A New Betulin Derivative Stimulates the Synthesis of Collagen in Human Fibroblasts Stronger than its Precursor

Materials and Methods

Betulin, betulinic acid and Betulin-Dab-NH₂. Betulin was extracted according to a previously published procedure (12) from the European white birch bark (Betula pendula, syn. Betula verrucosa) collected in the summer of 2012 in a forest near Wroclaw (Poland, Lower Silesia). The betulin derivative with the chemical name Betulin-Dab-NH₂ (betulin ester with L-2,4-diaminobutyl acid) was synthesised as part of the study of the National Science Centre project no. 0275/B/P01/2010/38. The synthesis method is described by Drag-Zalesinska et al. (10). Betulinic acid was purchased from Sigma company (Sigma, Poznan, Poland). Based on previous research (10), betulin (BE), betulinic acid (BA) and Betulin-Dab-NH₂ (BE-Dab-NH₂) were used in a concentration of 6 μM. The chemical structures of the compounds are shown in Figure 1.

Mass spectroscopy analysis. Mass spectrometry was conducted to confirm the molecular weights of synthesised Betulin-Dab-NH₂ compound with a high-resolution Waters LCT Premier XE mass spectrometer equipped with electrospray ionization (ESI) and time of flight (TOF) instruments.

Cell cultures. Primary fibroblast cultures were obtained from human gingival tissue collected from a healthy patient at the Department of Oral Surgery, Wroclaw Medical University. The experiment was conducted in accordance with the requirements of the Bioethics Committee of Wroclaw Medical University (No. 864/2012). Gingival biopsy, isolation and culture of human fibroblasts from healthy patients were performed as described by Saczko et al. (13). Isolated cells were cultivated in DMEM medium (Sigma, Poznan, Poland) supplemented by 5% foetal bovine serum (FBS), (EURx, Gdansk, Poland) and 1% of streptomycin/penicillin (Sigma) in a humidified atmosphere with 5% of CO₂. Before the experiments, cells were washed by Dulbecco's Phosphate Buffered Saline (DPBS), (Sigma) and trypsinized with 0.25% Trypsin-EDTA (Sigma).

Evaluation of collagen content. Sircol™ collagen assay. The Sircol™ collagen assay method (Biocolor Ltd., Carrickfergus, UK) is a color-chemical assay used to determine the content of mammalian collagen type I to V in biological material. It uses the properties of the Sirius Red dye, which binds to the helical structure [Gly-X-Y]_n characteristic for all these types of collagen. Primary fibroblasts were prepared and placed into 25 cm² culture flasks at 3×10⁵ cells/flask in 4 ml of DMEM with 5% serum. After 24 h of incubation at 37°C, the medium was collected and cells were refed with 4 ml of serum-free DMEM with the addition of the corresponding compound (BE 6 μM, BA 6 μM, Betulin-Dab-NH₂ 6 μM). After 3-day incubation at 37°C and 5% CO₂, the medium was collected and the collagen content was analysed using the Sircol™ Soluble Collagen Assay kit. For analysis 100 μl of media sample were collected, as well as 100 μl of control medium. Collagen solutions (Reference Standard, Biocolor Ltd.) were prepared at the same time to obtain a standard curve in the range of 50-200 μg/ml of collagen. Into test tubes containing medium, control medium, or standard collagen solutions, 500 μl of Sircol Dye Reagent was added (Biocolor Ltd.) and then the tubes were shaken for 30 min. Then, the samples were centrifuged for 30 min at 13000 RCF and 4°C. The supernatant was then removed and the pellets were resuspended in 500 μl Wash Reagent (Biocolor Ltd.) and centrifuged again at the same conditions. After the supernatant was removed again, the pellet was resuspended in 250 μl Alkali Reagent (Biocolor Ltd.), shaken for 5 min and seeded into a 96 well plate. The absorbance at 550 nm was measured using a multiwell scanning spectrophotometer (EnSpire Multimode Plate Reader, PerkinElmer, Inc. Waltham, MA, USA). Based on the standard curve, the collagen content was calculated in each sample. The effect of the compounds on collagen synthesis was calculated by subtracting from the values of samples those of the control.

