Growth of H. perforatum varieties and accessions
Hypericum field plots were established at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS) in Ames, Iowa. Three commercial cultivars and two unimproved populations of Hypericum perforatum were evaluated in this experiment. The 'Common' cultivar (Ames 28320, supplier's lot 16333) was obtained from Johnny's Selected Seeds (Winslow, ME), a seed company specializing in organic seeds, and the other cultivars, 'Helos' (Ames 27453, NCRPIS lot 04ncao01) and 'Medizinal' (Elixir™) (Ames 27452, NCRPIS lot 04ncao01), were grown from seeds supplied by Richter's Herb Specialists (Goodwood, ON, Canada). The cultivar 'Medizinal' was bred to contain a higher amount of napthodianthrones, and 'Helos' was bred for tolerance to anthracnose disease . Two unimproved populations from the former Soviet Union, PI 325351 (NCRPIS lot 85ncab01) and PI 371528 (NCRPIS lot 75ncai01) were obtained from the NCRPIS, a public germplasm collection.
Seeds were germinated in petri dishes. After germination, seedlings were transferred to plastic trays (72 plugs/tray) containing Sunshine LC-1 Mix™(Sun-Gro Horticulture, Bellevue, WA). These seedlings were transplanted into field plots on 11 June 2003, at 118 days after seeding, at a plant height of 6 to 10 cm.
Material of 'Common' used in the fractionation studies was harvested on 23 July 2004, when plants were at 50% flowering. The studies that assessed the antiviral activity and cytotoxicity associated with chloroform extracts from several H. perforatum cultivars and accessions used plant material that was harvested on 16 June 2005, also at 50% flowering. Three plants per plot were harvested by cutting aerial parts 30.5 cm above the soil surface and placing them in mesh bags. Bags were placed in drying racks with forced air at 40°C for 8 days . After the aerial parts were completely dry, dry weights were taken and tops were ground through a 40-mesh screen in a Wiley grinder.
Extraction of H. perforatum aerial material
For the studies that investigated antiviral activity associated with the extracts, 6 g of dried plant material from each cultivar or accession was chloroform extracted and dried by rotary evaporation. For the fractionation studies, 450 g of ground aerial parts of H. perforatum 'Common' were extracted with chloroform by Soxhlet extraction for 6 h. The extract was dried by rotary evaporation to yield a total of 64.93 g of material.
Fractionation of chloroform extracts
The crude extract (23.6 g) was dissolved in CHCl3 (150 mL) and activated charcoal was added. After filtration, the filtrate was concentrated in vacuo, and the residue was placed on a short (~5 cm) silica column. Sequential elution with hexane (2 L), CHCl3 (4 L), and CH3OH (1 L) afforded three fractions that were concentrated in vacuo and subjected to bioassay. The active methanol fraction (7.1 g) was dissolved in 50:50 CH3CN:CH3OH and then subjected to column chromatography (3 cm, 180 cm3 of silica). Sequential elution with 50:50 CH3CN:CHCl3 (600 mL), CH3CN (400 mL), 90:10 CH3CN:CH3OH (400 mL), 50:50 CH3CN:CH3OH (500 mL) gave four fractions (E1, 3.5 g; E2, 0.5 g; E3, 1.1 g; and E4, 2.0 g; ~100% recovery). After bioassay, the active fraction E4 (1.97 g) was subjected to column chromatography (3 cm, 100 cm3 silica). The fraction was dissolved in 5 mL 95:5 CH3CN:CH3OH and eluted with a step gradient consisting of 400 mL each of (95:5, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 0:100 CH3CN:CH3OH) affording eight new fractions (E4.1, 230 mg; E4.2, 240 mg; E4.3, 260 mg; E4.4, 240 mg; E4.5, 250 mg; E4.6, 140 mg; E4.7, 110 mg; E4.8, 90 mg; ~79% recovery). After bioassays revealed activity, samples E4.7 and E4.8 were further fractionated by reverse phase HPLC on a preparative scale C18 column. Samples E4.7 and E4.8 each were dissolved in 1:1:1:3 CH3CH2OH:CHCl3:H2O:CH3OH (3 mL), and purified by HPLC with a gradient elution (Solvent A. 10 mM aq NH4OC(O)CH3, Solvent B. 9:1 CH3CN:CH3OH). A gradient of 3% B:A to 100% B was used for sample E4.7, and a gradient of 15% B:A to 100% B was used for sample E4.8. The flow rate was set at 3 mL/min and tubes were collected every 45 sec. After concentration, 6 subfractions were obtained from each sample (E4.7a, 17 mg; E4.7b, 9 mg; E4.7c, 13 mg; E4.7d, 7 mg; E4.7e, 6 mg; E4.7f, 6 mg; ~54% total recovery) and (E4.8a, 34 mg; E4.8b, 8 mg; E4.8c, 3 mg, E4.8d, 4 mg; E4.8e, 4 mg; E4.8f, 4 mg; ~70% total recovery). These fractions were assayed as described above.
