(Sieb. Et Zucc.) Wood in LPS-Stimulated RBL-2H3 Cells

biomolecules

Article Investigation of Active Anti-Inﬂammatory Constituents of Essential Oil from Pinus koraiensis (Sieb. et Zucc.) Wood in LPS-Stimulated RBL-2H3 Cells

Jiyoon Yang 1,2 , Won-Sil Choi 3, Ki-Joong Kim 2 , Chang-Deuk Eom 4 and Mi-Jin Park 1,*

1 Division of Forest Industrial Materials, Department of Forest Products and Industry, National Institute of Forest Science, Seoul 02455, Korea; [email protected] 2 Division of Life Sciences, School of Life Sciences, Korea University, Seoul 02841, Korea; [email protected] 3 National Instrumentation Center for Environmental Management, Seoul National University, Seoul 08826, Korea; [email protected] 4 Division of Wood Industry, Department of Forest Products and Industry, National Institute of Forest Science, Seoul 02455, Korea; [email protected] * Correspondence: [email protected]

Abstract: In a previous study, we demonstrated the anti-inflammatory activity of the essential oil extracted from Korean pine (Pinus koraiensis, Sieb. et Zucc.) wood. This study aims to investigate the active anti-inflammatory constituents of P. koraiensis oil. The essential oil was extracted from P. koraiensis wood by hydrodistillation and was divided into six fractions (A–F) through fractional distillation. Then, the anti-inflammatory activities of the fractions (A–F) were determined. Fractions A and F markedly downregulated the production of pro-inflammatory cytokines as well as the Citation: Yang, J.; Choi, W.-S.; Kim, secretion of β-hexosaminidase in lipopolysaccharide (LPS)-stimulated RBL-2H3 cells. The main K.-J.; Eom, C.-D.; Park, M.-J. constituents of the active anti-inflammatory A and F fractions were (+)-α-pinene, (−)-β-pinene, Investigation of Active (+)-α-terpineol, 3-carene, (+)-limonene, and longifolene. These six single compounds decreased Anti-Inflammatory Constituents of Essential Oil from Pinus koraiensis the expression of inflammatory-related genes (i.e., IL-4 and IL-13) as well as the secretion of β- (Sieb. et Zucc.) Wood in hexosaminidase in LPS-stimulated RBL-2H3 cells. (+)-α-Pinene, (−)-β-pinene, (+)-α-terpineol, and LPS-Stimulated RBL-2H3 Cells. longifolene exhibited the strongest effects; these effects were comparable to those of the positive Biomolecules 2021, 11, 817. https:// control (i.e., dexamethasone). The findings indicate that the interactions between these components doi.org/10.3390/biom11060817 exhibit potential for the management and/or treatment of inflammatory conditions as well as base structures for the development of novel anti-inflammatory drugs. Academic Editor: Vladimir N. Uversky Keywords: Pinus koraiensis; essential oil; anti-inflammatory; active compound; α-pinene; β-pinene; α-terpineol; longifolene Received: 25 March 2021 Accepted: 26 May 2021 Published: 31 May 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in The Korean pine (Pinus koraiensis Siebold and Zuccarini) grows in Russia, Korea, published maps and institutional affil- China, and Japan at locations higher than 1000 m above sea level. It can reach 1 m in iations. diameter and 20–30 m in height [1]. The lignum of P. koraiensis is hard and has been widely used in North Asia and Korea as a furniture material for a long time. Further, the seeds of P. koraiensis contain high-quality fatty acids, amino acids, carbohydrates, and vitamins [2]. As a result, P. koraiensis has been used as a food supplement and in traditional Asian medicine for thousands of years. Studies have reported that the extract of P. koraiensis Copyright: © 2021 by the authors. P. Licensee MDPI, Basel, Switzerland. bark exhibits antitumor, antioxidant, antiaging, and antimutagenic activities. Therefore, This article is an open access article koraiensis is a species with very high economic value and has been selected as one of the 10 distributed under the terms and most economically and culturally important trees in Korea [3,4]. In addition, research has conditions of the Creative Commons recently been conducted in various fields, including breeding of P. koraiensis and its use as Attribution (CC BY) license (https:// a genetic resource [5]. creativecommons.org/licenses/by/ Inflammation is a physiological response to a variety of agents, including infectious 4.0/). microorganisms, toxic chemicals, and physical injury [6]. Many diseases are associated with

Biomolecules 2021, 11, 817. https://doi.org/10.3390/biom11060817 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 817 2 of 16

inflammation, including skin inflammation and auto-immune diseases, such as arthritis and diabetes, Alzheimer’s disease, and cancer [7]. Inflammation is mediated by a complex interaction of pro- and anti-inflammatory mediators, the perturbation of which is associated with an exacerbated release of proinflammatory cytokines and consequent deleterious effects [8] Inflammatory mediators produced from activated mast cells and T helper type 2 (Th2) cells drive allergic inflammation. Th2 cells induce allergic inflammation through the production of cytokines, such as interleukin (IL)-4, IL-10, and IL-13 [9]. IL-4 and IL-13 are central to type-2 inflammation; therefore, they represent targetable candidates for the amelioration of allergic disease [10]. For example, IL-4 and IL-13 are required to drive most of the key processes associated with type-2 inflammation, including immunoglobulin E (IgE) production, mucus production, and innate cell recruitment to sites of inflammation [11,12]. Given the key role of IL-4 and IL-13 in type-2 inflammation, anti-inflammatory activity can be confirmed via changes in the expression levels of these two cytokines. Many drugs that prevent or minimize the progression of inflammation are available, including non-steroidal anti-inflammatory drugs and corticosteroids. However, regular use of non-steroidal anti-inflammatory drugs results in a number of side effects, some of which can be very serious. Moreover, the prolonged use of corticosteroids can suppress pituitary– adrenal function, evoke hyperglycemia, and increase susceptibility to infections [13]. As a result, the use of natural medicine for the amelioration of inflammation has become an active research area. The biological activities of many plants have been exploited in ethnomedicine for the treatment of inflammatory diseases. These biological properties are often imparted by essential oils contained in medicinal plants. Such essential oils exhibit different biological activities, including anti-inflammatory, antioxidant, antibacterial, antiaging, and anti- melanogenic activities [14,15]. In a previous study, we highlighted the anti-inflammatory effect of the essential oil (10−7%) extracted from P. koraiensis wood on lipopolysaccharide (LPS)-stimulated rat basophilic leukemia (RBL)-2H3 cells [16]. We assessed the effect of wood oil on IL-4 and IL-13 mRNA production using the LPS-stimulated RBL-2H3 cell line. The essential oil extracted from P. koraiensis wood significantly decreased the relative mRNA expression of IL-4 (to 36.8%) and IL-13 (to 42.9%) at 10−7%, compared with the LPS-stimulated RBL-2H3 cells. The P. koraiensis oil showed an inhibitory effect on the β-hexosaminidase release (to 72.6%), but the effect was insignificant compared with other oils. These results suggest that the essential oil had immunostimulatory effects on T cells and meaningfully inhibited allergy-associated cytokines IL-4 and IL-13. Although the anti-inflammatory effect of the essential oil extracted from P. koraiensis wood has been confirmed, its anti- inflammatory constituents have not been identified. Accordingly, the present work aims to study the bioactive constituents of the essential oil from P. koraiensis wood in LPS-stimulated RBL-2H3 cells.

