Chemicals and solutions
All reagents were of analytical grade unless specified otherwise. Ultra-pure water (produced by arium® 611 VF Water System, Sartorius, Göttingen, Germany) was used for all solutions, media, and HPLC eluents.
Acetonitrile (HPLC gradient grade), calcium carbonate, Edamin® K, 30% hydrogen peroxide (H2O2), and d-mannitol were purchased from Sigma Aldrich (Steinheim, Germany).
Salts, Agar-Agar, bovine serum albumin (BSA), EDTA, α-d(+)-glucose monohydrate, soy peptone, sucrose, 3,3',5,5'-tetramethyl benzidine (TMB), Tween® 20, yeast extract, and organic solvents were purchased from Applichem (Darmstadt, Germany) or from Roth (Karlsruhe, Germany).
Malt extract was obtained from Fluka, Neu-Ulm, Germany. Molasses was obtained from Südzucker, Offstein, Germany. Oatmeal was provided from Dr. Oetker, Düsseldorf, Germany.
Standards of erinacines and striatals, originally isolated from the two selected producer strains, were obtained from the library of pure natural products of InterMed Discovery, Dortmund, Germany (IMD, formerly Bayer Healthcare) and their purity and identity was confirmed by 2D-NMR and HR-MS, prior to their use in the experiments. Additionally, striatals A and B (for immunization) and striatals C and D, striatins A and B, and erinacines C and P (for determination of cross reactivities) were extracted and purified from submerged cultures of the basidiomycetes Cyathus striatus and Hericium erinaceus. The structures were confirmed by comparison to the pure reference compounds and by NMR analysis (cf. Additional file 1: Supplementary material).
Solutions for ELISA:
Coating buffers:
-
1.
10 mM phosphate buffered saline (PBS), pH 7.4 mixed with DMSO, 1/1, v/v.
-
2.
0.1 M potassium acetate buffer, pH 4.0 mixed with DMSO, 1/1, v/v.
-
3.
0.1 M sodium carbonate buffer, pH 9.6 mixed with DMSO, 1/1, v/v.
Washing buffer: 10 mM PBS, pH 7.4 with 0.05% Tween® 20 (PBST).
Blocking reagent (1% gelatin) was purchased from Roche Diagnostics (Mannheim, Germany). Secondary antibodies (goat anti-rabbit lgG peroxidase conjugate) were obtained from Merck (Darmstadt, Germany).
Color development: 0.12 mg mL-1 TMB and 0.05% H2O2 in 0.05 M sodium acetate solution, pH 4.5 (TMB solution). This solution was freshly prepared before use.
Stopping reagent: 0.5 M sulfuric acid.
Fungal strains
Cyathus striatus (STMA07048, isolated from basidiospores of a specimen from a trunk of Picea collected in July 1997 in Stelzenberg, Rheinland-Pfalz, Germany) and Hericium erinaceus (FU70034, isolated from basidiocarp tissue) for submerged culture were obtained from IMD. Prior to the experiments, the identity of both strains was confirmed by microscopic studies and by comparison of their ITS nrDNA sequences with reference data in Genbank. The fungi were maintained on a solid medium containing 20 g L-1 malt extract and 15 g L-1 Agar-Agar.They are deposited in the culture collection of IMD Natural Solutions GbR (formerly InterMed Discovery GmbH under liquid nitrogen. A duplicate strain and the corresponding specimen of the C. striatus material is also maintained at the personal herbarium and culture collection of Marc Stadler.
Fruiting bodies of Hericium erinaceus were obtained from a commercial provider www.pilzgarten.de, Helvesiek, Germany.
Culture media
Soy peptone, yeast malt, and sugar molasses media were prepared according to [25].
Instruments
HPLC-DAD
The HPLC system was from Merck Hitachi, Darmstadt, Germany, and comprised a pump (L-7100), an auto sampler (L-7200), an interface (D-7000), and a diode array detector (L-7455, 200 – 600 nm). Column: reversed phase, Nucleosil® 100–5 C18, CC 125/3 mm with a guard column Nucleosil® 100–5 C18, CC 8/3 mm (Macherey Nagel, Düren, Germany).
Flow rate: 0.4 mL min-1 for striatal and striatin analysis and 0.6 mL min-1 for erinacine analysis.
Eluent: Acetonitrile (A) and ultra-pure water (B).
Wavelength: 210, 233 and 254 nm.
Gradient for striatal and striatin: 50% A (0 min) – 94% A (14 min) – 94% A (17 min) – 100% A (19 min) – 100% A (25 min) – 50% A (30 min) – 50% A (40 min).
Gradient for erinacine: 30% A (0 min) – 50% A (15 min) – 50% A (16 min) – 100% A (23 min) – 100% A (38 min) – 30% A (43 min) – 30% A (47 min).
