Article pubs.acs.org/jnp
Suberin Fatty Acids from Outer Birch Bark: Isolation and Physical Material Characterization Jyrki Heinam ̈ ak̈ i,*,† Minni M. Pirttimaa,‡ Sami Alakurtti,‡ H. Pauliina Pitkan̈ en,‡ Heimo Kanerva,‡ Janne Hulkko,‡ Urve Paaver,† Jaan Aruval̈ i,§ Jouko Yliruusi,⊥ and Karin Kogermann† †
Institute of Pharmacy, Faculty of Medicine, University of Tartu, Nooruse 1, 50411 Tartu, Estonia VTT Technical Research Centre Finland Ltd, VTT Industrial Synthesis, Biologinkuja 7, Espoo, P.O. Box 1000, FI-02044 VTT, Finland § Department of Geology, Institute of Ecology and Earth Sciences, University of Tartu, Ravila 14a, 50411 Tartu, Estonia ⊥ Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, P.O. Box 56 (Viikinkaari 5E), FI-00014 University of Helsinki, Finland ‡
S Supporting Information *
ABSTRACT: The isolation and physical material properties of suberin fatty acids (SFAs) were investigated with special reference to their potential applications as novel pharmaceutical excipients. SFAs were isolated from outer birch bark (OBB) with a new extractive hydrolysis method. The present simplified isolation process resulted in a moderate batch yield and chemical purity of SFAs, but further development is needed for establishing batch-tobatch variation. Cryogenic milling was the method of choice for the particle size reduction of SFAs powder. The cryogenically milled SFAs powder exhibited a semicrystalline structure with apparent microcrystalline domains within an amorphous fatty acids matrix. The thermogravimetric analysis (TGA) of SFAs samples showed a good thermal stability up to 200 °C, followed by a progressive weight loss, reaching a plateau at about 95% volatilization at about 470 °C. The binary blends of SFAs and microcrystalline cellulose (MCC; Avicel PH 101) in a ratio of 25:75 (w/w) displayed good powder flow and tablet compression properties. The corresponding theophylline-containing tablets showed sustained or prolonged-release characteristics. The physicochemical and bulk powder properties of SFAs isolated from OBB are auspicious in terms of potential pharmaceutical excipient applications.
B
Numerous studies have been published on solvent extraction of betulin and SFAs using a wide variety of solvents and extraction techniques. In principal, betulin (1) (Figure 1) and SFAs (2−9) can be extracted from OBB by two alternative methods. In a conventional method, betulin is first extracted with boiling organic solvent, followed by alkaline hydrolysis of the residual bark residue to liberate SFAs. In an alternative method, betulin and SFAs are both directly isolated from OBB using 2-propanol (IPA) and NaOH, followed by separation of betulin with a boiling aromatic solvent, leaving an SFA residue after acidification.5,7,13 Little work has been done to gain knowledge of the physicochemical and powder bulk properties of SFAs obtained with the present isolation techniques. The natural suberin polymer consists of a long-chain polyaliphatic (FAs containing) part and a polyphenolic lignintype part, which are linked via ester bonds via glycerol molecules.9,11 Ester bonds in the suberin biopolymer can be cleaved by hydrolysis to obtain SFAs. SFAs are a mixture of
irch bark is produced as a side product in large quantities in the wood refining industry, and is currently combusted to produce energy for the process. Birch (Betula spp.) contains between 12 and 14 w-% of bark.1,2 Birch bark consists of inner, brown phenolic-rich wood-like bark, and white outer bark.3 The proportion of outer birch bark (OBB) is between 3.4 and 5.4 w% of total biomass; thus the portion of OBB is roughly 25−40 w-% of the bark.2,4 Outer and inner bark can be separated after grinding by a water flotation technique, where water-absorbing inner bark sinks and water-repellent outer bark floats.5 Other separation techniques utilize screening: during grinding, more fragile inner bark is ground to smaller particles (98%, Pennsylvania, US), H2SO4 (Algol Chemicals, 93%, Espoo, Finland). The isolated SFAs mixture (Batch Pilot 1/14.01.2013 VTT, Espoo, Finland) was used as received. The chemical composition of the SFAs mixture was similar to the reported data (Figure 1 and Table 1). An established pharmaceutical direct compression excipient, microcrystalline cellulose, MCC (Avicel PH 101, FMC Biopolymer, Newark, DE, USA), was used as a reference material. Absolute EtOH (99.5% w/V, Etax Aa, Altia, Finland) was used for preparing ordered mixtures of SFAs and MCC. Theophylline anhydrate (Ph. Eur.)30 was used as a model drug and magnesium stearate (Ph. Eur.) as a lubricant in tablet compression. 921