2. Materials and Methods 2.1. Chemicals (+)-α-Pinene (Cat #. 80604, purity ≥97.0%), (+)-α-terpineol (Cat #. 83073, purity ≥97.0%), 3-carene (Cat #. 94415-5ML, purity ≥97.0%), and R-(+)-limonene (Cat #. 62118-5ML, purity ≥95.0%) were purchased from Fluka (Buchs, Switzerland). (−)-β-Pinene (Cat #. 402753-10G, purity ≥97.0%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). (+)-Longifolene (Cat #. 5395 S, purity ≥95.0%) was purchased from Extrasynthese (Rue Jacquard, Genay, France).

2.2. Plant Material The wood of P. koraiensis (Sieb. et Zucc.) was collected from Gapyeong, Gyeonggi Province of Korea, in January 2015. The wood was milled and the sawdust obtained was used for the extraction of essential oil. Biomolecules 2021, 11, 817 3 of 16

2.3. Extraction of Essential Oil and Its Fractioanl Distillation The obtained sawdust of the P. koraiensis wood was hydrodistilled at atmospheric pressure using a Clevenger-type apparatus. A 10 L round-bottom flask containing 1.0 kg of sawdust was placed on a digital heating mantle (MTOPS) and 6.0 L of distilled water was poured into the flask, which was then connected to the Clevenger-type apparatus. The sawdust was extracted for 7 h. The obtained glass green (Pantone color. 11-0205 TPG) essential oil was dried over anhydrous sodium sulfate (Samchun) and filtered through a 0.45 µm membrane disk filter (ADVANTEC). For the separation of essential oil, fractional distillation was performed under atmospheric pressure [17]. The oil fractions (A, B, C, D, E and F) obtained at different temperatures are presented in Table1.

Table 1. Temperature conditions for the fractionation of the essential oil of P. koraiensis wood.

Oil Fraction Temperature Range (◦C) A 50–65 B 65–75 C 75–81 D 81–82 E 82–94 F Residues

The six oil fractions were then transferred to dark vials and stored at 4 ◦C until further analysis. The yield of the oil fractions was calculated using the following equation (Equation (1)):

Recovered mass of the essential oil (g) Oil fraction yield (%) = × 100 (1) Initial mass of the essential oil (g)

2.4. GC–MS Analysis To identify the volatile components in the oil fractions, the six oil fractions (A–F) were analyzed by GC–MS (Thermo Fisher Scientific, Waltham, MA, USA) using the DB-5MS capillary column (30 m × 0.25 mm; 0.25 µm; Thermo Scientific, Waltham, MA, USA) and a flame ionization detector (FID) as described previously (Table2)[16].

Table 2. Operating parameters of GC–MS.

DB-5MS capillary column Column (30 m × 0.25 mm; 0.25 µm; Thermo Scientific, USA) 50 ◦C, hold 5 min 10 ◦C/min to 65 ◦C, hold 30 min 5 ◦C/min to 120 ◦C, hold 10 min GC oven conditions 5 ◦C/min to 180 ◦C 5 ◦C/min to 210 ◦C, hold 10 min 20 ◦C/min to 325 ◦C, hold 10 min Carrier gas He (1 mL/min, 25 psi) Linear velocity 19.8 cm/s Injection mode Split 1:20 Injection temperature 250 ◦C MS parameters FID parameters MS ionization mode EI FID temperature 300 ◦C Scan time 0.2 s Hydrogen flow 35.0 mL/min Mass range 35–550 amu Air flow 350.0 mL/min Ion source 270 ◦C Make up flow 40.0 mL/min temperature Interface temperature 250 ◦C Biomolecules 2021, 11, 817 4 of 16

The components of the six oil fractions were identified based on peaks with the highest spectral matching using the National Institute of Standards and Technology (NIST; Gaithers- burg, MD, USA) library search program (version 11). The Kovats retention index (KI) values of the individual compounds were determined by comparing their relative retention times with those of an n-alkane mixture (C8–C20, Sigma-Aldrich, Missouri, USA) using the DB-5MS column (Thermo Scientific). The essential oil components were identified by comparing their calculated KI values with literature values (e.g., NIST Chemistry WebBook).

2.5. Cell Culture The rat basophilic leukemia cell line (RBL-2H3, KCLB no. 22256) was obtained from Korea Cell Line Bank (Seoul, Korea). RBL-2H3 cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM, Welgene, Gyeongsan-si, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, Co Dublin, Ireland), 1% penicillin–streptomycin (Gibco), and ◦ 0.4 µL/mL Plasmocin (Invivogen, San Diego, CA, USA) at 37 C in a 5% CO2 incubator (Panasonic, Osaka, Japan).

2.6. Cell Cytotoxicity The microdilution method of CLSI M07-49 was modified and used to evaluate the cell cytotoxicity (10−5–10−7%) [18]. Cell cytotoxicity was measured using Cell Counting Kit (CCK)-8 (DoGenBio, Seoul, Korea) according to the manufacturer’s instructions. Briefly, the oil fractions and single compounds were prepared in DMSO. A stock solution of the ingredients to be tested were prepared by diluting 100 µL ingredients in 900 µL DMSO. The cells were seeded into 96-well plates at a density of 8 × 103 cells/well, and then, stock solutions were added to the well plate to give a final concentration of 10−5–10−7% for 24 h. After treatment, 10 µL of CCK-8 solution was added to each well and the 96-well plate was incubated at 37 ◦C for 1 h. Then, the optical density (OD) value of each well was determined using a microplate reader (Epoch, Winooski, VT, USA) at a wavelength of 450 nm to obtain the cell viability data. The percentage of viable cells was calculated using the following equation (Equation (2)):

(At − Ab) Cell viability(%) = × 100 (2) (Ac − Ab)

where, At is the absorbance of treated cells, Ab is the absorbance of the blank, and Ac is the absorbance of the control.

2.7. Determination of the Levels of Cytokines IL-4 and IL-13 via Quantitative Real-Time PCR Total RNA was isolated from RBL-2H3 cells as done in a previous study [19]. RBL-2H3 cells were grown on 6-well plates (3 × 105 cell/well) for 24 h. The cells were stimulated with 1 µg/mL LPS (Sigma-Aldrich) for 1 h and then washed twice with DPBS (Gibco). The cells were treated with 200 µL dexamethasone, oil fraction (A–F), and the single compounds (10−7%) for 24 h. Total RNA was isolated using the TRIzol reagent (Invitrogen) and quantified using a spectrophotometric microplate reader at 260 and 280 nm. Total RNA was reverse transcribed into first-strand complementary DNA (cDNA) using M-MLV reverse transcriptase (Invitrogen) and random primers (Takara Bio Inc., Shiga, Japan). Each cDNA sample was amplified with 2× SYBR® Premix Ex Taq (Takara Bio Inc.) and 3 pmol of each primer. The oligonucleotide primers used for PCR (Foster) amplification that were obtained from target cellular RNA are listed in Table3. Biomolecules 2021, 11, 817 5 of 16

Table 3. Oligonucleotide primer sequences used for quantitative real-time polymerase chain reaction.

Gene Primer Sequence (50–30) Accession No. F: TGA TGT ACC TCC GTG CTT GA IL-4 X16058 R: AGG ACA TGG AAG TGC AGG AC F: CTG GAA TCC CTG ACC AAC AT IL-13 L26913 R: CCA TAG CGG AAA AGT TGC TT F: CCA CAG TCC ATG CCA TCA C GAPDH NM_017008.4 R: TCC ACC ACC CTG TTG CTG TA F; forward, R; reverse.

