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Research Article pubs.acs.org/journal/ascecg

Bioresource of Antioxidant and Potential Medicinal Compounds from Waste Biomass of Spruce František Kreps,*,† Zuzana Burčová,† Michal Jablonský,‡ Aleš Ház,‡ Vladimír Frecer,§,∥ Jan Kyselka,⊥ Štefan Schmidt,† Igor Šurina,‡ and Vladimír Filip⊥ †

Department of Food Science and Technology, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, 812 37 Bratislava, Slovak Republic ‡ Department of Wood, Pulp and Paper, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, 812 37 Bratislava, Slovak Republic § International Centre for Applied Research and Sustainable Technology, Jamnickeho 19, Bratislava 841 04, Slovak Republic ∥ Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Odbojarov 10, Bratislava 832 32, Slovak Republic ⊥ Department of Dairy, Fat and Cosmetics, University of Chemistry and Technology, Technická 5, 166 28, Prague, Czech Republic S Supporting Information *

ABSTRACT: The bark of Norway spruce (Picea abies) has been a source of medications for centuries. Despite that, bark is a waste biomass produced by wood industry in million tons annually. The most abundant compounds obtained from hexane extracts of bark were sterols and terpenes subjected to in silico analysis. Their absorption, distribution, metabolism and excretion properties and possible types of biological activities were predicted. Abietic acid, dehydroabietic acid and β-caryophyllene oxide originated from bark of Norway spruce had most likely antihypercholesterolemic, mucomembranous protecting and antineoplastic activity, respectively. An important discovery was estimation of their pharmaceutically relevant properties. Isolated fractions that were rich in these compounds (contained them up to 30%) displayed excellent trolox equivalent antioxidant capacity (15 mM/mg dm), which was 2.5 times greater than the source of powerful antioxidant, pycnogenol. Antioxidant activity of extracts and fractions determined using a DPPH assay were moreover investigated by EPR analysis of prolongation ascorbyl radical lifetime and rapid oxidation in lard. We found out that bark is an alternative feedstock of compounds with a potential antioxidant and medicinal activity that may replace synthetic drugs. Healing effect, antioxidant capacity, prolongation of ascorbyl radical lifetime and stabilization of lipids with compounds of spruce bark have not yet been published. KEYWORDS: Waste biomass, Bark, Picea abies, Antioxidant capacity, Biological activity, Antineoplastic

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and flavonoids. Some compounds identified in the bark of spruce display health-promoting properties including anticancer activity.4,7−9 According to Li et al.,8 more than 60 phenolic compounds of various types have been isolated and identified from the bark of Norway spruce. Stilbenes, the most studied compounds of bark of the conifer Picea abies, may occur both as aglycones and glucosides.10,11 Resveratrols are stilbenes, whose presence was well documented in the grapevine,12 but are still under investigation in the tree bark. Some authors8 showed that stilbenes in the bark serves as a chemical defense of the spruce against herbivores and pathogens. The root fungus Heterobasidion annosum is a serious pathogen of Norway spruce (Picea abies). It was found that a high level of stilbene in the bark and

INTRODUCTION Bark of the trees is a renewable source of biologically active compounds and represents a versatile alternative to fossil-based raw materials. The bark is a part of residual biomass produced by wood industry in the European Union in the amount of 6−8 million tons annually.1 From the environmental point of view, the bark is currently used mainly ineffectively as a source of heat, energy and biogas. The bark of the various tree species contains noteworthy compounds with medicinal effects and therefore has been used as a source of medications for centuries.2−4 A significant discovery was made by Stone,5 who described the usage of willow bark for treatment of malaria. On the basis of this observation, salicin was isolated from the willow bark, which gave rise to the production of aspirin.6 The Norway spruce (Picea abies) contains potentially bioactive compounds such as fatty acids, terpenes, waxes, sterols, saccharides and phenolic compounds like stilbenes, tannins © 2017 American Chemical Society

Received: June 6, 2017 Revised: July 17, 2017 Published: July 25, 2017 8161

DOI: 10.1021/acssuschemeng.7b01816 ACS Sustainable Chem. Eng. 2017, 5, 8161−8170

Research Article

ACS Sustainable Chemistry & Engineering sapwood caused resistance to this fungus.11 Many studies also confirmed the beneficial effects of stilbenes on human health. It has been also shown that stilbenes are potent antiinflammatory, anticancer, antioxidant, antiaging and chemoprotective agents.3,9,10,12,13 Current research shows that the bark of spruce contains hydrophobic substances with biological activity like stilbenes.4,14−18 It has been found out that the biosynthesis of terpenes (C10−C20) contributed increasingly to the protection of Norway spruce (Picea abies) against fungal pathogens and bark beetle.14,19 The bark and resin of Norway spruce contains resin acids such as abietic, dehydroabietic, neoabietic, pimaric, isopimaric, levopimaric, sandrakopimaric and palustric acids. Mainly because of their antimicrobial properties, they show wound-healing properties and show also a skin regeneration enhancing effect.17,20−22 Jokinen et al.17 observed that the exposure of Staphylococcus aureus to the resin caused agglomeration and destruction of the cell wall of bacteria. This is associated with a disruption of proton transport in the membrane-bound adenosine triphosphatase resulting in the uncoupling of oxidative phosphorylation (graphical abstract). The energy supply of the cell metabolism is inhibited and the mitosis repressed subsequently.17,21 It can be assumed that extracts and compounds obtained from the bark of Norway spruce (Picea abies) may exert antimicrobial and anticancer activities as well as other beneficial properties to human health.