DOI: 10.1021/acs.jnatprod.6b00771 J. Nat. Prod. 2017, 80, 916−924
Journal of Natural Products
Article
Extractive Hydrolysis of SFAs from Outer Birch Bark. A schematic diagram of the isolation process is shown in Figure 2. Three separate extraction experiments were performed with OBB (10.0 g), which was added in small portions via a funnel to a boiling mixture of NaOH (3.0 g) and 2-propanol (150 mL). Extractive hydrolysis was continued under reflux for 1 h. Nondissolved residue (Solid 1) was filtered off and the filtrate (Solute 1) was stored at 4 °C overnight to precipitate betulin and the sodium salts of suberin fatty acids (NaSFAs). The resulting orange precipitate was filtered and dried in vacuum to give a crude mixture of betulin and NaSFAs (Solid 2, 4.17 g, 42 w-%; 4.76 g, 48 w-%; and 4.71 g, 47 w-%). NaSFAs were extracted from Solid 2 by refluxing with H2O (40 mL) for 1 h and filtered while hot. NaSFAs were immediately precipitated out of the filtrate when the temperature decreased. The procedure was repeated on the nondissolved residue. Water-insoluble precipitate (Solid 3) was dried in vacuum at 40 °C to obtain betulin 1 (Betulin_lab 1−3: 1.70 g, 17 w-%; 1.94 g, 19 w-%; and 1.57 g, 16 w-%) as off-white solid. NaSFAs in water (Solute 2, 80 mL) were acidified to pH 2 with aqueous H2SO4 (2%, 20 mL) to precipitate SFAs. Precipitate (Solid 4) was filtered, washed with H2O, and freeze-dried to give SFAs (SFAs_lab 1−3: 1.17 g, 12 w-%; 1.38 g, 14 w-%; 1.40 g, 14 w-%). Extractive hydrolysis was repeated with a larger amount of OBB by using the following chemical amounts: OBB (47.5 g), NaOH (14.0 g), and (IPA, 700 mL). The yield of the mixture of betulin and NaSFAs (Solid 2) was 35 w-% (16.7 g). A portion of the crude mixture (8.44 g) was extracted with H2O (2 × 75 mL) as described above to yield betulin (solid 3, Betulin_lab 4, 2.90 g, 12 w-%). The filtrate (Solute 2) was acidified with H2SO4 (2%, 30 mL); the precipitate was filtered and freeze-dried to afford Solid 4 (SFAs_lab 4), yield 15 w-% (3.51 g). A pilot demonstration was performed with two batches (12 and 11 kg OBB). First, IPA (130 L) and NaOH (3.6 kg) were heated to 80 °C in a reactor (De Dietrich 200 dm3 glass-lined reactor) and finely ground OBB (12 or 11 kg) was placed in a pressure filter unit (Seitz Terra EF60/250) and preheated to 70 °C. Hot NaOH−IPA solution was pumped to a preheated pressure filter unit and hydrolysis was continued at 90 °C for 1 h. Insoluble bark cake (Solid 1) was filtered under pressure (3 bar) and the hot brown solute (Solute 1) containing NaSFAs and betulin was collected. When the solute was cooled to room temperature overnight, NaSFAs and betulin precipitated. The slurry including NaSFAs and the betulin precipitate was pumped back to a pressure filter unit, filtered, and air-dried at room temperature for 1 week to obtain NaSFAs and betulin as off-white precipitate (Solid 2, yield 9.5 kg, 41 w-%). Separation of SFAs and betulin was performed using a pressure filter unit (Seitz Terra EF 60/250) and a 3-phase separator (GEA, Westfalia Separator Process GmbH, Model ISC 6 01−576). Water (100 L) and the NaSFAs and betulin mixture (7.7 kg) were mixed (pH = 11) in a pressure filter unit and heated to 90 °C. The hot water-soluble NaSFAs (Solute 2) were filtered through wire and the process was continued for ca. 4 days. The pressure during filtration was 3 bar. Owing to slow filtration, only approximately half of the filtrate (Solute 2, including NaSFAs) was collected. Betulin (Solid 3) on top off the filtration wire was collected and dried in vacuum (Betulin_Pilot_1, yield 450 g). Filtrate (Solute 2, including NaSFAs) was allowed to cool to room temperature and was acidified with aqueous 10% H2SO4 to pH 2.0. The SFAs precipitate was partially washed from excess H2SO4 by running it three times through a pilot scale GEA 3-phase separator collecting the solids as off-white slurry and reslurrying solids twice after runs 1 and 2 with H2O (approximately 60 L/run) to remove residual H2SO4. The end pH after the third run was 3.0. The recovery of the off-white aqueous SFAs slurry was 14.3 kg (Solid 4, dry solid content 8.6%). The SFAs slurry was frozen and freeze-dried to obtain SFAs (SFAs_Pilot_1, yield 1.23 kg). Because of severe difficulties in filtration when using batch-type filtration, nonfiltered leftover basic solute (Solute 1, including NaSFAs and betulin) from the first batch was treated separately. Nonfiltered alkaline solute was heated to 75 °C to increase the solubility of NaSFAs. Water-insoluble betulin (Solid 3) was separated using a separator at 75 °C, collected as off-white slurry, and dried in vacuum to give betulin (Betulin_Pilot_2, yield 1.64 kg). The solute (Solute 2)
