 days after planting. Results indicated that a high level of N supply increased flower numbers and promoted continuous new rhizome formation, but storage root growth was depressed. The N supply to the plants increased the N concentrations both in the rhizomes and in the storage roots. The predominant nitrogenous compounds related to total N increase were proteins in the rhizomes. The N of the insoluble fraction of 80% ethanol or the N of the soluble fraction of 10% trichloroacetic acid was the predominant fraction of N that accumulated in the storage roots. A lack of N supply increased the starch concentration both in the rhizomes and in the storage roots. These results suggested that a high level of N supply to the curcuma plant increased new rhizome formation because of increased flower numbers, but depressed new storage root formation because of reduced starch accumulation.     

Dynamic aspects of N.P.K. uptake and O2 secretion in relation to the metabolic pathways within the plant roots

As previously reported^1)-4) the inhibition of nutrients uptake by rice plant with such respiration inhibitors as hydrogen sulfide, butyric acid, cyanide, and azide differed strikingly from element to element. The order of suppression, however, was similar in these respiratory inhibitors, and the Ce I Itlon coefficients as revealed in terms of percents of decrease were grouped in the order of K²O·P²O⁵>SiO²·SO³·Br>MnO·NH⁴-N·H²O2> MgO·CaO. This established order attracted the authors interest to elucidate the essential mechanism of the uptake of each element, possibly linked specifically with metabolic pathways, particularly of respiration, within the plant roots. In view of the prime importance of three major fertilizer elements N.P.K., the specific metabolic linkages of these three elements were subjected to a energetic study.     

Relationship between in vitro production of auxins by rhizobacteria and their growth-promoting activities in Brassica juncea L.

Rhizobacteria were isolated from the rhizosphere of different Brassica species and assayed for their ability to produce auxins in vitro. The isolates varied greatly in their potential for auxin production (ranging from 0.33 to 11.40 µg ml^-1). L-Tryptophan (an auxin precursor) addition to the media increased the auxin production by several fold. Based upon in vitro auxin production and growth promotion of B. juncea seedlings caused by various isolates under gnotobiotic conditions, promising isolates were selected and tested in pot trial to observe their effects on growth, yield and oil content of the same Brassica species. Results showed that seed inoculation with different isolates of rhizobacteria significantly increased plant height (up to 56.5%), stem diameter (up to 11.0%), number of branches (up to 35.7%), number of pods per plant (up to 26.7%), 1,000-grain weight (up to 33.9%), grain yield (up to 45.4%) and oil content (up to 5.6%) over the uninoculated control. Isolate S54 gave the most promising and consistent results. Highly significant correlations between L-TRP-derived auxin production by plant growth-promoting rhizobacteria (PGPR) in vitro and grain yield (r =0.77^**), number of pods (r =0.78^**) and number of branches per plant (r =0.77^**) of B. juncea were found. It was hypothesized that these PGPR may influence the growth and yield of inoculated plants by production of auxins in the rhizosphere of inoculated plants from the L-TRP present in the root exudates, although other mechanisms of action might have also contributed.     

Influence of plant water status on the production of C13-norisoprenoid precursors in Vitis vinifera L. Cv. cabernet sauvignon grape berries

The influence of irrigation strategy on grape berry carotenoids and C13-norisoprenoid precursors was investigated for Vitis vinifera L. cv. Cabernet Sauvignon. Two irrigation treatments were compared, one in which vines received reduced irrigation applied alternately to either side of the vine (partial rootzone drying, PRD) and a second control treatment in which water was applied to both sides of the vine. Over the two years of the experiments, PRD vines received on average 66% of the water applied to the controls. Initially, the PRD treatment did not alter midday leaf (psiL) and stem (psiS) water potential relative to the control, but decreased stomatal conductance (gs). Continued exposure to the PRD treatment resulted in treated grapevines experiencing hydraulic water deficit relative to the control treatment and induced lowered midday psiL and psiS, which was also reflected in decreased berry weight at harvest. In both irrigation treatments, the most abundant grape berry carotenoids, beta-carotene and lutein, followed the developmental pattern typical of other grape varieties, decreasing post-veraison. At certain points in time, as the fruit approached maturity, the concentration of these carotenoids was increased in fruit of PRD-treated vines relative to the controls. This effect was greater for lutein than for beta-carotene. PRD consistently caused increases in the concentration of hydrolytically released C13-norisoprenoids beta-damascenone, beta-ionone, and 1,1,6-trimethyl-1,2-dihydronaphthalene in fruit at harvest (24 degrees Brix) over two seasons. The effect of the PRD treatment on the concentration of hydrolytically released C13-norisoprenoids was greater in the second of the two seasons of the experiment and was also reflected in an increase in total C13-norisoprenoid content per berry. This suggests that the increases in the concentration of the C13-norisoprenoids in response to PRD were independent of water deficit induced changes in berry size and were not the result of an altered berry surface area to volume ratio.     