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Antioxidant Capacity of Extracts and Obtained Fractions. The antioxidant capacity/scavenging activity of spruce bark extract fractions and commercial pycnogenol extract of pine were determined using DPPH as a free radical. From a mixture that contained 1 mg of the sample and 6 mL of ethanol (UV purity) was taken away 3 mL, which served as a blank. 300 μL of DPPH (0,5 mM in ethanol) was added to the remaining 3 mL of solution in a second test tube. The absorbance of blank at 517 nm was subtracted from the absorbance of the sample with DPPH after 5 min, which is the optimal reaction time. The resulting absorbance was compared with the calibration curve of the Trolox equivalent antioxidant capacity (TEAC) and expressed in mmol/L Trolox on 1 mg of dry matter (dm) of sample. EPR Analysis of Prolongation Ascorbyl Radical Lifetime. The antioxidant activity of hexane extract at 80 °C as well as its fraction 4 has been investigated and compared to the activity of commercially available pycnogenol. The samples were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 μg of sample in 1 mL of DMSO. They were added to 0.5 mM ascorbic acid and dissolved in 0.1 M phosphate buffered saline (pH 7.5). To initiate the ascorbic acid oxidation, 0.1 UN/mL ascorbate oxidase (100 UN/mg, Sigma-Aldrich, Germany) was added. DMSO was used as the control sample. A Bruker 200D EPR spectrometer (adjusted temperature 23 °C) was used for measuring the ascorbate radical. Experimental EPR spectra were simulated using WinEPR and SimFonia programs (Bruker, Bruker BioSpin, Karlsruhe Germany). The conditions of the EPR experiment were selected according to literature.24 Protective Factor in Lipid Oxidation. Antioxidant activity of samples and prolongation of lipid resistance to oxidation was defined as protective factor. According to an AOCS method,25 it was determined as the proportion stability of lard with and without addition of antioxidants. The lipid oxidation was measured at the constant temperature 110 °C, in the presence of air (20 mL/h bubbled through the oils) with a Rancimat 743. (Metrohm Ltd., Herisau, Switzerland). For the protective factor (PF) in lard lipid oxidation of the spruce bark extract obtained at 80 °C and its fraction 4, the concentration of 200 mg/kg was determined and compared to that of pycnogenol at the same concentration. The addition of 200 mg/kg was chosen according to the most commonly used supplement of synthetic antioxidants in food. GC-FID/MS Chromatography of Obtained Fractions. Samples were derivatized according to literature23 to form trimethylsilyl ethers derivates that were identified by gas chromatography with combined FID (GC-7890A, Agilent) and MS detector (MS-8975C, Agilent). About 60% of the mixture of substances was captured by the FID detector, whereas the remaining 40% was seized by the GC−MS detector. Analytes were separated by column HP-5MS (30 m × 0.25 mm × 0.25 μm). The conditions of analysis were as follows: hydrogen carrier gas (40 mL min−1), pressure 220 kPa, injection temperature 230 °C, split ratio 10:1, injection loop 1 μL, column temperature set to 100 °C during the first 2 min, then a temperature gradient program at 10 °C min−1 up to 150 °C during 2 min and the last temperature gradient program was set to 10 °C min−1 up to 250 °C during 30 min. Internal standards squalene, α-tocopherol, oleic acid, cholesterol, caffeic acid (99%, Sigma-Aldrich, Germany) were used for the quantitative analysis. The FID detector was flushed with hydrogen and air at 40 and 450 mL min−1, respectively. The flow of makeup nitrogen gas was set to 40 mL min−1. Qualitative analysis was carried out with GC−MS. The transfer line between GC and MS was heated at 280 °C. MS detector was adjusted to 250 °C, MS quadrupole 150 °C, electron ionization energy 70 eV and m/z range 30−780. Prediction of Activity Spectra for Substances (PASS). The probable biological activity profiles of fractionated compounds were estimated from their structural formulas based on the method of Poroikov.26−28 This in silico analysis was carried out with Prediction of Activity Spectra for Substances software (PASS version 2.0, geneXplain GmbH, Wolfenbüttel, Germany, 2012). The list of predictable biological activities consists of over 4000 activity types and includes pharmacologic effects, biochemical mechanisms, toxicity, metabolism, gene expression regulation and transporter-related

MATERIAL AND METHODS

Chemicals. The bark of Norway spruce (Picea abies) was obtained as a waste product, which is produced during industrial debarking of spruce wood in Bioenergo Inc. (Ružomberok, Slovakia). Pycnogenol, extract from the bark of French maritime pine (Pinus pinaster), was purchased from Pharma Nord (Czech Republic). Solvents and reagents used for the extraction, isolation and fractionation (n-hexane, diethyl ether) were of analytical grade (90−99%) and were purchased from Sigma-Aldrich (Slovakia) and VWR (Austria). Purity of nitrogen was 99.998%. The spraying agent phosphomolybdic acid hydrate was 99% and Trolox had the same purity 99%. Flash chromatography cartridge (4 × 15 cm) was filled with silica gel (40−63 μm, Merck, Slovakia). Sample Preparation. The bark was disintegrated by a hammer mill. A fractionation system of sieves separated bark particles with the particle size from 0.5 to 1 mm. Accelerated solvent extraction was carried out with hexane in a Dionex ASE 350. Most conditions of extraction were adjusted according to literature;7 only the temperatures were changed and bark particles were kept for 30 min at the temperature of 60, 80 or 120 °C. A small amount of insoluble sediment in the extract was eliminated by centrifugation (3500 min−1, for 10 min). The solvent was evaporated and the residue was stored under nitrogen atmosphere in a refrigerator. Isolation/Fractionation of Compounds from Extracts. Compounds present in the 1.5 g residue of the bark hexane extract were separated by flash chromatography (BÜ CHI C-601 pump, C-610 controller) with a cartridge (diameter, 4 cm; length, 15 cm; BÜ CHI 44883) that contained 80 g of silica gel (40−63 μm). Analytes were eluted by the mobile phase consisting of 300 mL of hexane, 300 mL of the mixture hexane:diethyl ether (95:5), 200 mL of hexane:diethyl ether (90:10), 300 mL of hexane:diethyl ether (80:20) and 300 mL of hexane:diethyl ether (60:40). Separation was stopped after elution of 200 mL of methanol. According to the test model shown, the average retention of compounds in the column was 22−30% on weight of model sample/crude extract. According to literature,23 fractions (Figures S1−S3) eluted from flash chromatography were qualitatively analyzed by TLC and characterized by retention factors (Rf): 0.71− 0.75, 0.19, 0.09 and 0.01−0.03. Then the fractions were collected in descending order and numbered. 8162


