3.3.1 Extraction of herbal substances and propolis
Herbal substances: For 1H-NMR-measurements of herbal substances, plant material was powdered by using a mortar and pistil. Samples were prepared by weighing 300 mg of the plant powder in case of herbal substances from TCM provided by the EDQM (due to the limited availability of samples). Concerning all other herbal substances, 500 mg of powdered plant material was used for the extraction with DCM. After this, dichloromethane extracts were made according to a protocol established in a former research project (Daniel, 2009): The powdered sample material was extracted with respectively 10 mL DCM for 15 minutes under occasional shaking. Subsequently, DCM extracts were filtrated through folded filters (Schleicher/Schüller), and the resulting filtrates were evaporated under reduced pressure with a rotary evaporator.
Concentrated samples were stored at -20 °C or directly investigated under the specific conditions described in 3.3.2. The extractions were performed by triple determination.
Propolis: Samples of propolis were extracted according to the extraction of herbal substances with small variations in sample preparation. Respective samples were prepared according to the properties of the samples. Crude propolis samples were milled very shortly, if possible. If necessary, powdered samples were grinded additionally using a mill, but in most cases, they were directly applied for the extraction with DCM. Crude propolis samples that were too soft for milling were grinded in a mortar adding a small amount of DCM. The extractions were performed by triple determination. Per sample, 300 mg propolis was used and partially pretreated.
3.3.2 1H-NMR-analysis
Dried plant extracts were resolved in 500 µL deuterated chloroform. 1H-NMR spectra were recorded on a Bruker Avance 300 DPX operating at 300 MHz at a constant temperature of 25°C.
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1H-NMR spectra were processed using Bruker TOPSPIN 1.3 Amix-Software. Spectra were referenced to residual solvent signals with resonances at 7.26 for CDCl3.
3.3.3 Principal component analysis (PCA)
The 1H-NMR-spectra were imported into the Bruker BioSpin’s AMIX Software, for evaluation by PCA. The following settings were adjusted for generation of the bucket table:
For 1H-NMR-spectra: Spectroscopy: 1D NMR; 1D-NMR bucket method: simple rectangular buckets; Left border: 10.0 ppm or 8.0 ppm; Right border: 0 ppm; Bucket width: 0.05 ppm; Integration mode: sum of intensities; Scaling: scale to total intensity;
Exclusions: DCM-extracts: 7.25-7.3 ppm (CHCl3-signal)
The bucket table was created after selection of the spectra. After that, a PCA was calculated. The following settings were adjusted:
Basic PCA; scaling of bucket variables: no scaling; suppress small variances: min.
variance level 5 %; set default confidence level: 99 %; select number of PCs by: min.
explained variance; min. explained variance: 95 %
Score plots were chosen for the graphical depiction of the results from PCA. The lengths of the PC-axes represent the percentage share of the respective PC for explanation of the total variance of the original data.
3.3.4 Thin layer chromatography (TLC)
TLC was carried out using TLC aluminium sheets silica gel 60 F254 (Merck). Two different protocols were applied:
TLC of Salviae trilobae/officinalis folium (Pachaly, DC-Atlas, 3. Aufl.): The test solution was prepared by extracting 0.5 g of the powdered herbal substance with 5 mL methanol under occasional shaking. The filtrate served as test solution. As reference solutions, 20 µL borneole and bornylacetate, 25 µL cineole and 20 µL thujone were solved in 5 mL methanol. Chromatograms of plant extracts were prepared by applying 20 µL of solution to the TLC plate and using toluol/ethyl acetate as mobile phases under saturated conditions. The migration distance was about 12 cm. Detection was performed by spraying with anisaldehyde-H2SO4 (0.5 mL anisaldehyde are mixed with 10 mL glacial acetic acid , 85 mL methanol and 5 mL H2SO4) and heating of the TLC
47 plate at 105 °C for 5 minutes. The result of the TLC was evaluated by daylight and under UV365.
TLC of Salviae trilobae/officinalis folium (European Pharmacopoeia 2007):
Salviae officinalis folium The test solution was prepared by extracting 0.5 g of the powdered herbal substance with 5 mL anhydrous ethanol under shaking. The filtrate served as test solution. As reference solutions, 25 µL cineole and 20 µL thujone were solved in 5 mL anhydrous ethanol. Chromatograms of plant extracts were prepared by applying 20 µL of solution to the TLC plate and using toluol/ethyl acetate (95:5 [V/V]) as mobile phases under saturated conditions.The migration distance was about 15 cm.