Preparative HPLC
A semi-preparative HPLC system (Young Lin, Hongye Anyang, Korea) equipped with quaternary pump (YL9110S) and dual wavelength UV/Vis detector (YL 9120S), combined with a fraction collector (CHF 122SC, Advantec, Osaka, Japan) was used. A column Kromasil 100 C18, 7 μm, 250 – 20 mm (MZ Analysentechnik, Mainz) with a guard column Kromasil 100 C18, 7 μm, 50 – 20 mm (MZ) was used to isolate the target compounds.
Flow rate: 15 mL min-1.
Eluent: Acetonitrile (A) and ultra-pure water (B).
Gradient for striatals and striatins: 50% A (0 min) – 50% A (10 min) – 80% A (55 min) – 80% A (70 min) – 100% A (80 min) – 100% A (95 min). Wavelengths: 210 and 254 nm.
Gradient for erinacines: 25 A% (0 min) – 25% A (10 min) – 48% A (35 min) – 48% A (45 min) – 55% A (52 min) – 55% A (60 min) – 100% A (90 min) – 100% A (110 min). Wavelengths: 210 and 233 nm.
ELISA
The ELISA was performed in microtiter plates Immuno Plate Maxisorp F96 (Nunc, Denmark). Incubation steps and color development were performed in an incubator INE 500 (Memmert, Schwabach, Germany). Washing was done by an eight-channel pipette Research Pro 50–1200 μL (Eppendorf, Wesseling-Berzdorf, Germany). The extinction was measured by a microtiter plate reader Synergy 2 (BioTek, Bad Friedrichshall, Germany).
Biosynthesis and isolation of cyathane diterpenoids
Submerged cultivation
Pre-cultures of C. striatus were grown submerged in 250 mL Erlenmeyer flasks containing 100 mL soy peptone medium at 24°C and 150 rpm for 7 days. After that, the mycelium was homogenized with an Ultra-Turrax (IKA, Staufen, Germany) at 10,000 rpm for 10 sec. 40 mL homogenized mycelium were inoculated into a new 1,000 mL Erlenmeyer flask containing 400 mL soy peptone medium and incubated at 24°C and 150 rpm for further 12 days.
Pre-cultures of H. erinaceus were grown submerged in 250 mL Erlenmeyer flasks containing 100 mL yeast malt medium at 24°C and 150 rpm for 7 days. Afterwards, 40 mL homogenized mycelium were inoculated into 400 mL sugar molasses medium in 1,000 mL Erlenmeyer flasks and incubated at 24°C and 150 rpm for further 4 days to obtain erinacine P or 12 days to obtain erinacine C.
Extraction and isolation
After submerged cultivation, the mycelia from 2.2 L culture media were separated from the supernatants by centrifugation at 2,880 g and 4°C for 10 min (Allegra® X-15R, BECKMAN COULTER™, Krefeld, Germany), and extracted twice with 400 mL ethyl acetate. The combined organic extracts were dried over sodium sulfate, and afterwards evaporated to dryness. A brown residue was obtained after evaporation, which was dissolved in acetonitrile and subjected to preparative HPLC. The extracts of Cyathus striatus contained the striatals A, B, C and D. The striatins A and B were prepared by conversion of the striatals A and B by stirring in methanol at room temperature overnight, respectively. The erinacines P and C were isolated from the extracts of Hericium erinaceus.
Production of polyclonal antibodies
The aldehyde function of the striatals and NH2 functions of BSA were used for synthesis of striatal A/B (1/1, w/w)-BSA conjugates. The ratio of striatals to BSA was determined by MALDI-TOF-MS. About 17 striatal molecules were coupled to one BSA molecule (Eurogentec S.A, Seraing, Belgien). The production of rabbit pAbs against striatal A/B was performed by Eurogentec S.A. Two rabbits with ID SA6928 and SA6929 were immunized four times with 200 μg of the striatal A/B-BSA conjugate on day 0, 14, 28 and 56. An additional immunization was done on day 91with 400 μg striatal A/B-BSA conjugate. The final antisera were collected on day 115 and stored at −20°C without further purification until use. The pre-bleed sera of these two rabbits on day 0 before immunization were used as negative control in ELISA. The final antiserum from rabbit SA6928 was used in the following assays because of its higher specificity against striatal B compared to SA6929 (data not shown).
Development of an indirect competitive ELISA
Optimization of coating conditions
Striatal B was directly attached to the plate in several different concentrations. The optimal conditions (buffer, temperature and time) for direct coating of striatal B were studied by an indirect non-competitive ELISA. The incubation temperatures were 4°C, 24°C and 37°C, and the coating time was 2 h or overnight.
Checkerboard titration
The optimal dilution of pAbs and the optimal coating amount of striatal B were determined simultaneously by checkerboard titration (a two-dimensional titration method) by indirect non-competitive ELISA. Striatal B with different concentrations (0.04 to 20 μg mL-1, two fold dilution) was coated to the microtiter plate and then bound to pAbs with serial dilutions (1:50 to 1:6,400, stepwise in two fold dilution).