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ACS Sustainable Chemistry & Engineering activities. PASS prediction is based on structure−activity relationships of a training set of more than 260 000 compounds with known biological activities. Average accuracy of prediction estimated for the training set by leave-one-out cross-validation is about 95%. Prediction of Absorption, Distribution, Metabolism and Excretion (ADME). Computational prediction of ADME-related properties of considered compounds was carried out by the QikProp software (QikProp version 4.0, release 17, Small-Molecule Drug Discovery Suite 2014-2, Schrödinger, LLC, New York, NY, 2014) which uses the method of Jorgensen.29,30 QikProp predicts physically significant descriptors and pharmaceutically relevant properties of organic molecules. Besides the Lipinski parameters,31 QikProp computes altogether 39 descriptors, such as hydrophobic molecular surface, octanol/water partitioning coefficient, aqueous solubility, brain/blood partition coefficient, Caco-2 cell permeability, serum protein binding, number of likely metabolic reactions, predicted blockage of potassium channels, and many others. This in silico analysis eliminate bioactive molecules with insufficient ADME profiles from further pharmaceutical development. Statistical Analysis. It was carried out with the Statgraphics Plus, software version 3.0 for Windows (Manugistic Inc., Rockville, MD). Isolation of fractions was carried out with identical samples in triplicate. The figures were prepared by using Origin software, version 8.5 (Origin Lab Corporation, Northampton, MA).

Figure 1. Trolox equivalent antioxidant capacity (TEAC) of hexane extracts of spruce bark (Picea abies) and their fractions.

equivalent antioxidant activity than pycnogenol. Devaraj et al.36 reported that pycnogenol had mainly in vivo antioxidant capacity. Regular intake of pycnogenol (150 mg/d) for 6 weeks significantly increases antioxidant capacity of plasma, as determined by ORAC, and exerts favorable effects on the lipid profile. In clinical studies, it was found to counteract increased platelet aggregation in smokers37 and act as an antiinflammatory agent38 in addition to more health promoting properties. Prolongation of Ascorbyl Radical Lifetime of Extracts and Their Fractions from Bark. It was proved that extracts from bark of Norway spruce and their fractions significantly prolonged ascorbyl radical lifetime that was generated by ascorbate oxidase. Results of EPR analysis on Figure 2 shows

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RESULTS AND DISCUSSION We focused on the bark of Norway spruce (byproduct), which according to our study represents a new alternative feedstock of antioxidant and potential medicinal compounds. Hexane extract was rich in sterols and terpenes. Many lipophilic terpenes display anticancer activity.3,15,16 Therefore, it was our aim to isolate nonpolar substances and determine their unknown antioxidant potential and other biological properties. Trolox equivalent antioxidant capacity (TEAC) (Figure 1), prolongation ascorbyl radical lifetime (Figure 2) and stabilization of lipids (Figure 3) with an addition of bark extracts were investigated. We also found out that the compounds of bark displayed significant biological and ADME related properties. Preparation of Bark. Hammer mill was used for grinding of bark to yield particle with size from 0.5 to 1 mm. According to the patent,32 this method ensures similar fraction size and prevents overheating of the material. Nonpolar compounds in amount of 3.8, 4.1 and 5.3% were obtained from milled bark by using the accelerated solvent extraction (ASE) with hexane at 60, 80 and 100 °C. This result was in good correlation with literature.33 The extracts were subjected to fractionation with flash chromatography and similar compounds were combined according to the results of TLC analysis. Antioxidant Capacity of Extracts and Their Fractions from Bark. The finding that isolated fractions had greater antioxidant activity than extract itself was an important discovery. The greatest antioxidant activity of 15 mM/mg dm had fraction 4 obtained at 80 °C (Figure 1). The bark of Chir pine (Pinus roxburghii) has greater antioxidant potential than wood and needles.34 It is known that the bark of Norway spruce (Picea abbies) is a source of antioxidants; however, its antioxidant activity has not been examined sufficiently. Currently, pycnogenol, the extract from bark of French maritime pine (Pinus pinaster), is the most discussed for its antioxidant capacity. Packer et al.35 found out its antioxidant capacity is 6 mM/mg dm, which is consistent with our results (Figure 1). Our results show that extracts from the bark of Norway spruce had greater antioxidant activity than pycnogenol. Even more, isolated fractions 2 and 4 had 2−2.5 times greater trolox

Figure 2. Influence of pycnogenol, hexane extract of Picea abies and its fraction on the lifetime of ascorbate radical.

that lifetime of ascorbyl radical increased from 20 min (solvent DMSO) to 40 and 60 min due to the addition of 200 μg/mL of extract of bark (80 °C) and its fraction 4, respectively. Pycnogenol (200 μg/mL) increased lifetime of ascorbyl radical to 80 min. These results are in accord with the results of Cossins et al.24 that confirmed that pycnogenol may regenerate 8163