PCR ampliﬁcation was performed under 40 cycles of denaturation at 95 ◦C for 15 s, annealing at 60 ◦C for 30 s, extension at 72 ◦C for 30 s, and ﬁnal elongation at 72 ◦C for 10 min. The IL-4 and IL-13 gene expression Ct values were normalized to the corresponding values for the 18S gene expression using the RQ software (version 1.3, Applied Biosystems, Bedford, MA, USA).

2.8. β-Hexosaminidase Secretion Assay The degranulation response of RBL-2H3 cells was quantiﬁed by measuring the level of β-hexosaminidase released in the supernatants [20]. RBL-2H3 cells were grown on 6-well plates at a density of 2 × 105 cells for 24 h. The cells were then treated with 800 ng/mL DNP-speciﬁc IgE (Sigma-Aldrich) overnight. To remove the excess IgE, the cells were washed twice with Tyrodes’ assay buffer (pH 7.3). The cells were then stimulated with DNP-BSA (Invitrogen), suspended in 500 µL of extracellular buffer with 0.1% BSA, and incubated at 37 ◦C for 1 h. After incubation, 50 µL of the supernatant was incubated with 200 µL of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) in 0.05 M citrate buffer (pH 4.5) at 37 ◦C for 1 h. The enzyme reaction was terminated by adding 500 µL of sodium carbonate buffer. The OD of each reaction was recorded at 405 nm. The percentage of viable cells was calculated using the following equation (Equation (3)):

Am β − hexosaminidase sec retion (%) = × 100 (3) (Al − Am)

where, Al is the absorbance of the cell lysate and Am is the absorbance of the medium.

2.9. Statistical Analysis The results are expressed as mean ± standard deviation (n = 3). Differences between vehicle control and treatment groups were tested using two-way ANOVA followed by multiple comparisons by the Tukey test. The p values lower than 0.05 were considered statistically signiﬁcant.

3. Results 3.1. Yield and Chemical Composition P. koraiensis wood oil was divided into six fractions (A–F) using fractional distillation. The yields of the fractions are presented in Table4.

Table 4. Yields of the fractions (A–F) isolated from P. koraiensis wood oil.

Weight of Oil Yield (%) Fraction (g) Fraction A 4.0 15.2 Fraction B 0.7 2.6 Fraction C 6.1 23.4 Fraction D 1.4 5.5 Fraction E 1.7 6.4 Fraction F 12.3 46.9 Biomolecules 2021, 11, 817 6 of 16

The chemical compositions of the oil fractions as determined via GC–MS are presented in Table5.

Table 5. Chemical compositions of six fractions identiﬁed by GC–MS analysis.

Area % KI a Compound Name b Wood Oil Fraction A Fraction B Fraction C Fraction D Fraction E Fraction F Monoterpene hydrocarbons 913 β-Ocimene - - - - 0.01 - - 919 Tricyclene 0.08 0.23 0.24 0.17 0.05 - - 921 α-Thujene - 0.11 0.12 0.09 - - - 928 (+)-α-Pinene 27.00 58.26 62.99 54.66 22.78 3.34 - 935 (−)-β-Citronellene - 0.08 0.09 0.08 0.04 - - 941 (+)-Camphene 0.14 0.25 0.29 0.29 0.20 0.05 - 943 (−)-Camphene 1.10 1.93 2.27 2.40 1.80 0.45 - 946 2,4(10)-Thujadien 0.13 0.17 0.20 0.18 0.10 0.07 - 973 (−)-β-Pinene 11.15 13.19 16.54 21.95 29.67 6.65 - 986 β-Myrcene 0.23 0.15 0.21 0.26 0.27 0.06 - 1013 α-Phellandrene - 0.03 0.05 0.08 0.16 0.11 - 1017 3-Carene 3.05 2.49 3.32 5.44 11.67 4.30 - 1030 α-Terpinene 0.33 0.17 0.27 0.48 1.05 0.68 - 1040 Cymene 1.32 0.77 1.09 1.91 5.71 6.66 - 1045 (+)-Limonene 3.47 1.93 2.68 4.92 14.54 13.32 - 1047 Sabinene 0.16 0.05 0.08 0.16 0.40 0.32 - 1063 (1S,3S)-(E)-4-Carene - - - - 0.03 - - 1072 γ-Terpinene 0.12 0.04 0.06 0.11 0.32 0.38 - 1092 Terpinolene 0.65 0.14 0.21 0.44 1.41 3.70 - 1109 (+)-Verbenone - - - - - 0.04 - 1114 1,5,8-p-Menthatriene - - - - - 0.15 - 1158 Camphene hydrate 0.15 0.01 0.02 0.03 0.06 0.79 0.22 1213 3,6,6-Trimethylnorpinan-2-one - - - - - 0.08 - 1250 Carvotanacetone - - - - - 0.06 - Oxygenated monoterpenes (1S,2S,3R,5S)-(+)-2,3- 982 - - - - 0.04 - - Pinanediol 1027 1,4-Cineole - 0.02 0.03 0.06 0.14 0.09 - 1049 1,8-Cineole - 0.01 0.02 0.04 0.09 0.10 - 1083 ρ-Menth-1-ene-3β,7-diol 0.15 0.04 0.06 0.12 0.36 1.10 - 1101 α-Pinene oxide - 0.03 0.02 - - - - 1121 Fenchol 0.77 0.05 0.12 0.25 0.61 5.36 0.72 1128 α-Campholenic aldehyde 0.23 0.04 0.05 0.09 0.18 1.28 - 1142 (−)-(E)-Pinocarveol 0.71 0.03 0.08 0.15 0.28 1.92 0.18 1149 (+)-Camphor 0.25 0.03 0.04 0.06 0.12 1.33 0.54 1152 α-Phellandren-8-ol 0.25 0.01 0.01 0.02 0.02 0.06 - 1163 (E)-Pinocamphone 0.10 0.02 0.04 0.07 0.12 1.26 0.50 1165 Pinocarvone 0.13 - - 0.01 0.01 - - 1166 Isoborneol - - - - - 0.14 - 1180 Isopinocamphone 0.31 0.01 0.01 0.02 0.02 0.30 - 1183 Terpinen-4-ol 1.55 0.07 0.12 0.20 0.32 5.51 3.17 1190 ρ-Cymen-8-ol 0.28 - 0.02 0.02 0.04 0.74 0.60 1200 (+)-α-Terpineol 7.07 0.23 0.40 0.66 0.84 16.69 16.98 1202 (−)-Myrtenol 0.81 0.02 0.03 0.06 0.10 - - 1202 Estragole - - - - - 1.04 0.47 1212 (−)-Verbenone 0.30 0.01 0.02 0.02 0.02 - - 1218 (−)-β-Fenchyl acetate - - - - - 0.05 - 1220 (R)-Carveol 0.08 - - - - 0.19 0.27 1228 Methyl thymol - - - - - 0.14 - 1244 (+)-Carvone - - - - - 0.06 0.16 1286 Bornyl acetate 1.26 0.03 0.04 0.06 0.07 1.63 3.51 1304 Perillyl alcohol ------0.21 1343 Piperitenone ------0.31 Biomolecules 2021, 11, 817 7 of 16

Table 5. Cont.