Cinnamic acid inhibits growth but stimulates production of pathogenesis factors by in vitro cultures of Fusarium oxysporum f.sp. niveum

Long-term monoculture of watermelon leads to frequent occurrence of watermelon fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON). Some allelochemicals contained in watermelon root exudates and decaying residues are possibly responsible for promoting the wilt disease. The purpose of this study was to evaluate the allelopathic effect of artificially applied cinnamic acid on FON. Results demonstrated that hyphal growth of FON was strongly inhibited by cinnamic acid. At the highest concentration of cinnamic acid, the biomass in liquid culture was decreased by 63.3%, while colony diameter, conidial germination on plates, and conidial production in liquid culture were completely inhibited. However, mycotoxin production and activity of phytopathogenic enzymes were greatly stimulated. Mycotoxin yield, pectinase activity, proteinase activity, cellulase activity, and amylase activity were increased by 490, 590, 760, 2006, and 27.0%, respectively. It was concluded that cinnamic acid dramatically stimulated mycotoxin production and activities of hydrolytic enzymes by FON but inhibited growth and germination of FON. The findings presented here indicate that cinnamic acid is involved in promoting watermelon fusarium wilt.     

Effect of phosphorus supply on plant productivity,photosynthetic efficiency and bioactive-component production in Prunella vulgaris L. under hydroponic condition

We have examined the influence of four phosphorus (P) levels on growth performance, photosynthetic efficiency and bioactive phytochemical production of Prunella vulgaris L. in hydroponic culture. Results that 0.20 mM P was enhanced the dry weight, shoot height, spica and root weights, spica length and number, total chlorophyll and carotenoid contents, net photosynthetic rate, transpiration rate, and stomatal conductance were determined after three months' treatment. A supply of surplus P resulted in a higher concentration of foliar P and was negatively correlated with biomass. Both P-deficient (0 mM) and high P (10.00 mM) resulted in increased concentrations of ursolic acid and oleanolic acid, with the exception of flavonoids. An increase in water extract from P. vulgaris spicas was noted with the application of increasing P concentrations. Our results indicated that the application of 0.20 mM P improves the biomass production and the yield of bioactive constituents of P. vulgaris.     

Nutrio-physiological studies on the tomato plant III. Photosynthetic rate of individual leaves in relation to the dry matter production of plants

The apparent photosynthetic rate at 70 klux of a leaf attains a peak (33-46 mg CO₂-dm^-3.hr^-1) at a very early stage of its development, decreases rapidly, and then maintains a low constant value (5mg CO₂.dm^-3.hr^-1).

The photosynthetic rate is positively correlated with the content of nitrogen, phosphorus, chlorophyll and starch, and is negatively correlated with the content of calcium, manganese, silicon, and moisture. However, the physiological significance of these correlations is doubtful.

The photosynthetic potential exceeds the requirement of the sink as a whole plant. The surplus of photosynthates of young, upper leaves is considered to depress the photosynthetic activity of old, lower leaves. This is a conceivable reason for the rapid decline of photosynthetic activity of a leaf with age.

A low but constant photosynthetic rate (5mg CO₂.dm^-3.hr^-1) of old leaves at later stages is due, most probably, to a gradual increase in the weight of these leaves. The leaves lower than L₆ are considered to be the source for roots, and the photosynthetic rate of these leaves remains higher (10 mg CO₂.dm^-3.hr^-l than the upper leaves.