Tolerance against organic solvents
The tolerance against organic solvents (DMSO, acetonitrile, methanol) used to dissolve the cyathane diterpenoids was examined by indirect competitive ELISA. Different proportions of organic solvent (5 - 50%, v/v, 0% as blank) were added to PBST with a final dilution of pAbs 1:200 and pre-incubated without competing molecules at 24°C for 1 h. The effects of organic solvents on the ELISA system were evaluated by comparing the measured extinctions to those of the blanks.
ELISA
Coating
100 μL of 5 μg mL-1 striatal B in coating buffer 1 were pipetted in wells of a microtiter plate. 100 μL of 1 μg mL-1 BSA diluted in PBS were pipetted in two wells as positive control. The incubation was performed overnight at 24°C.
Blocking
200 μL of 1% gelatin were added to the wells and incubated at 24°C for 3 h.
Pre-incubation
Serial dilutions of analyte were prepared in a concentration range of 0.0003 - 20 μg mL-1 in PBST containing 5% acetonitrile or DMSO and a final pAbs dilution of 1:200. The incubation was performed for 1 h at 24°C in 2 mL Eppendorf® cups.
Incubation with pAbs
100 μL pre-incubated analyte were added to each well (in triplicate) and incubated for 1 h at 24°C. For the indirect non-competitive ELISA, 100 μL pAbs with a 1:100 dilution in PBST were used without competitors instead of the pre-incubated analyte.
Incubation with secondary antibodies
100 μL secondary antibodies with a 1:5,000 dilution in PBST were added to each well and incubated for 1 h at 24°C.
Color development
100 μL of the TMB solution were added to each well and incubated for 15 min at 24°C. Without washing step, 100 μL of 0.5 M sulfuric acid were added to each well to stop the color development.
Measurement
The extinctions were measured immediately by a microtiter plate reader at 450 nm and 630 nm.
Unless otherwise specified, the wells were washed with 300 μL washing buffer PBST three times after each incubation step. The indirect non-competitive ELISA involved all of the steps of the indirect competitive ELISA, except for the pre-incubation of pAbs with competitors.
Data analysis
Striatal B standards and samples were analyzed in triplicates. The differences between extinctions at 450 nm and 630 nm were used for calculation and plotting the sigmoidal curves. %B/B0-values for calibration curves were calculated as follows:
ΔE: the extinction differences of samples at 450 nm and 630 nm.
ΔE
0
: the extinction differences of the upper asymptote of the sigmoidal curve between 450 nm and 630 nm.
ΔE
excess
: the extinction differences of the lower asymptote of the sigmoidal curve between 450 nm and 630 nm.
Cross reactivity (IC50)
The assay specificity was evaluated by obtaining sigmoidal curves for several structurally related compounds (striatals A, C and D, striatins A and B, and erinacines C and P) as competitors against the striatal B standard. The calibration curve of striatal B standard was measured individually for each plate. The IC50 were estimated by using a linearization of the calibration curves with a logit-transformation of %B/B0-values:
Cross reactivity (IC50) = A/B × 100%.
A: concentration of striatal B standard at logit (%B/B0) =0.
B: concentration of competitors at logit (%B/B0) =0.
The linear regression of logit (%B/B0) was used for determination of concentrations in unknown samples.
Recovery
Acetonitrile was spiked with striatal B at several concentrations (0.001 - 10 μg mL-1) and analyzed by indirect competitive ELISA as well as by HPLC-DAD. The concentrations of striatal B of spiked samples were calculated from linear regression of logit (%B/B0) in ELISA and linear calibration for striatal B in HPLC-DAD. The recoveries of striatal B were obtained by:
Analysis of H. erinaceussamples
The supernatants and extracts of supernatants from submerged cultures, as well as extracts of fruiting bodies of H. erinaceus were analyzed by indirect competitive ELISA:
Sample preparation for pre-incubation of supernatants in ELISA
50 μL of supernatants from submerged cultures (culture day 1 to day 8) were added to 950 μL PBST with a final DMSO proportion of 5% and a final pAbs dilution of 1:200. Culture medium without H. erinaceus was used as negative control.
Sample preparation for pre-incubation of extracts of supernatants
20 mL of supernatants from submerged cultures (culture day 1 to 8) were extracted once with 20 mL ethyl acetate. 15 mL of the ethyl acetate phase was evaporated to dryness, and the residues were redissolved in 1 mL acetonitrile for ELISA and HPLC-DAD analysis. 5 μL of the extracts were added to 995 μL PBST with a final acetonitrile proportion of 5% and a final pAbs dilution of 1:200. Extracts of culture media without H. erinaceus were used as negative controls.
Sample preparation for pre-incubation of extracts of fruiting bodies
32.6 g fruiting bodies of H. erinaceus were homogenized and then extracted with 200 mL ethyl acetate under stirring overnight. The ethyl acetate phase was dried over sodium sulfate and evaporated to dryness after filtration. 1 mg crude extract was dissolved in 1 mL acetonitrile. Several concentration levels (50.0, 10.0, 5.0, 2.5, 1.0 and 0.5 μg mL-1) of the extracts were used in the pre-incubation solution. Acetonitrile was used as blank.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional file.