Area % KI a Compound Name b Wood Oil Fraction A Fraction B Fraction C Fraction D Fraction E Fraction F Sesquiterpene hydrocarbons 1349 (−)-α-Cubebene - - - - - 0.06 - 1354 α-Longipinene 1.09 0.01 0.01 0.02 0.02 0.60 2.24 1373 (+)-Cyclosativene 0.18 - - - - 0.10 0.64 1379 α-Copaene 1.34 0.01 0.01 0.02 0.02 0.55 2.35 1396 (+)-Sativene 0.22 - - - - 0.08 0.73 1405 (+)-Aromadendrene ------0.15 1406 (−)-α-Gurjunene ------0.24 1408 γ-Elemene ------0.68 1415 Longifolene 8.77 0.04 0.05 0.07 0.06 2.61 27.87 1423 β-Caryophyllene 1.08 - - - - 0.19 1.82 1436 α-Guaiene ------0.29 1452 (E)-β-Famesene 0.24 ------1457 α-Humulene 0.26 - - - - - 0.78 1475 γ-Muurolene ------0.21 1489 α-Ylangene ------0.38 1491 β-Selinene 0.07 - - - - - 0.20 1495 (−)-β-Chamigrene ------0.32 1498 α-Selinene ------0.16 1501 α-Muurolene 0.51 - - - - 0.05 1.77 1504 β-Himachalene 0.07 - - - - - 0.30 1512 β-Bisabolene 0.19 - - - - - 0.62 1514 (+)-Cuparene ------0.16 1526 (+)-δ-Cadinene 0.47 - - - - - 1.46 1531 Calamenene 0.09 - - - - - 0.64 1559 α-Calacorene ------0.14 1984 β-Elemene ------1.10 Oxygenated sesquiterpenes 1578 (E)-Nerolidol 0.24 - - - - - 0.30 1593 (−)-Caryophyllene oxide 0.24 ------1598 Viridiﬂorol ------0.29 1609 Longiborneol 0.27 - - - - - 1.19 1639 Cubenol 0.16 - - - - - 0.63 1659 δ-Cadinol 0.08 - - - - - 0.31 1668 α-Cadinol 0.08 ------1694 α-Bisabolol ------0.22 Diterpene hydrocarbons 1951 Cembrene 8.36 - - - - - 0.28 2036 Manoyl oxide 0.07 ------2043 Pimara-8(14),15-diene ------0.28 2151 Sclarene ------0.15 2293 Isopimara-7,15-dien-3-one 0.12 - - - - - 0.22 Oxygenated diterpenes 2083 Thunbergol 2.38 ------2092 Verticiol ------0.32 2199 Sclareol ------0.32 Triterpene hydrocarbons 1855 Squalene ------0.18 Monoterpene hydrocarbons 49.09 80.00 90.74 93.66 90.30 41.22 0.22 Oxygenated monoterpenes 14.27 0.65 1.11 1.91 3.39 38.99 27.62 Sesquiterpene hydrocarbons 14.58 0.06 0.07 0.11 0.10 4.25 45.27 Oxygenated sesquiterpenes 1.08 - - - - - 2.93 Diterpene hydrocarbons 8.56 - - - - - 0.93 Oxygenated diterpenes 2.38 - - - - - 0.63 Triterpene hydrocarbons ------0.18 Others 10.03 19.29 8.08 4.32 6.21 15.54 22.20 Total identiﬁed 99.99 100.00 100.00 100.00 100.00 100.00 99.98 a b Kovats retention index was experimentally determined using DB-5MS column with a homologous series of C8–C26 alkanes. Compounds are listed in order of their elution from the DB-5MS column. BiomoleculesBiomolecules2021 2021, 11, 11, 817, x 118 ofof 1619

As shown in Table5, the main components of fractions A, B, C, and D were α-pinene, β-pinene, 3-carene, and limonene. Furthermore, the contents of monoterpenes in fractions Biomolecules 2021, 11, x A, B, C, and D were higher than those of sesquiterpenes. By contrast, the major constituents11 of 19

of fraction E were α-terpineol (16.06%) and limonene (12.82%). Longifolene (27.27%) and α-terpineol (16.61%) were the predominant components in fraction F (Figure1).

Figure 1. Chemical structures of the main components in the six oil fractions.

3.2. Cell Cytotoxicity The CCK‐8 assay was used to determine the cytotoxicity of the oil fractions against RBL‐2H3 cells. The results of cell cytotoxicity are shown in Figure 2. The individual oil FigureFigurefractions 1. 1. ChemicalChemical exhibited structuresstructures toxicity of ofin the thea dose main main‐dependent components components manner. in in the the six six We oil oil fractions. fractions.found that no sample exhib‐ ited a general cytotoxicity, defined as a cell viability below 80%, as compared with vehicle, 3.2. Cell Cytotoxicity 3.2.in the Cell RBL Cytotoxicity‐2H3 cells [21]. The CCK-8 assay was used to determine the cytotoxicity of the oil fractions against TheFraction CCK A‐8 exhibitedassay was an used IC50 to value determine of 0.063%, the cytotoxicitywhereas IC50 of values the oil of fractions 0.113%, 0.121%,against RBL-2H3 cells. The results of cell cytotoxicity are shown in Figure2. The individual RBLand ‐0.06%2H3 cells. were The recorded results for of fractionscell cytotoxicity B, C, and are D, shown respectively. in Figure IC50 2. values The individual of fractions oil E oil fractions exhibited−4 toxicity in a dose-dependent manner. We found that no sample fractionsand F were exhibited >10 %. toxicity in a dose‐dependent manner. We found that no sample exhib‐ exhibited a general cytotoxicity, deﬁned as a cell viability below 80%, as compared with ited aTherefore, general cytotoxicity, to evaluate defined the anti ‐asinflammatory a cell viability effects below of 80%, the oil as fractions,compared RBL with‐2H3 vehicle, cells vehicle, in the RBL-2H3 cells [21]. −7 inwere the treatedRBL‐2H3 with cells oil [21]. fractions at a concentration of 10 %. Fraction A exhibited an IC50 value of 0.063%, whereas IC50 values of 0.113%, 0.121%, and 0.06% were recorded for fractions B, C, and D, respectively. IC50 values of fractions E and F were >10−4%. Therefore, to evaluate the anti‐inflammatory effects of the oil fractions, RBL‐2H3 cells were treated with oil fractions at a concentration of 10−7%.

FigureFigure 2. 2. Effects of the the six six oil oil fractions fractions on on RBL RBL-2H3‐2H3 cell cell viability. viability. RBL RBL-2H3‐2H3 cells cells were were seeded seeded in 96 in‐ 96-wellwell plate plate and and treated treated with with a aseries series of of concentration concentration of of the the six six oil oil fractions fractions (10 −5,5 ,10 10−6−, 6and, and 10 10−7%)−7 %)for for24 24h. Cell h. Cell viability viability was was assessed assessed by bycell cell counting counting kit‐ kit-88 assay. assay. VE: VE: vehicle, vehicle, DMSO. DMSO. The The results results are are presentedpresented as as mean mean± ± standard deviations. * p < 0.05 and ** p < 0.01 0.01 compared to VE.