The detection was carried out by spraying with phosphomolybdic acid in anhydrous ethanol and heating of the TLC plate at 105°C for 10 minutes. The result of the TLC was evaluated by daylight.
Salviae trilobae folium The TLC was prepared according to the protocol for Salviae officinalis folium (European Pharmacopoeia 2007), with the exception that 0.3 g of the powdered herbal substance was used for the preparation of the test solution.
3.3.5 Gaschromatography (GC)
A steam distillation was performed to obtain the essential oils of Salvia species. For this purpose, respectively 50 g of sample material was powdered with a mill and subjected to the steam distillation apparatus after adding 500 mL water. The herbal material was extracted for 1.5 h.
The essential oils were diluted with DCM (Salvia officinalis TD 047: 1:240; Salvia triloba TD 051: 1:720; Salvia officinalis IG 008: 1:240 [essential oil : DCM ]) and measured by GC. The measurements were performed using a Chrompack CP-9003 apparatus. As reference solutions, cineole (1:1000), thujone (1:1000), borneole (1:50) and bornylacetate (1:50) were respectively solved in DCM (reference substance / DCM [V/V]). (The reference substances borneol and bornylacetate were solid substances [0.01 g was solved respectively in 5 mL DCM; then diluted 1:50 in DCM [V/V]]. The spectra were integrated with the supplied Maestro software. Manual peak integration was selected.
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3.3.6 GC-MS analysis of propolis samples
GC-MS analysis performed in cooperation with Prof. Chinou, University of Athens
Extraction and sample preparation:
Propolis samples were extracted for 24 h with 70% ethanol (1:10, w/v) at room temperature. The extracts were evaporated to dryness in vacuo. About 5 mg of each residue was mixed with 50 μL of dry pyridine and 75 μL of BSTFA and heated at 80 °C for 20 min. Reference compounds were subjected to the same procedure for silylation as about 1 mg of the pure compound was mixed with 10 μL of dry pyridine and 15 μL of BSTFA. The silylated ethanolic extracts and reference compounds were analyzed by GC-MS.
GC-MS analysis
The GC-MS analysis was performed with a Hewlett-Packard gas chromatograph 5890 series II Plus linked to a Hewlett-Packard 5972 mass spectrometer system equiped with a 30 m long, 0.25 mm i.d., and 0.5 μm film thickness HP5-MS capillary column.
The temperature was programmed from 100 to 300 °C at a rate of 5 °C/ min. Helium was used as a carrier gas at a flow rate of 0.7 mL/min. The split ratio was 1:20, the injector temperature 280 °C, the interface temperature 300 °C and the ionization voltage 70 eV. Hexane solution of n-alkanes was separated under the above conditions. Linear temperature programmed retention indices (LTPRI) were calculated.
3.3.7 Microscopic analyses of propolis samples
Microscopic analyses of propolis samples carried out by Dr. Sophia Karabournioti, University of Athens
Method of analysis (Graikou et al., 2011):
200 mg of propolis were dissolved and homogenized in a solution of ethylic alcohol, chloroform and acetone (1:1:1), the sediment obtained by centrifugation and separated is dissolved in 20 cm3 of 10% KOH solution. It is then boiled for 2 minutes, centrifuged and separated for a second time and then treated using the alcohol. The sediment is centrifuged and separated for a second time. The compound is prepared on a slide.
49 3.3.8 Microscopic analyses of dried plant material
The dried plant leaves were cut in cross section with a sharp razor blade, then boiled up in trichloromethane, covered with a cover glass and then examined under the microscope.
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4 Results & Discussion
Authentication of herbal substances by complementary methods
In two former research projects (Daniel, 2009; Kersten, 2013), methods for the characterization of herbal substances by metabolic- and ITS barcoding were established. To elaborate specific applications and also limitations provided by these two fingerprint methods, different species from the Lamiaceae family, herbal substances from TCM, the herbal substance Cimicifugae racemosae rhizoma and the plant derived bee product propolis were investigated within the framework of this study.
Particular emphasis was placed on the analysis of mixtures of herbal substances. For this purpose, three PCR-based approaches were established and the efficiency of identification of herbal components in mixtures was examined based on defined plant mixtures and finished herbal medicinal products.
4.1 Case study 1: Authentication of herbal substances belonging to