Immunocytochemical assay. Expression of type I and III collagen was assessed by immunocytochemistry. For this purpose, human normal fibroblasts were placed onto 8-well microscope slides. After 24 h when the cells were attached to the surface, test compounds were added at a concentration of 6 μM. After a 24-h incubation, the preparations were washed in PBS and then fixed with 4% formaldehyde. The slides were rinsed in PBS buffer and placed in 1% H₂O₂ for 30 min. Then, they were rinsed 3×5 min in PBS with 1% Triton X 100 (Sigma). Next, the slides were drained and a goat polyclonal anti-collagen type I antibody (Santa Cruz BT COL1A1, CA, USA) or a goat polyclonal anti-collagen type III antibody (Santa Cruz BT COL3A1) were applied. After a 24-h incubation at 4°C, they were rinsed 3x 5 min in PBS with 1% Triton X100. Then the procedure was performed using the DAKO LSAB kit (Carpinteria, CA, USA) + System HRP, successively with Biotinylated Link Universal, Streptavidin −HRP, DAB + substrate buffer with DAB + Chromogen, as described by the manufacturer. For the staining of cell nuclei, hematoxylin (Carl Roth, Karlsruhe, Germany) was used for 30 sec – 1 min. Next, the preparations were rinsed in water for 30 min, carried out by the ascending series of alcohol (50-100%) and dehydrated in xylene. Finally, the preparations were closed with DPX synthetic resin (Aqua Medica, Poland) using cover slides. A coloured immunocytochemical reaction was assessed using an Olympus BX51 microscope (Japan). The percentage of cells that underwent a staining reaction and the intensity of the immunocytochemical reaction were estimated. The counting was performed by two independent investigators. The intensity of immunohistochemically stained reaction was evaluated as (−) negative (no reaction), (+) weak, (++) moderate, and (+++) strong.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

The chemical structures of betulin (BE), betulinic acid (BA) and betulin ester with L-2,4-diaminobutyl acid (BE-Dab-NH₂). Mass spectroscopy (MS) analysis: MS calculated m/z+=543.4526; MS measured m/z+=543.4528.

Proliferation test. MTT assay. The effect of the BE-DAB-NH₂, BE and BA on human normal fibroblasts was assessed by the MTT test. The MTT test is a colorimetric method designed to determine the metabolic activity of living cells. Its action is based on the reduction of the tetrazolium salt by mitochondrial dehydrogenase to the coloured crystals of formazanes. The product of this reaction is insoluble in water and therefore requires the delivery of an organic solvent, e.g. DMSO or isopropanol. The intensity of the obtained solution translates proportionally to the cell proliferation and is measured spectrophotometrically in the 492-570 nm wavelength range. The cells were seeded into a 96-well culture plate. After 24 h and when the cells were adherent, the culture fluid was removed and the test compounds were added in the appropriate concentration (1 μM, 3 μM, 6 μM, 12 μM, 20 μM). The cells were then incubated 24 h with the test compounds. After this period, the MTT test was performed according to the manufacturer's protocol (Sigma Aldrich, Darmstadt, Germany). After the dissolution of the formazan crystals, the absorbance at 570 nm wave was measured using the Multimode EnSpire Reader (PerkinElmer, Inc.). The absorbance results were converted to a percentage of control cells. The test was performed in triplicate and the percentage of viable cells was determined for individual concentrations of Betulin-DAB-NH₂, BA and BE.

Statistical analysis. Statistical analysis was performed using Statistica 13.0 (Dell Inc., USA). Student's t-test, Mann-Whitney U-test and nonlinear estimation were used for the calculations.