3.3. EffectsFraction of the A exhibited Oil Fractions an IC on50 thevalue Expression of 0.063%, of IL whereas‐4 and IL‐ IC1350 values of 0.113%, 0.121%, Figure 2. Effects of the six oil fractions on RBL‐2H3 cell viability. RBL‐2H3 cells were seeded in 96‐ and 0.06%The modulation were recorded of inflammatory for fractions B, cytokines C, and D, from respectively. mast cells IC is50 avalues key indicator of fractions of re E‐ well plate and treated with a series of concentration of the six oil fractions (10−5, 10−6, and 10−7%) for and F were >10−4%. 24duced h. Cell allergic viability symptoms. was assessed In byparticular, cell counting the regulationkit‐8 assay. VE:of IL vehicle,‐4 and DMSO. IL‐13 is The considered results are the Therefore, to evaluate the anti-inﬂammatory effects of the oil fractions, RBL-2H3 cells presentedmost important as mean therapeutic ± standard deviations. strategy for * p allergic< 0.05 and inflammatory ** p < 0.01 compared conditions to VE. [22]. Therefore, were treated with oil fractions at a concentration of 10−7%. 3.3. Effects of the Oil Fractions on the Expression of IL‐4 and IL‐13 The modulation of inflammatory cytokines from mast cells is a key indicator of re‐ duced allergic symptoms. In particular, the regulation of IL‐4 and IL‐13 is considered the most important therapeutic strategy for allergic inflammatory conditions [22]. Therefore,

Biomolecules 2021, 11, 817 9 of 16

3.3. Effects of the Oil Fractions on the Expression of IL-4 and IL-13 Biomolecules 2021, 11, x 12 of 19 The modulation of inflammatory cytokines from mast cells is a key indicator of reduced allergic symptoms. In particular, the regulation of IL-4 and IL-13 is considered the most important therapeutic strategy for allergic inflammatory conditions [22]. Therefore, wewe determined determined whether whether the the oil oil fractions fractions from from P.P. koraiensis koraiensis woodwood oil oil regulate regulate the the levels levels of of proinflammatoryproinflammatory cytokines cytokines IL IL-4‐4 and and IL IL-13‐13 in in RBL RBL-2H3‐2H3 cells cells (Figure (Figure 3).3).

Figure 3. Effect of the six oil fractions on (a) IL-4 and (b) IL-13 mRNA expression levels in LPS- Figure 3. Effect of the six oil fractions on (a) IL‐4 and (b) IL‐13 mRNA expression levels in LPS‐ stimulatedstimulated RBL RBL-2H3‐2H3 cells. cells. VE: VE: vehicle, vehicle, DMSO; DMSO; NC: NC: negative negative control, control, 1‐μ 1-g/mLµg/mL LPS LPS treated treated group; group; −7 PC:PC: positive positive control, control, 100 100-nM‐nM dexamethasone dexamethasone treated treated group, group, an oil an fraction oil fraction (A–F) (A–F) (10− (107%). Values%). Values areare presented presented as as mean mean ± standard± standard deviation. deviation. * p *

StimulationStimulation of of RBL RBL-2H3‐2H3 cells cells with with LPS LPS significantly significantly increased increased the the relative relative expression expression levellevel of of IL IL-4‐4 (Figure (Figure 3a).3a). However, However, this this increase increase in in IL IL-4‐4 expression expression was was suppressed suppressed in in LPS LPS-‐ stimulatedstimulated RBL RBL-2H3‐2H3 cells cells treated treated with with dexamethasone dexamethasone (~77.5% (about 77.5% reduction), reduction), a steroidal a steroidal in‐ −7 flammatoryinflammatory drug. drug. Anti Anti-inflammatory‐inflammatory activities activities of offractions fractions at at a a10 10−7%% concentration concentration showedshowed 27.6–55.5% 27.6–55.5% inhibition inhibition compared compared with negative control.control. Inhibition Inhibition of of IL-4 IL‐4 expression expres‐ sionby by fractions fractions are are as as follows: follows: 42.1%, 42.1%, 40.9%, 40.9%, 48.3%,48.3%, 55.5, 27.6%, 27.6%, and and 45.3% 45.3% inhibition inhibition for for fractionsfractions A–F, A–F, respectively. respectively. Fractions Fractions A, A, C, C, D, D, and and F Fshowed showed a ahigher higher inhibitory inhibitory effect effect of of ILIL-4‐4 expression expression than than other other fractions. fractions. However, However, these these fractions fractions had had a lower a lower inhibitory inhibitory effect effect thanthan dexamethasone, dexamethasone, positive positive control. control. A Aremarkable remarkable increase increase in in IL IL-13‐13 expression expression compared compared with with that that of of vehicle vehicle (VE) (VE) was was alsoalso observed observed when when RBL RBL-2H3‐2H3 cells cells were were treated treated with with LPS. LPS. However, However, this this increase increase was was significantlysignificantly suppressed suppressed following following treatment treatment of thethe cellscells with with dexamethasone dexamethasone and and fractions frac‐ (Figure3b) as follows: 74.1% inhibition for dexamethasone and 43.0%, 35.4%, 44.7%, 21.8%, tions (Figure 3b) as follows: 74.1% inhibition for dexamethasone and 43.0%, 35.4%, 44.7%, 20.4%, and 48.8% inhibition for fractions A–F, respectively. Fractions A, C, and F showed a 21.8%, 20.4%, and 48.8% inhibition for fractions A–F, respectively. Fractions A, C, and F higher inhibitory effect of IL-13 expression than other fractions. showed a higher inhibitory effect of IL‐13 expression than other fractions. 3.4. Effects of the Oil Fractions on β-Hexosaminidase Release 3.4. Effects of the Oil Fractions on β‐Hexosaminidase Release β-Hexosaminidase is present in the granules of basophils or mast cells and is se- cretedβ‐Hexosaminidase by these cells upon is present allergic in the reactions. granulesβ-Hexosaminidase of basophils or mast secretion cells and can is be secreted used to byindicate these cells and upon quantify allergic the reactions. extent of degranulationβ‐Hexosaminidase [23]. secretion The effects can of be the used oil to fractions indicate on andβ-hexosaminidase quantify the extent release of degranulation are shown in Figure [23]. 4The. effects of the oil fractions on β‐hex‐ osaminidase release are shown in Figure 4.

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FigureFigure 4. 4.Inhibitory Inhibitory effect effect of theof the six six oil fractionsoil fractions on β -hexosaminidaseon β‐hexosaminidase release. release. VE: vehicle, VE: vehicle, DMSO; DMSO; NC:NC: negative negative control, control, 800 800 ng/mL ng/mL DNP-IgE DNP‐IgE and and 10- µ10g/mL‐μg/mL DNP-BSA DNP‐BSA treated treated group; group; PC: positive PC: positive control,control, 100-nM 100‐nM dexamethasone dexamethasone treated treated group, group, an oil an fraction oil fraction (A–F) (10(A–F)−7%). (10 Values−7%). Values are presented are presented asas meanmean± ± standardstandard deviation. deviation. * p*