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ACS Sustainable Chemistry & Engineering

addition of extracts from the bark. The oil stability index and the different scanning calorimetry best describe the stability of oils extended by the addition of extracts.25 For comparison of ́ et al.39 oxidation stability of our results with literature, Esquivel investigated sunflower oil with addition 1350 mg/kg of supercritical fluid extract (CO2) from Summer savory (Satureja hortensis, L.). Addition of this nonpolar extract was 6.8 times greater than in our experiment (200 ppm). Nevertheless, the protective effect of the extract of Summer savory was negligible with value PF 1.1. Hydrophobic compounds present in the bark of Norway spruce showed better protective activity then extracts form Summer savory and pycnogenol, extract from French maritime pine. Compounds Obtained from Bark Extracts. The most abundant biologically active compounds identified in the hexane extract of the bark of Picea abies were thunbergol > manoyl oxide > β-sitosterol > dehydroabietic acid > Δ3-carene > α-pinene > α-cadinol = docosanol > β-caryophyllene oxide > abietic acid > campesterol > sclareolide > longifolene > quebrachamine in the descending order. In Table 1 and 2 are listed only compounds which according to our in silico analysis and current literature knowledge4,15−18 possess various biological activities especially anticancer and antimicrobial effects. Identification of mentioned compounds in bark of Norway spruce with GC−MS and quantification by GC-FID (Table 1 and 2) have not yet been published. Fractionated compounds were subjected to in silico analysis for prediction pharmacokinetic profiles and biological activities. This information may be critical in the selection of extracts and fractions that could be utilized in functional foods, pharmaceuticals and cosmetics. Predicted Biological Activity and Pharmacokinetic Profiles of Compounds in Bark. The most abundant compounds derived from the hexane extracts of Norway spruce bark were subjected to prediction of biological activities in silico, using the software for PASS. To discover potential and new drugs in the bark of Norway spruce, isolated compounds should not only possess specific biological activities but also display drug-like properties and favorable pharmacokinetic profiles (Table 3). We have confirmed that all analyzed compounds were well absorbed orally in humans and did not show toxic or oxidation activity. In silico profiling of bioactive substances was calculated with different probability thresholds, and results are summarized in Tables 4 and 5. For instance, docosanol displayed almost 140 predicted bioactivities with estimated

ascorbic acid better than most herbal extracts. According to Packer et al.,35 this effect occurs especially in biological systems, where it may also prevent the formation of the ascorbyl radical in a tissue homogenate system challenged by Cu2+. Protective Factor of Extracts and Their Fractions from Bark. The hexane extract obtained at 80 °C from bark of Norway spruce and its fraction 4 had the highest antioxidant capacity. They were added to lard (200 mg/kg) to examine their protection factor (PF), which is expressed as a resistance of lard with antioxidant to accelerated oxidation at 110 °C (Figure 3). Pycnogenol as a commercial extract from the bark

Figure 3. Influence of commercial pycnogenol, hexane extract and one isolated fraction from bark of Norway spruce (Picea abies) on protective factor (PF) of lard before oxidation.

of French maritime was used to compare its antioxidant effect with extractive compounds from the bark of Norway spruce. The best protective factor 1.40 had fraction 4 and hexane extract had protective factor 1.29. Pycnogenol demonstrated only negligible protective factor 1.07. It is probably because it is composed of polyphenols, which protect lipids from oxidation in vivo.36 Butylated hydroxytoluene (BHT) with protective factor 1.87 was the best stabilizer of lard (data not show on figure). Current scientific knowledge does not contain any relevant results of oil stability with the

Table 1. Biologically Active Substances of the First Hydrophobic Fractionsa GC−MS/EI Compounds

a

Retention time [min]

α-Cubebene α-Ylangene Isoledene Longifolene α-Cedrene

9.71 10.09 10.64 10.77 10.89

Manoyl oxide Methyl dehydroabietate

19.02 22.00

Longi-camphenylone β-Caryophyllene oxide

13.45 13.76

Molecule/base ion [m/z] Fraction 1 204/161 204/161 204/161 204/161 204/161 Fraction 2 290/275 359/314 Fraction 3 206/191 220/205

Yield of compounds Other ions [m/z] 119, 119, 119, 119, 119,

105, 105, 105, 107, 105,

81 93 91 91 93

Fractions [%]

Bark [%]

0.2 0.7−1.1 2.0−3.3 18.2−20.7 3.1−5.2

0.01 0.07 0.2 1.4 0.4

257, 192, 177 299, 239, 141

18.0−30.1 0.2−0.7

4.3−7.2 0.2

145, 107, 93 93, 79, 41

1.2−6.2 7.9−13.1

0.1−0.7 0.9−1.6

Obtained from hexane extracts of Picea abies. 8164


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ACS Sustainable Chemistry & Engineering Table 2. Biologically Active Substances of the Second Hydrophobic Fractionsa GC−MS/EI

a

Compounds

Retention time [min]

Borneolb Citronellolb Sclareolide δ3-Carene α-Pinene Spathulenol α-Cadinol Docosanolb Thunbergol Abietic acidb Dehydroabietic acidb Borneolb

6.76 7.56 10.92 11.60 11.70 17.61 18.40 21.13 22.70 27.01 27.66 6.76

α-Terpineol Etiocholanoneb Campesterolb β-Sitosterolb

11.60 23.21 39.21 42.74

Isolongifolol Quebrachamine

20.55 33.66

Yield of compounds

Molecule/base ion [m/z] Fraction 4 226/211 228/143 250/235 136/121 136/122 250/235 222/204 398/383 290/229 374/359 372/357 226/211 Fraction 5 154/136 362/347 472/382 486/396 Fraction 6 222/204 282/253