HeLa37 cells were used for HIV studies . This HeLa cell line expresses both CD4 and CXCR4 ectotopically and are permissive for HIV strains that use CCR5 or CXCR4 for entry. HeLa37 cells were maintained in high glucose DMEM with 10% fetal calf serum and pen/strep. Equine dermis cells (ED cells)(ATCC CCL57) used for the EIAV studies were also maintained in high glucose DMEM with 10% fetal calf serum and pen/strep.
Generation of viral stocks
Stocks of HIV-1 were generated by transfecting a 150 cm plate of 80% confluent HEK 293T cells with 75 μg of the HIV molecular clone pNL4-3 by using the CaPO4 procedure . Supernatants were collected at 48-h post-transfection, clarified to remove cell debris and frozen at -80°C until needed. Virus production was assessed by reverse transcriptase activity in the viral stocks and by the single round of infection assay in HeLa37 cells described below. RT assays were performed as previously described .
Viral stocks of EIAVMA-1 were produced in ED cells. Supernatants were harvested from cells that were >95% positive for EIAV antigen as determined by EIAV antigen immunostaining. Supernatants were centrifuged for 5 min at 13,500 × g to remove cell debris, aliquoted, and frozen at -80°C until needed. Viral titers were determined by infection of ED cells by using the single round of infection assay described below.
All extracts or fractions were resuspended in DMSO. 2.5 × 102 infectious particles of HIV (MOI = 0.01) were combined with the concentrations of extracts or fractions noted in the figures. The amount of DMSO was adjusted so that equivalent concentrations of DMSO were used in all wells. No more than 0.5% DMSO was used, as HeLa37 cytotoxicity was observed at higher DMSO concentrations. The extract and HIV mixture was added to 2.5 × 104 cells/well of HeLa37 cells in a 48-well format. The cells were maintained for 40 h at 37°C in a CO2 incubator. Cells were fixed in 75% acetone/25% water and immunostained for HIV antigens with human anti-HIV antisera (1:500) followed by HRP-conjugated goat anti-human IgG (1:500). 3-amino-9ethyl-carbazole was used as the horse radish peroxidase substrate. Plates were dried and wells were counted for the number of HIV antigen-positive cells. Numbers of HIV antigen-positive cells in the presence of extract, fraction or fatty acid were divided by the number of HIV antigen-positive cells present in control wells that did not contain extracts, and these values are expressed as % control.
All studies were performed in ED cells. All extracts or fractions were resuspended in DMSO. 2.5 × 102 infectious particles of EIAV were combined with the concentrations of extracts or fractions noted in each experiment. The amount of DMSO was adjusted so that equivalent concentrations of DMSO were used in all wells. No more than 1% DMSO was used, as ED cell cytotoxicity was observed at higher DMSO concentrations. The extract and virus mixture was added to 5 × 104 cells/well of ED cells in a 48-well format to yield a MOI of ~0.005. The infections were maintained for 40 h. Cells were fixed with75% acetone/25% water at 40 h following initiation of the infection, and anti-EIAV immunostaining of the cells was performed as previously described . The EIAV antigen-positive cells within the monolayer were enumerated. Numbers of EIAV antigen-positive cells in the presence of the fractions were divided by the number of EIAV antigen-positive cells present in control wells that did not contain extracts, and these values were expressed as % control.