ββ-Hexosaminidase‐Hexosaminidase secretion secretion in the in negative the negative control (NC) control group, (NC) stimulated group, with stimulated DNP- with BSA and with promoted degranulation, was approximately three times higher than that of DNP‐BSA and with promoted degranulation, was approximately three times higher than the VE group. However, treatment of the inflammatory-induced RBL-2H3 cells with dex- amethasonethat of the resultsVE group. in a significant However, decrease treatment in the of relative the inflammatory secretion of β‐-hexosaminidaseinduced RBL‐2H3 cells comparedwith dexamethasone with the NC results (about 69.2%in a significant decrease). Similarly,decrease βin-hexosaminidase the relative secretion secretion of β‐hex‐ wasosaminidase significantly compared reduced with following the NC treatment (about with 69.2% the decrease). fractions. Similarly, Subsequently, β‐hexosaminidase the oil fractionssecretion exhibited was significantly considerable reduced anti-inflammatory following activity treatment by inhibiting with theβ fractions.-hexosaminidase Subsequently, secretionthe oil fractions in LPS-induced exhibited RBL-2H3 considerable cells as follows: anti‐inflammatory 50.1%, 23.4%, 23.1%, activity 45.1%, by 23.9%, inhibiting and β‐hex‐ 44.6%osaminidase inhibition secretion for fractions in LPS A–F,‐induced respectively. RBL Of‐2H3 all the cells fractions, as follows: A, D, and50.1%, F were 23.4%, the 23.1%, most45.1%, potent. 23.9%, and 44.6% inhibition for fractions A–F, respectively. Of all the fractions, A, 3.5.D, and Effects F were of Single the Compounds most potent. on Cell Viability The anti-inflammatory effects of six standard substances [(+)-α-pinene, (−)-β-pinene, (+)-3.5.α Effects-terpineol, of Single 3-carene, Compounds limonene, on longifolene], Cell Viability the main components of fractions A, D, and F,The which anti have‐inflammatory excellent anti-inflammatory effects of six standard activity, substances were investigated. [(+)‐α‐pinene, In particular, (−)‐β‐pinene, to(+) determine‐α‐terpineol, the 3 effect‐carene, of six limonene, standard longifolene], substances on the the main survival components and proliferation of fractions of A, D, RBL-2H3and F, which cells, thehave CCK excellent assay was anti performed‐inflammatory by treating activity, the were cells with investigated. a concentration In particular, −7 −5 rangeto determine of 10 –10 the effect% (Figure of six5). standard substances on the survival and proliferation of RBL‐ Cell viability of six single compounds is shown in Figure5. The six single com- 2H3 cells, the CCK assay was performed by treating the cells with a concentration range pounds did not show any cytotoxicity at the investigated concentration in this study. −7 −5 Therefore,of 10 –10 the% anti-inflammatory(Figure 5). effects of the six single compounds were evaluated at 10−7% concentration. Both (+)-α-pinene and (−)-β-pinene exhibited an IC50 value of 0.033%. The IC50 value of (+)-α-terpineol was 0.030%. However, IC50 values of 0.021%, 0.021%, and 0.028% were recorded for 3-carnene, limonene, and longifolene, respectively.

3.6. Effects of Single Compounds on the Expression of IL-4 and IL-13 The inﬂammatory response induced RBL-2H3 cells were treated with the six single compounds. The results are shown in Figure6.

Biomolecules 2021, 11, x 13 of 19

Figure 4. Inhibitory effect of the six oil fractions on β‐hexosaminidase release. VE: vehicle, DMSO; NC: negative control, 800 ng/mL DNP‐IgE and 10‐μg/mL DNP‐BSA treated group; PC: positive control, 100‐nM dexamethasone treated group, an oil fraction (A–F) (10−7%). Values are presented as mean ± standard deviation. * p < 0.05 and ** p < 0.01 compared with VE; # p < 0.05, ## p < 0.01 com‐ pared with NC.

β‐Hexosaminidase secretion in the negative control (NC) group, stimulated with DNP‐BSA and with promoted degranulation, was approximately three times higher than that of the VE group. However, treatment of the inflammatory‐induced RBL‐2H3 cells with dexamethasone results in a significant decrease in the relative secretion of β‐hex‐ osaminidase compared with the NC (about 69.2% decrease). Similarly, β‐hexosaminidase secretion was significantly reduced following treatment with the fractions. Subsequently, the oil fractions exhibited considerable anti‐inflammatory activity by inhibiting β‐hex‐ osaminidase secretion in LPS‐induced RBL‐2H3 cells as follows: 50.1%, 23.4%, 23.1%, 45.1%, 23.9%, and 44.6% inhibition for fractions A–F, respectively. Of all the fractions, A, D, and F were the most potent.

3.5. Effects of Single Compounds on Cell Viability The anti‐inflammatory effects of six standard substances [(+)‐α‐pinene, (−)‐β‐pinene, (+)‐α‐terpineol, 3‐carene, limonene, longifolene], the main components of fractions A, D, and F, which have excellent anti‐inflammatory activity, were investigated. In particular, to determine the effect of six standard substances on the survival and proliferation of RBL‐ Biomolecules 2021, 11, 817 2H3 cells, the CCK assay was performed by treating the cells with a concentration11 of 16 range Biomolecules 2021, 11, x −7 −5 14 of 19 of 10 –10 % (Figure 5).

Figure 5. Effect of single compounds on the RBL‐2H3 cell viability. RBL‐2H3 cells were seeded in 96‐well plate and treated with a series of concentration of the six single compounds (10−5, 10−6, and 10−7%) for 24 h. Cell viability was assessed by cell counting kit‐8 assay. VE: vehicle, DMSO. The results are presented as mean ± standard deviation. * p < 0.05 and ** p < 0.01 compared with VE.

Cell viability of six single compounds is shown in Figure 5. The six single compounds did not show any cytotoxicity at the investigated concentration in this study. Therefore, the anti‐inflammatory effects of the six single compounds were evaluated at 10−7% con‐ centration. Both (+)‐α‐pinene and (−)‐β‐pinene exhibited an IC50 value of 0.033%. The IC50 value of (+)‐α‐terpineol was 0.030%. However, IC50 values of 0.021%, 0.021%, and 0.028% were recorded for 3‐carnene, limonene, and longifolene, respectively.

Figure 5. Effect of single compounds on the RBL-2H3 cell viability. RBL-2H3 cells were seeded in 3.6. Effects of Single Compounds on the Expression of IL‐4 and IL‐13 96-well plate and treated with a series of concentration of the six single compounds (10−5, 10−6, and 10−7%)The for inflammatory 24 h. Cell viability response was assessed induced by cell RBL counting‐2H3 kit-8 cells assay. were VE: treated vehicle, with DMSO. the The six single compounds.results are presented The results as mean ±arestandard shown deviation. in Figure * p 6.< 0.05 and ** p < 0.01 compared with VE.

Figure 6.6. EffectsEffects of the single compounds on on ( a(a)) IL-4 IL‐4 and and ( b(b)) IL-13 IL‐13 mRNA mRNA expression expression in in LPS- LPS‐stim‐ ulatedstimulated RBL RBL-2H3‐2H3 cells. cells. VE: VE: vehicle, vehicle, DMSO; DMSO; NC: NC: negative negative control, 1- 1µ‐μg/mLg/mL LPS LPS treated treated group; group; PC: − positivePC: positive control, control, 100 100-nM‐nM dexamethasone dexamethasone treated group, group, the the single single compounds compounds (10 (107%).−7%). Values Values are presentedare presented as asmean mean ± ±standardstandard deviation. deviation. * p << 0.05 and and ** **p p< < 0.01 0.01 compared compared with with VE; VE;# p < # 0.05,p < 0.05, ## p <## 0.01p < 0.01 compared compared with with NC. NC. As shown in Figure6a, LPS treatment increased the relative expression level of IL-4 by >3.8 timesAs shown compared in Figure with VE 6a, treatment. LPS treatment Upon treatment increased with the dexamethasone relative expression (PC), the level IL- of IL‐4 by4 gene >3.8 expression times compared level in NC with was VE similar treatment. to that in Upon VE (about treatment 77.5% reduction). with dexamethasone In addition, (PC), the relativeIL‐4 gene IL-4 expression expression level levels in upon NC was treatment similar with to thethat six in single VE (~77.5% compounds reduction). were In ad‐ dition,decreased the in relative VE compared IL‐4 expression with that in levels NC as upon follows: treatment 78.4% inhibition with the for six (+)- singleα-pinene, compounds were9.0% inhibition decreased for in (− VE)-β -pinene,compared 43.3% with inhibition that in for NC (+)- asα-terpineol, follows: 78.4% 49.7% inhibitioninhibition for for (+)‐α‐ pinene,3-carnene, 9.0% 48.2% inhibition inhibition for for ( limonene,−)‐β‐pinene, and 43.3% 52.4% inhibitioninhibition for for longifolene. (+)‐α‐terpineol, (+)-α-Pinene 49.7% inhibi‐ tion for 3‐carnene, 48.2% inhibition for limonene, and 52.4% inhibition for longifolene. (+)‐ α‐Pinene was found to be more effective than the other single compounds, followed by longifolene, 3‐carnene, limonene, and (+)‐α‐terpineol. Similar results were observed regarding the relative expression level of IL‐13 (Figure 6b). The six single compounds exhibited anti‐inflammatory activity compared with NC.