Other ions [m/z]

Fractions [%]

Bark [%]

108, 95, 73 123, 95, 81 149, 123, 95 105, 93, 77 93, 91, 79 206, 123, 109 161, 121, 95 103, 75, 43 147, 107, 81 256, 241, 73 255, 239, 73 108, 95, 73

0.2−0.6 0.1 5.3−6.1 7.5−9.9 9.2−10.2 0.4−1.3 3.4−7.4 2.0−6.0 28.0−35.0 3.0−5.0 3.5−8.0 0.2−0.6

0.2 0.03 1.7 2.2−2.9 2.9 0.1−0.4 1.0−2.0 0.6−2.0 8.1−10.2 0.9−1.5 3.6−4.4 0.2

121, 272, 343, 381,

0.5−2.6 0.1−0.4 2.2−10.8 23.3−38.9

0.5 0.07 0.4−1.9 3.9−6.8

1.3−3.9 7.3−12.2

0.1−0.4 0.8−1.3

93, 59 244, 75 129, 73 357, 129

109, 75, 41 157, 125, 110

Obtained from hexane extracts of Picea abies. btrimethylsilyl derivates (TMS).

Table 3. Predicted ADME-Related Properties of Obtained Compounds from Bark Substances

Log Po/wa

CI Log Sb

Log B/Bc

Log Kpd

Log Khsae

Log HERGf

#metabg

#starsh

Abietic acid Campesterol Dehydroabietic acid Docosanol Longifolene Manoyl oxide Quebrachamine Sclareolide Thunbergol α-Cadinol α-Pinene β-Caryophyllene oxide β-Sitosterol Δ3-Carene

4.91 7.16 4.92 7.55 4.81 4.20 4.01 2.86 5.50 3.96 3.61 2.51 7.39 3.61

−4.72 −6.74i −4.80 −5.14 −5.87 −7.57i −3.38 −3.07 −4.23 −3.41 −3.98 −4.30 −7.03i −3.98

−0.21 −0.27 −0.19 −1.32 1.03 0.87 0.75 0.08 0.42 0.16 0.87 0.11 −0.33 0.87

−2.54 −1.73 −2.44 −0.41j −1.45 −1.36 −3.20 −2.70 −1.30 −1.92 −1.42 −1.44 −1.65 −1.41

0.78 1.97k 0.77 1.46 0.87 1.27 0.81 0.25 1.23 0.66 0.34 0.37 1.97k 0.34

−1.90 −4.51 −2.11 −5.95l −2.72 −2.93 −4.90 −2.52 −3.00 −3.20 −2.77 −2.43 −4.42 −2.77

4 3 2 1 1 1 3 1 8m 4 3 2 3 3

0n 6 0n 9 7 6 0 2n 4n 3n 7 4n 6 7

Log Po/w: predicted octanol/water partition coefficient, range of values of 95% of known drugs is −2.0−6.5. bCI Log S: conformation-independent predicted aqueous solubility, range of values of 95% of known drugs is −6.5−0.5. clog B/B: predicted brain/blood partition coefficient for orally delivered substances, range of values of 95% of known drugs is −3.0−1.2. dLog Kp: predicted skin permeability, range of values of 95% of known drugs is −9.0 − −1.0. eLog Khsa: prediction of binding to human serum albumin, range of values of 95% of known drugs is −1.5−1.5. fLog HERG: predicted IC50 value for blockage of HERG K+ channels, concern below −5. g#metab: number of likely metabolic reactions, range of values of 95% of known drugs is 1−8. h#stars: number of property or descriptor values that fail outside the 95% range of similar values for known drugs (based on 24 QikProp descriptors), range of values of 95% of known drugs is 0−5. iCompounds that fail the CI log S test. jCompound over limit of log B/B test. kCompounds over limit of log Khsa test. lPotential cardiac toxicity. mCan easily gain access to the target site after entering the bloodstream. n Compounds that display favorable pharmacokinetic properties. a

Biological Activity of Compounds in Fraction 1. According to the results shown in Figure 1, this fraction had low antioxidant activity, but contained compounds (Rf 0.71− 0.75) with probably interesting health promoting properties. The most abundant compound (18−21%) in this fraction was longifolene, although it constituted only one percent of all compounds in the bark (Table 1). It was the most hydrophobic fraction, obtained by hexane elution only. The same conclusion was also made by Gordien et al.40 who isolated in the same way the first fraction from roots of Juniperus communis containing

probabilities larger than 90% (in Table 4 is shown only the 15 most probable activities). On the other hand, thunbergol was suggested only carminative activity (probability 80−90%) by PASS software. Computer predictions of thunbergols further biological activities were calculated with lower probability threshold (70−80%). These included antieczematic, CYP3Ax substrate, antineoplastic activity, then H+ exporting ATPase inhibitor and Alzheimer’s disease treatment. Therefore, thunbergol provides space for further research. 8165


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ACS Sustainable Chemistry & Engineering Table 4. Biological Activities of Obtained Compounds with Probability 90−100%a Substances Abietic acid Campesterol Dehydroabietic acid Docosanol

Longifolene Manoyl oxide Quebrachamine Sclareolide Thunbergol α-Cadinol α-Pinene β-Caryophyllene oxide β-Sitosterol Δ3-Carene a