including NaSFAs was collected and treated in a similar fashion as earlier. The end pH after water washings was 3.9. Recovery of offwhite aqueous SFAs slurry was 9.08 kg (Solid 4, dry solid content 7.2%). SFAs slurry was frozen and freeze-dried to obtain SFAs (SFAs_Pilot_2, yield 0.66 kg). The main chemical compositions of laboratory and pilot batches are shown in Table 1. Cryogenic Milling and Preparation of Binary Powder Mixtures. The particle size distribution of the isolated SFAs powder was wide ranging from a few micrometers to a few millimeters (Figure 4). Therefore, it was necessary to reduce the particle size of the material for the subsequent experiments. Since SFA powder was plastic, 5 g of powder was manually milled for 5 min in a mortar and pestle in liquid nitrogen. During cryogenic milling, the material was fully immersed in the liquid nitrogen. The ordered binary mixtures of the cryogenically milled SFAs and MCC for bulk powder tests were prepared by manually spraying the ethanol solution of SFAs (50 g) onto the MCC powder particles. For preparing the binary mixtures containing 2%, 5%, or 10% (w/w) SFAs, the concentration of SFAs in the absolute ethanol solution (99.5% w/ V) was 4%, 10%, and 20% (w/w), respectively. During the entire procedure, the MCC powder (90.0−98.0 g) was periodically mixed to avoid the formation of agglomerates. Each batch comprised 100.0 g of ordered binary powder mixture (surface-treated MCC with SFAs). The sprayed binary mixtures were gently sieved through a 1 mm sieve and allowed to dry for at least 4−6 h at 40 °C. The mixtures were evaluated for bulk and tapped densities and flowability (Table 2). Physicochemical Material Characterization. SFAs and betulin samples were analyzed quantitatively by gas chromatography with a flame ionization detector (GC-FID) and qualitatively by GC-MS. Samples for GC and GC-MS were silylated by dissolving 2−15 mg sample in 0.7 mL pyridine and 0.6 mL N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and heating at 70 °C on a heatblock for 30 min. Qualitative analysis of samples was carried out with a Shimadzu GCMS-QP 2010 Ultra GC-MS spectrometer (Column: Agilent DB-5 30 m × 0.25 mm, start temperature: 50 °C, 15 °C/min, end temperature: 325 °C). A similarity search in library NIST11 and exact mass calculations were used to identify the components. Quantitative analysis was performed by GC-FID with an Agilent 6890N spectrometer (Method for SFAs samples: Column: Agilent HP-5 30 m × 0.32 mm, start temperature: 100 °C, 5 °C/min, and end temperature 300 °C; method for betulin samples: Column: Agilent HP5 30 m × 0.32 mm, start temperature 200 °C, 2 °C/min, and end temperature: 320 °C). External standards used for calibration were 16hydroxyhexadecanoic acid (CAS 506−13−8, Sigma-Aldrich, 98%, Milwaukee, US) and betulin (CAS 473−98−3, Sigma-Aldrich, 98%, Saint Louis, US). The 1H and 13C NMR spectra were measured on a Bruker Avance III 500 MHz NMR spectrometer (Billerica, MA, USA). 1H and 13C NMR spectra were recorded in DMSO-d6. Chemical shifts (δ) are given as ppm relative to the NMR solvent signals (DMSO-d6 2.50 and 39.50 ppm for 1H and 13C NMR, respectively). Particle size, shape, surface morphology, and microstructure of SFAs powder were investigated with a high-resolution scanning electron microscope, SEM (Zeiss EVO 15 MA, Germany). Samples were mounted on aluminum stubs with a conductive carbon film and were magnetron-sputter coated with a 3 nm gold layer in an argon atmosphere prior to microscopy. Moisture content analysis was done by determining the powder sample capacity to absorb water vapor in controlled conditions. Preweighed powder samples of raw nonmilled and cryogenically milled SFA were kept as thin layers under controlled temperature and humidity conditions at 21 ± 2 °C and 75% RH for 72 h, and the initial weight was determined. The powder samples were transferred into silica gel-containing desiccators (21 ± 2 °C and 0% RH) and stored for a subsequent 72 h before reweighing. The moisture content of the powders was obtained by calculating the weight difference between the initial and end weight of the sample (n = 6). X-ray powder diffraction (XRPD) patterns of SFAs were obtained by using an X-ray diffractometer (D8 Advance Bruker AXS GmbH, Karlsruhe, Germany). The XRPD experiments were carried out in 922
DOI: 10.1021/acs.jnatprod.6b00771 J. Nat. Prod. 2017, 80, 916−924
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ORCID