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Biomolecules 2021, 11, x 15 of 19

was found to be more effective than the other single compounds, followed by longifolene, 3-carnene, limonene, and (+)-α-terpineol. Similar results were observed regarding the relative expression level of IL-13 (Figure6b) . TheInhibitions six single ofcompounds IL‐13 expression exhibited by anti-inﬂammatory single compounds activity were compared as follows: with 74.1% NC. Inhibi- for dexame‐ tionsthasone, of IL-13 70.6% expression for (+)‐ byα‐pinene, single compounds 38.4% for were (+)‐α as‐terpineol, follows: 74.1% 12.2% for for dexamethasone, (−)‐β‐pinene, 44.1% 70.6%for 3‐carnene, for (+)-α -pinene,9.5% for 38.4% limonene, for (+)- andα-terpineol, 54.7% for 12.2% longifolene for (−)- βagainst-pinene, NC. 44.1% Inhibition for 3- of IL‐ carnene,13 expression 9.5% for by limonene,(+)‐α‐pinene and was 54.7% more for effective longifolene than against the other NC. Inhibition compounds. of IL-13 The activity expressionof (+)‐α‐pinene by (+)- wasα-pinene similar was to more that effectiveof dexamethasone than the other (PC). compounds. Longifolene The activityand 3‐carene of also (+)-hadα an-pinene inhibitory was similar effect to on that the of IL dexamethasone‐13 expression. (PC). Longifolene and 3-carene also had an inhibitory effect on the IL-13 expression.

3.7. EffectsEffects of of Active Active Compounds Compounds on βon-Hexosaminidase β‐Hexosaminidase Release Release TheThe effects effects of theof the six singlesix single compounds compounds on β-hexosaminidase on β‐hexosaminidase secretion are secretion graphically are graph‐ illustratedically illustrated in Figure in7 .Figure 7.

Figure 7. 7.Inhibition Inhibition effect effect of theof the single single compounds compounds on β-hexosaminidase on β‐hexosaminidase release. VE: release. vehicle, VE: DMSO; vehicle, NC:DMSO; negative NC: negative control, 800-ng/mL control, 800 DNP-IgE‐ng/mL and DNP 10-‐IgEµg/mL and DNP-BSA 10‐μg/mL treated DNP‐BSA group; treated PC: positive group; PC: −7 control,positive 100-nM control, dexamethasone 100‐nM dexamethasone treated group, treated the single group, compounds the single (10 compounds%). Values are (10 presented−7%). Values are # ## aspresented mean ± asstandard mean ± deviation. standard **deviation.p < 0.01 compared** p < 0.01 with compared VE; p with< 0.05, VE; # p < 0.05, 0.01 compared## p < 0.01 com‐ withpared NC. with NC. The RBL-2H3 cells stimulated with DNP-BSA promoted degranulation and signif- icantlyThe increased RBL‐2H3β-hexosaminidase cells stimulated secretion with DNP (NC).‐BSA The promoted tested single degranulation compounds hadand signifi‐ inhibitorycantly increased effects on β‐βhexosaminidase-hexosaminidase secretionsecretion from (NC). DNP-BSA-stimulated The tested single RBL-2H3 compounds cells. had in‐ Inhibitionhibitory effects of β-hexosaminidase on β‐hexosaminidase secretion secretion by single compounds from DNP were‐BSA as‐stimulated follows: 69.2% RBL for‐2H3 cells. dexamethasone,Inhibition of β‐hexosaminidase 37.2% for (+)-α-pinene, secretion 57.5% by for single (+)-α-terpineol, compounds 53.3% were for as (− )-follows:β-pinene, 69.2% for 36.5%dexamethasone, for 3-carnene, 37.2% 43.8% for for (+) limonene,‐α‐pinene, and 57.5% 47.3% for longifolene(+)‐α‐terpineol, against 53.3% NC. Moreover, for (−)‐β‐pinene, the36.5% inhibitory for 3‐carnene, effects of 43.8% (+)-α-terpineol for limonene, and (− and)-β-pinene 47.3% werefor longifolene high. These against results indicate NC. Moreover, thatthe inhibitory (+)-α-terpineol effects and of (− (+))-β‐α-pinene‐terpineol in the and essential (−)‐β‐pinene oil from wereP. koraiensis high. Thesewood results are the indicate highest contributors of the inhibition in β-hexosaminidase secretion. that (+)‐α‐terpineol and (−)‐β‐pinene in the essential oil from P. koraiensis wood are the 4.highest Discussion contributors of the inhibition in β‐hexosaminidase secretion. Inflammatory diseases are generally treated with steroidal or non-steroidal anti- inflammatory4. Discussion drugs. However, both have significant negative side effects, thereby limiting theirInflammatory use in certain sections diseases of theare populationgenerally treated [24]. As with a result, steroidal the development or non‐steroidal of new anti‐in‐ drugsflammatory with novel drugs. modes However, of action both and have fewer significant side effects negative is warranted. side Essentialeffects, thereby oils are limiting secondarytheir use in metabolites certain sections that are extensivelyof the population used in aromatherapy[24]. As a result, and variousthe development traditional of new medicinal systems. Many of these oils possess different pharmacological properties. drugs with novel modes of action and fewer side effects is warranted. Essential oils are secondary metabolites that are extensively used in aromatherapy and various traditional medicinal systems. Many of these oils possess different pharmacological properties. Immune responses are regulated by a highly complex and intricate network of con‐ trol elements. This reaction causes degranulation of mast cells and facilitates the release of primary chemical mediators such as histamine. At this time, cell membranes are acti‐ vated and then secondary chemical mediators are secreted. Due to this, cytokines (i.e., IL‐