Possible types of biological activities with probability 90−100% mucomembranous protector, antihypercholesterolemic DELTA14-sterol reductase inhibitor, prostaglandin-E2 9-reductase inhibitor, antihypercholesterolemic, cholesterol antagonist, alkenylglycerophosphocholine hydrolase inhibitor, alkylacetylglycerophosphatase inhibitor, UGT1Ax substrate, testosterone 17β-dehydrogenase (NADP+) inhibitor, CYP7 inhibitor and CYP2C substrate, acylcarnitine hydrolase inhibitor mucomembranous protector, testosterone 17β-dehydrogenase (NADP+) inhibitor sugar-phosphatase inhibitor, alkenylglycerophosphocholine hydrolase inhibitor, carboxypeptidase Taq inhibitor, alkylacetylglycerophosphatase inhibitor, alkylglycerone-phosphate synthase inhibitor, dextranase inhibitor, glucan 1,4-α-maltotriohydrolase inhibitor, fucosterol-epoxide lyase inhibitor, pullulanase inhibitor, gluconate 5-dehydrogenase inhibitor, alkanal monooxygenase (FMN-linked) inhibitor, peptide-N4-(N-acetyl-βglucosaminyl) asparagine amidase inhibitor, ubiquinol-cytochrome-C reductase inhibitor, polyporopepsin inhibitor, sphinganine kinase inhibitor dermatologic, CYP2J substrate growth hormone agonist − − − − − antineoplastic antihypercholesterolemic, DELTA14-sterol reductase inhibitor, prostaglandin-E2 9-reductase inhibitor, cholesterol antagonist, CYP7 inhibitor, alkenylglycerophosphocholine hydrolase inhibitor, alkylacetylglycerophosphatae inhibitor, hypolipemic, acylcarnitine hydrolase inhibitor, testosterone 17β-dehydrogenase (NADP+) inhibitor −

Possible types of biological activities (PASS) of obtained compounds from the bark of Norway spruce.

Table 5. Biological Activities of Obtained Compounds with Probability 80−90%a Substances Abietic acid Campesterol Dehydroabietic acid Docosanol Longifolene Manoyl oxide Quebrachamine Sclareolide Thunbergol α-Cadinol α-Pinene β-Caryophyllene oxide β-Sitosterol Δ3-Carene a

Possible types of biological activities with probability 80−90% oxidoreductase inhibitor, testosterone 17β-dehydrogenase (NADP+) inhibitor, hypolipemic, alkenylglycerophosphocholine hydrolase inhibitor, CYP2J substrate, antieczematic UDP-glucuronosyltransferase substrate, oxidoreductase inhibitor, CYP3Ax inducer and substrate, cholestanetriol 26-monooxygenase inhibitor, linoleate diol synthase inhibitor, Dextranase inhibitor, CYP4Bx substrate, hypolipemic, UGT2Bx substrate, anesthetic general CYP2J substrate, antihypercholesterolemic, oxidoreductase inhibitor, alkenylglycerophosphocholine hydrolase inhibitor, urease inhibitor, antieczematic − testosterone 17β-dehydrogenase (NADP+) inhibitor, antineoplastic, antieczematic ecdysone 20-monooxygenase inhibitor 5 hydroxytryptamine release stimulant antineoplastic, CYP2J substrate carminative antieczematic, CYP2C12 substrate testosterone 17β-dehydrogenase (NADP+) inhibitor, CYP2J substrate, cardiovascular analepti − UDP-lucuronosyltransferase substrate, UGT2Bx substrate, oxidoreductase inhibitor, anesthetic general, CYP3Ax, CYP4Bx and other CYPs substrate and inducer, adenomatous polyposis treatment, dextranase inhibitor, linoleate diol synthase inhibitor, chemopreventive −

Possible types of biological activities (PASS) of obtained compounds from the bark of Norway spruce.

mainly longifolene. We predicted that longifolene had most likely the dermatologic and CYP2J substrate activity (Table 4). Less likely properties (80−90%) generated by PASS software are presented in Table 5. It even has a number of positive pharmacokinetic properties, but fails in #stars test (Table 3). This means they will most probably fail in the course of pharmaceutical development due to the inadequate pharmacokinetics. Gordien et al.40 found out that these compounds inhibited in 97.7% Mycobacterium tuberculosis at 100 μg/mL. The next result indicates that longifolene was relatively toxic toward mammalian cells, especially when compared to the high selectivity of the antibiotic control.40 Hence longifolene probably exhibits significant biological properties, but its real pharmacokinetic properties required further examination.

This fraction contained following compounds with only minor concentration and therefore have not been subjected to PASS and QikProp software analysis. In other plants are higher concentration with important biological properties. Like αcubebene, which is in trace concertation in bark of spruce, bud essential oil from leaves of Ocimum basiliscum contained 5.7% of this compounds with significant antifungal activity on Aspergillus flavus.41 Then we confirmed the presence of ylangene in the bark of Norway spruce (Table 1). According to Xio et al.,42 it possesses cytotoxic and anti-inflammatory activity. Bark extract contained only 0.2% of isoledene, but cedar hardwood is rich in isoledene (12%).43 The same authors found this compound had selective cytotoxic effects toward human 8166