ED or HeLa37 cells were plated and treated with extracts, fractions or fatty acid as described above. Cell viability was monitored at 40 h after treatment initiation by ATPLite Assay (Packard Biosciences) per manufacturer's instructions.
Aseptic growth of H. perforatum seedlings
Hypericum perforatum (Accession Ames 28320, lot 06ncao01) seeds were surface sterilized by treating for 7 min with a solution consisting of 50% (v/v) Bleach and 0.05% (v/v) TritonX-100. After washing the seeds 3 times with sterile water, the seeds were placed on sterile wet 3 MM Whatman paper filters in sterile Petri plates. After germination, seedlings were aseptically transferred to individual Magenta boxes containing 25 ml of sterile 1% agar prepared in 1× Murashige & Skoog Basal Medium containing Gamborg Vitamins with macro and micronutrients (PhytoTechnology lab) Boxes were placed in a growth room maintained at 21°C, and under a 16-h light cycle, illuminated at 50 mmol m-2 s-1.
Lipid-bound fatty acids were extracted by a modification of a previously published method . Approximately 0.1 g fresh weight of aerial tissue or 0.05 g of root tissue, from 4-week-old H. perforatum plants, spiked with a known quantity of nonadecanoic acid as an internal standard, was homogenized with 1 mL of 10% (v/v) barium hydroxide and 0.55 mL of 1,4-dioxane, and the mixture was heated at 100°C for 24 h. After acidification with 6 M hydrochloric acid, fatty acids were extracted with two aliquots of hexane, which were pooled and taken to dryness under a stream of N2 gas.
Derivatization and GC/MS analysis
All samples were silylated [42, 43] by dissolving the dried extracts in 1 mL of acetonitrile, and adjusted to 6% of bis-trimethylsilyl-trifluoroacetamide and 10% trimethyl-chlorosilane. Samples were incubated at 65°C for 20 min, cooled, and filtered through a polytetrafluoroethylene filter. Silylated samples were analyzed by using an Agilent GC series 6890 equipped with an HP-5ms capillary column (30 m × 0.32 μm, inner diameter) using helium as the carrier gas. The GC was coupled to an Agilent 5973 mass detector. The injector was held at 250°C, the oven was initially at 70°C for 4 min, then ramped at 5°C/min to 320°C and held at that temperature for 6 min. Resulting chromatograms were integrated with Agilent's HP enhanced ChemStation TM G14701 BA version D.02.00.275.software. Peaks were identified by comparing acquired mass spectra with the Agilent NIST05 mass spectrum library.
Synthesis of hydroxy fatty acids
(1) To a solution of diisopropyl amine (3.3 mL, 24 mmol) in THF (20 mL) at 0°C, n-BuLi was added (8.8 mL, 2.5 M solution in hexane). The solution was cooled to -78°C with stirring, and then acetic acid (0.6 g, 10 mmol) in 5 mL of THF was added. After 30 min, decanal (0.56 g, 10 mmol) in 5 mL of THF was added. The mixture was stirred for 1 h and then brought to RT slowly. The reaction was diluted with dichloromethane and washed with ammonium chloride solution and the layers then were separated. The organic layer was dried with sodium sulfate and then concentrated. The solid mass was crystallized from dichloromethane to give 3-hydroxydodecanoic acid (mp 141°C). This compound has previously been prepared from the beta-keto ester .
1H NMR (400 MHz, CDCl3) δ 4.05 (m, 1H), 2.65 – 2.48 (m, 2H), 1.59 – 1.26 (m, 16H), 0.89 – 0.85 (t, J = 6.6 Hz, 3H)
All studies were performed at least three independent times except where noted in the figure legends. Means and standard errors of the mean are shown. To obtain IC50 and IC90 values for dose response curve data, the results were evaluated in the software Table Curve by using a best fit logistic dose response curve equation. Student's t-test was used to evaluate the statistical differences between treatments, utilizing the two-tailed distribution and two-sample equal variance conditions. P-values were accessed by comparing the level of infectivity with treatment to the level of cytoxicity seen with that treatment. A significant difference was determined by a p-value of < 0.05, and significance levels were identified in each figure. If the p-value was > 0.05, the data were not considered significantly different.