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Immune responses are regulated by a highly complex and intricate network of control elements. This reaction causes degranulation of mast cells and facilitates the release of primary chemical mediators such as histamine. At this time, cell membranes are activated and then secondary chemical mediators are secreted. Due to this, cytokines (i.e., IL-3, IL-4, and IL-5, etc.) are secreted from mast cells. Among these, the key regulatory components are anti-inflammatory cytokines and specific cytokine inhibitors [25]. IL-4 and IL-13 share a common cellular receptor (IL-4 type 1 receptor); this accounts for many of the similarities between the actions of these two anti-inflammatory cytokines [26]. The principal functional difference between IL-4 and IL-13 lies in their effect on T cells. β-Hexosaminidase is used as a typical marker of mast cell degranulation. Approximately 85% of β-hexosaminidase contained in mast cells is localized in the granules [27]. Based on this, β-hexosaminidase is commonly used as a biomarker of mast cell degranulation caused by various stimuli. Therefore, active anti-inflammatory constituents may be different depending on the target due to these differences in anti-inflammatory mechanisms [28]. The most well-known endotoxin for inducing an anti-inflammatory response in vitro is lipopolysaccharides (LPS). LPS stimulates monocytes/macrophages through TLR4, resulting in the activation of a series of signaling events that potentiate the production of inflammatory mediators [29]. However, LPS is a model that does not correctly reproduce the actual process [30]. Nevertheless, many studies have been used because it is a strong immunostimulant when tested in vitro. In this study, LPS was also used as an inflammatory stimulator to search for anti-inflammatory constituents. The anti-inflammatory effect of essential oil from the wood of P. koraiensis was demonstrated in our previous study [16]. The present study investigates the active anti- inflammatory constituents in the essential oil. Fractional distillation was used to separate essential oil into several fractions. As shown in Table5, the main chemical compositions of each fraction were found to be different. Early fractions obtained by fractional distillation mainly comprised monoterpenes, which typically have low boiling points. However, sesquiterpenes, which typically have high boiling points, were the main components of late fractions. According to the results of GC–MS analysis, the main components of fractions A, B, C, and D were α-pinene (22.78–58.26%), β-pinene (13.19–29.67%), 3-carene (2.49–11.67%), and limonene (1.93–14.54%). By contrast, the major constituents of fraction E and F were α-terpineol (16.69, 16.98%) and longifolene (2.61, 27.87%). To investigate active anti-inflammatory constituents of P. koraiensis oils, the effects of fractions on the expression of inflammation-related genes (i.e., IL-4 and IL-13) and secretion of β-hexosaminidase were examined. As mentioned earlier, IL-4 and IL-13 have many similarities in the inflammatory response. Fractions that effectively inhibit IL-4 and IL-13 expression were the same as A, C, and F. There was no statistical significance detected in the expression of inflammation-related genes (i.e., IL-4 and IL-13) between fractions. In contrast, a result of β-hexosaminidase secretion showed a different trend. In the inhibition of β-hexosaminidase secretion, fraction A, D, and F were more active than other fractions. In conclusion, the fractions that most effectively regulated the anti-inflammatory cytokines and the secretion of β-hexosaminidase were A and F. As a result of GC-MS, the main constituents of fraction A were (+)-α-pinene (58.26%) and β-pinene (13.19%). Additionally, the main components of fraction F were α-terpineol (16.98%) and longifolene (27.87%). The anti-inflammatory activity with the major components of each fraction was evaluated. The gene expression level of IL-4 and IL-13 were reduced significantly by six single compounds used in this study. In particular, (+)-α-pinene among the six compounds, effectively suppressed the gene expression of both IL-4 and IL-13. (+)-α-Pinene and (−)-β-pinene showed statistical significance in IL-4 and IL-13 gene expression (p = 0.001; p = 0.048). In addition, (+)-α-pinene and limonene showed a statistical significance of p = 0.034 in IL-13 gene expression. Other single compounds showed no statistical significance. From the results, although six single compounds had anti-inflammatory activities, (+)-α-pinene had the best inhibitory effects on IL-4 and IL-13 gene expression. Additionally, it was found that inhibitory effects of α-terpineol, 3-carene, and longifolene on IL-4 and IL-13 gene ex- Biomolecules 2021, 11, 817 14 of 16

pression were similar. All six single compounds also had an inhibitory effect on the release of β-hexosaminidase. There was no statistical significance between the single compounds. The single compounds that suppressed β-hexosaminidase secretion very efficiently were (−)-β-pinene and (+)-α-terpineol. Based on these results, it is suggested that six single compounds had anti-inflammatory activities. Fractions A and C, active anti-inflammatory fractions, were mainly consisted of (+)-α- pinene, (−)-β-pinene, (+)-α-terpineol, 3-carene, and limonene, which had anti-inflammatory activities. Faction A and C contained these constituents in high portions of 76.14% and 87.7%, respectively. For this reason, fractions A and C exhibited positive activities in gene expression of IL-4 and IL-13. The major constituents of fraction F were α-terpineol (16.98%) and longifolene (27.87%). Although active anti-inflammatory constituents such as (+)-α-pinene, (−)-β-pinene, 3- carene were not contained in fraction F, fraction F had inhibitory effects on gene expression of IL-4, IL-13 and β-hexosaminidase secretion. Fraction E contained α-terpineol (16.69%) and longifolene (2.61%) like fraction F but had less anti-inflammatory activity than fraction F. These results seem to be due to the difference in the portion of major constituents in each fraction. The proportions of (+)-α- terpineol in fractions E and F were similar, but the proportions of longifolene in F fraction was higher than fraction E. It was accordingly inferred that longifolene showed a significant effect on anti-inflammatory activity. Additionally, longifolene was expected to be an active anti-inflammatory compound. In the present study, (+)-α-terpineol and β-pinene were identified as the potent con- stituent of the essential oil against β-hexosaminidase secretion. Previous studies have revealed the various biological properties of (+)-α-terpineol and β-pinene. (+)-α-Terpineol exerts a wide range of biological actions in humans, animals, and plants [31,32]. Further, it possesses a wide range of biological applications as anti-hypertensive, antiprolifera- tive effects, and potent inhibitor of superoxide production [33]. For instance, it has been identified as one of the most important bioactive constituents of many essential oils. β- pinene displayed antimicrobial, anti-tumor, anti-inflammatory, antioxidant, and analgesic effects [34]. According to the results of this study, (−)-β-pinene activity was not prominent. In previous studies, there was a substantial difference in the activity of the two enantiomers; the two isomers behaved differently in vitro. In a chiral environment, one enantiomer may display different chemical and pharmacologic behavior than the other enantiomer [35]. Consequently, it is necessary to evaluate the anti-inflammatory activity of (+)-β-pinene, the enantiomer of (−)-β-pinene. Taken together, the results presented in this study suggest the applications of essential oils or their components as anti-inflammatory agents and might accelerate the development of new drugs for different inflammatory diseases. Essential oils could be exploited as effective alternatives or complementary compounds of the chemical industry without inducing the same secondary effects.

5. Conclusions Although several previous studies have demonstrated the anti-inflammatory effects of P. koraiensis wood oil, the active anti-inflammatory constituents of the oil have not been identified. Therefore, this study explored the in vitro anti-inflammatory effects of the fractions obtained from P. koraiensis wood oil as a means to investigate the anti-inflammatory constituents in the essential oil. GC–MS analysis revealed that the chemical compositions of the six oil fractions were different and identified the six most effective single components. The six single compounds were found to exhibit anti-inflammatory activities; among them, (+)-α-pinene, (−)-β-pinene, α-terpineol, and longifolene showed the highest activity. There- fore, four single compounds were assumed to be the most contributing compounds to the anti-inflammatory activity of P. koraiensis wood oil. Therefore, we decided that (+)-α-pinene, (−)-β-pinene, α-terpineol, and longifolene have a high contribution to anti-inflammatory Biomolecules 2021, 11, 817 15 of 16

activity in essential oil from P. koraiensis wood. Additionally, the minor components are critical to the synergistic activity [36]. Thus, (+)-α-pinene, (−)-β-pinene, α-terpineol, and longifolene may be useful for the prevention and treatment of inflammatory diseases. Furthermore, our findings provide a scientific rationale for the future utilization of P. koraiensis, (+)-α-pinene, (−)-β-pinene, α-terpineol, and longifolene.

Author Contributions: All authors contributed to the research. J.Y. performed the experiments and wrote the manuscript. W.-S.C. performed data analysis using GC–MS. K.-J.K. designed and coordi- nated the experiments. C.-D.E. selected the P. koraiensis trees and processed them for experimental use/application. M.-J.P. provided discussion and reviewed the thesis. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Institute of Forest Science (FP0700-2015-02). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analyzed during this study are included in this published article. Additional data are available from the corresponding author upon request. Acknowledgments: This work was supported by the National Institute of Forest Science (FP0700-2015-02). Conﬂicts of Interest: The authors declare that they have no competing interests.

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