symmetrical reflection mode (Bragg−Brentano geometry) with Cu Kα radiation (1.5406 Å). The scattered intensities were measured with the LynxEye one-dimensional detector with 166 channels. The angular range was from 5° 2-θ to 40° 2-θ with steps of 0.02° 2-θ. The total measuring time was 166 s/step. The operating current and voltage were 40 mA and 40 kV, respectively. Simultaneous thermal analysis combining thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) was performed by using a Netzsch STA449 F1 (Netzsch-Gerätebau GmbH, Selb, Germany) thermal analysis system. The sample weight was 8.0−11.0 mg and calcined alumina was used as a sample of comparison. A nitrogen purge with a flow rate of 40 mL/min was used in the furnace. The scans were obtained by heating from 30 to 800 °C at a rate of 10 °C/min. Each run was performed in duplicate. Bulk and tapped densities of the binary powder mixtures of cryogenically milled SFAs and MCC were determined by a tapped density tester (Erweka SVM1, Erweka GmbH, Heusenstamm, Germany) and according to the standard method described in the European Pharmacopoeia.30 The Hausner ratio and Carr’s consolidation index were calculated from the bulk and tapped densities.18 The physical mixtures for the tableting test were prepared by blending 1% (w/w) of theophylline with the pure excipients (MCC, SFAs) or with the binary mixtures of cryogenically milled SFAs and MCC (Table 3). The powder mixtures were blended in a laboratoryscale Turbula mixer (Turbula System Schatz, Willy A. Bachofen AG Mascinenfabrik, Switzerland) for 10−15 min. The actual batch size used for tablet compression was 2.4 g. The tablets were compressed in an instrumented eccentric tablet machine (Korsch EK-0, Erweka Apparatebau GmbH, Berlin, Germany) using 9 mm flat-faced punches. The tablet height under maximum load was held constant at 2 mm. The die and punches were prelubricated with a magnesium stearate in acetone suspension prior to each compression. The die was filled manually with a powder mixture (175 ± 1 mg) and compressed into the tablet. The lower punch position was experimentally fixed in such a position that the powder samples under compression produced tablets with acceptable mechanical strength. In order to minimize the statistical variation, the tablets were compressed individually, and subsequently numbered from 11 to 510. The first number refers to the tablet compression batch (1−5) and the second one (1−10) refers to the ranking number of tablet inside the batch (e.g., code “510” indicates the tablet prepared in batch “5”, and it has been compressed as the 10th tablet in order). From the force−displacement data obtained, maximum upper punch force (Fup), maximum lower punch force (Flp), effective force (Feff), and minimum height of a tablet during compression cycle (h min ) were determined to estimate the compression properties of the samples. A modified in vitro tablet disintegration test for tablet formulations was performed in 250 mL Erlenmeyer flasks at an ambient room temperature (23 ± 1 °C). The flasks were first filled with 100 mL of purified H2O and tablets were then immersed into H2O. The flasks were shaken manually in regular intervals in order to prevent the formation of a static diffusion layer around the particles, and the in vitro disintegration of tablets was followed for 2 h. The present preliminary test was performed to characterize and visualize the disintegration behavior of the tablets.
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Jyrki Heinämäki: 0000-0002-5996-5144 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the European Social Fund’s Doctoral Studies and Internationalisation Programme DoRa and GREASE Woodwisdom-ERANET (Tekes decision 40375/ 11). This work is part of the ETF grant project no ETF7980 and IUT34-18. The Estonian Ministry of Education and Research is acknowledged for financial support. M. Jumppanen, T. Pietarinen, O. Antikainen, and L. Rammo are acknowledged for the support in laboratory experiments.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00771.
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REFERENCES
NMR spectra for suberin fatty acids (SFAs) (PDF)
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DOI: 10.1021/acs.jnatprod.6b00771 J. Nat. Prod. 2017, 80, 916−924
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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on March 23, 2017, with an error in Figure 1. The corrected version was reposted on April 10, 2017.
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DOI: 10.1021/acs.jnatprod.6b00771 J. Nat. Prod. 2017, 80, 916−924