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protection factor (Figure 3). It was collected compounds with Rf 0.19 (Figures S1−S3). The most abundant (8−10%) hydrophobic compound in the bark is thunbergol (Table 2), which had 80−90% probability of carminative effect (Table 3). Our results of ADME-related properties confirmed that it is a pharmacologically important substance. Thunbergol was only verified compound that reached the maximum points in the #metab test, indicating the number of likely metabolic reactions. It is interesting to note that current research did not focus on the biological activity of thunbergol. Our study confirmed thunbergol to be responsible for the antioxidant capacity of isolated fraction number 4 (15 mM/mg dm for the 80 °C fraction). This result is in conformity with the literature.50 The bark of Norway spruce contained 2.9% of α-pinene, which was with 80−90% probability predicted to be a testosterone 17β-dehydrogenase (NADP+) inhibitor, CYP2J substrate and cardiovascular analeptic properties. ADMErelated properties fail in their pharmacokinetic characteristics only in #stars test (Table 3). Hafizoglu et al.51 found out that the bark of young tree Picea orientalis contained 4.7% of αpinene. During aging of trees, its content decreases until complete decomposition. Silva et al.4 found out that only the (+)-enantiomers of the α- and β-isomers of pinene were antimicrobial active. Time-kill curves showed that α-pinene and β-pinene were highly toxic to Candida albicans, killing 100% of inoculum within 60 min. The bactericidal effect was manifested after 6 h in methicillin-resistant S. aureus (MRSA). Δ3-Carene was one of the dominant monoterpenes (2.2− 2.9%) in the bark of spruce. According to Hafizoglu et al.,51 Δ3carene was the dominant monoterpene (14.1%) present in the bark of Picea orientalis. From in silico analysis resulted that Δ3carene showed significant biological activities and ADMErelated properties were in the same relation as in α-pinene (Table 3). Literature52 confirmed that Δ3-carene had antiinflammatory activity and antispasmodic activity against oxytocin. The content of diterpene sclareolide found in bark was 1.7%. Sclareolide demonstrated with 80−90% probability antineoplastic and CYP2J substrate activity (Table 5) with acceptable ADME-related properties (Table 3). Jasiński et al.53 found out that sclareolide is one of the most abundant diterpenes of the leaf surface of Nicotiana spp. with antimicrobial and growthregulating activity. Another no less interesting substance whose presence was confirmed in the bark of spruce was α-cadinol. It was represented 1−2% in bark, which is consistent with the literature.18 We confirmed that compounds had relevant pharmacokinetic profiles (Table 3). PASS software predicted antieczematic and CYP2C12 substrate activity with 80−90% probability. It has been found,54 that exceptionality of α-cadinol lies in its in vivo hepatoprotective effect due to the retardation reaction of viral hepatitis. Then according to Tung et al.54 αcadinol may be used against resistant tuberculosis as a substance with drug potential. Saturated alcohol docosanol (C22) was identified in hexane extract of bark. The amount 0.6−2% in bark extract made it suitable for use in cosmetics and food nutrient supplement. Despite the fact that this compound had multiple biological properties, Table 4 shows only 15 most probable activities. It should be noted, that docosanol may be potential cardiac toxins (log HERG below −5) according to in silico analysis. Katz et al.55 concluded that docosanol had an inhibitory effect of the

CRC lines. It can be noted that CRC is one of the most common human malignant tumors.44 The last compound of this fraction was α-cedrene with a small concentration in bark of Norway spruce. This compound had the favorable pharmacokinetic characteristics to be further tested as an antiobesity drug in clinical studies.45 Biological Activity of Compounds in Fraction 2. This fraction represents a promising source of antioxidants, and biologically active substances that had a response factor 0.68 on TLC. It is the second isolated fraction representing on average one-fifth of the entire bark mass. The results displayed in Figure 1 show that this fraction has the second highest antioxidant activity of all fractions. It contained the second most represented (4.3−7.2%) biologically active substance, manoyl oxide, in the bark (Table 1). The PASS software predicted manoyl oxide to be a growth hormone agonist with high probability threshold (Table 4). But its Ci log S and #stars result (Table 3) were on the edge of acceptability of pharmacokinetic ADME-related properties. Demetzo et al.15 isolated and identified manoyl oxide and its derivative in the resin of Cistus creticus. In the study, the authors confirmed that these compounds exhibited a cytotoxic effect against on cell lines MOLT 3, cell lines T, H9 cells originating from a patient with acute lymphoblastic leukemia. Other authors also confirmed, that manoyl oxide and its derivatives had in vitro cytotoxic activity against nine leukemic cell lines.46 Methyl dehydroabietate was in bark of Norway spruce minor compounds 0.2%, so the in silico analysis was not carried out. But Kinouchi et al.16 showed potent inhibitory effects on Epstein−Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. Biological Activity of Compounds in Fraction 3. This fraction did not excel in antioxidant capacity, but contains compounds with important biological properties. We identified excellent biologically active compounds in the fraction 3 with Rf 0.35 (Table 1). The first one was βcaryophyllene oxide (0.9−1.6% in the bark), which had antineoplastic properties with 90−100% probability (Table 4). In silico profiling of β-caryophyllene oxide based on a lower probability threshold suggested additional biological activities such as apoptosis agonist, antieczematic, HIF1A expression inhibitor (Table 5). These results were supported with promising ADME-related properties. Most likely, β-caryophyllene oxide can find application as an antineoplastic used in the treatment of metastases. In compliance with our results, Shahwar et al.47 showed 85% cytotoxicity of β-caryophyllene oxide against A-2780 human ovarian cancer cell lines. Kim et al.48 came up with a breakthrough confirming that βcaryophyllene oxide was a novel STAT3 signaling cascade blocker and thus is an enormous potential for the treatment of various cancers harboring constitutively activated STAT3. Authors identified these compounds in essential oils of guava (Psidium guajava), and oregano (Origanum vulgare L.). Finally, this fraction contained longicamphenylone, which represented 0.7% of hydrophobic compounds of bark. Yang et al.49 found out that longicamphenylone exhibited potent antidepressant activity in vitro by inhibiting reuptake in rat brain synaptosomes by 55%. Biologically Active Compounds in the Fraction 4. This fraction represents one-third of the total extract and had the highest antioxidant potential as demonstrated by TEAC (Figure 1), extension the lifetime of ascorbate radical (Figure 2) and 8167


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α-Terpineol and etiocholanone are the next biological active compounds, which were in minor concentration. It has been reported by Hassan et al.61 that α-terpineol enhances the permeability of the skin to the lipid soluble compounds. According to the authors, α-terpineol has been shown to have also antibacterial, antifungal, anti-inflammator and anticancer activities. Then, etiocholanone increases the activity of GABAA receptors which inhibit the effect of conduction, as borneol, which was also isolated.62 Biologically Active Compounds in Fraction 6. The last (most polar) fraction was not so rich in biologically active substances. First, the most abundant compound (0.8−1.3%) was alkaloid quebrachamine. Its name was derived from the name of tree Quebracho, which is rich in alkaloid quebrachamine. Among its biological properties is 5-hydroxytryptamine release, predicted only with 80−90% probability. Its ADMErelated properties were successful for pharmacology application. According to Deutsch et al.,63 aspidospermine and quebrachamine from Quebracho tree have been found to possess adrenergic blocking activities for a variety of urogenital tissues. Isolongifolol was the minor biological active compound of this fraction (Table 2). It has been found out that this compound is butyrylcholinesterase inhibitor, which could contribute to the discovery of clinically useful agents by reducing amnestic disorders in patients with Alzheimer’s disease.64

replication of herpes simplex viruses and respiratory virus. Even docosanol was approved by the Food and Drug Administration (FDA) as a pharmaceutical antiviral agent to reduce the duration of cold sores caused by herpes simplex virus. Abietic acid and dehydroabietic acid belong to the diterpene family, which are primary compounds of resin acids. The content of abietic acids (0.9−1.5%) and dehydroabietic acids (3.6−4.4%) in bark of spruce correspond to literature.51 Abietic acid has antibacterial activity attributed to the mechanism which we have proposed in graphical abstract. Our hypothesis is in accordance with the predicted biological properties. It serves as a mucomembranous protector and antihypercholesterolemic agent with 90−100% probability (Table 4). Biological results with 80−90% indicate their versatile application (Table 5). In Table 3 is described their excellent ADME-related properties. Because of their widespread effects on the microorganisms, they have potential application in tissue and skin regeneration where conventional medicines do not work by creating resistances in microorganisms.17 Dehydroabietic acid shows similar biological activity like abietic acid (Table 4). Interesting is also its antihypercholesterolemic activity with 90−80% probability. If we take into account positive ADME-related properties of dehydroabietic acid (Table 3), it can be effective against elevated serum cholesterol levels and the level of LDL particles. This information was in accordance with literature.56 The authors found out that with administration reduced blood glucose, insulin and triglycerides in plasma as well as the hepatic triglycerides. Dehydroabietic acid further reduced the accumulation of macrophages in adipose tissue. The results indicate that dehydroabietic acid is a useful compound related to prevention of diseases that may be formed during diabetes.56 Spathulenol, borneol and citronellol were further compounds in bark of Norway spruce that were found in minor concentration, thus they were not included in the in silico analysis. According to literature,57 spathulenol enhances the effects of chemotherapy in cancer patients. Borneol is a very well-known compound, used in Chinese medicine as an anesthetic and analgesic.58 Biologically Active Compounds in Fraction 5. Campesterol was present at 0.4−1.9% in the extract of bark. The most interesting predicted biological properties were prostaglandinE2 9-reductase inhibitor, antihypercholesterolemic, cholesterol antagonist activity with 90−100% probability. Its biological properties are in accord with the literature.59 It is postulated that the molecules of campesterol “compete” with cholesterol for binding place and thus reduce the absorption of cholesterol in the intestine. But failed in CI log S, log Khsa and #stars (Table 3). From this can be concluded that it is challenging for sterols to get to the target site via the bloodstream. Therefore, they should be ingested in slight excess. β-Sitosterol is the next interesting substance present at 3.9− 6.8% in the extract of bark. Predicted biological properties (Tables 4 and 5) and ADME-related properties (Table 3) were similar as in campesterol. At present, the need for phototherapeutic preparations containing β-sitosterols grows. It has been shown to be effective in clinical trials for medical benign prostatic hyperplasia (BPH) therapy regarding symptoms and uroflow parameters.60 Moreover, the authors reported that βsitosterols had anti-inflammatory effects, alteration of cholesterol metabolism, antiandrogenic and antiestrogenic effects and decrease in sex-hormone-binding globulin.

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CONCLUSION This study is the first investigation of a novel bioresource of potential antioxidant, medicinal compounds from the waste biomass of spruce. The most abundant and biologically valuable ones were terpenoids like abietic acid, dehydroabietic acid and β-caryophyllene oxide. The fractions 2 and 4 were rich in these compounds and possessed 2−2.5 times greater trolox equivalent antioxidant activity than noted pycnogenol extract. Moreover, abietic and dehydroabietic acids have high probability mucomembranous protectors and show significant antihypercholesterolemic effect. Most likely, β-caryophyllene oxide could have application as an antineoplastic used in the treatment of metastases. Its promising biological activity may have application in food, packaging and pharmaceutical development.

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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/acssuschemeng.7b01816.

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TLC analysis of obtained compounds eluted form column chromatography (PDF)

AUTHOR INFORMATION

Corresponding Author

*F. Kreps. E-mail: [email protected]. Tel.: (+421) 2 52493198. ORCID

František Kreps: 0000-0002-5607-4821 Notes

The authors declare no competing financial interest. 8168


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ACKNOWLEDGMENTS This work was supported by the Slovak Scientific Grant Agency contract no. VEGA-1/0353/16. This work was supported by the Slovak Research and Development Agency under the contract No. APVV-16-0088, No. APVV-15-0052 and under the contract No. APVV-14-0393. The work was further supported by a grant for young scientists at STU under number 1675.

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