Extractives elucidation of Taiwania cryptomerioides sapwood

Taiwania (Taiwania cryptomerioides Hayata) has long been regarded as a living fossil from the Tertiary period of Mesozoic Era for its distinguished yellowish-red color with purplish-pink streaks presented in its heartwood. With this elegant appearance that matches the color “red” for good fortune in the Taiwanese culture, Taiwania is supposed to be a popular wood in Taiwan where it is a native species of. Extractives contribute to the properties of wood. It is a fascinating subject to investigate extractives biosynthesis in the process of heartwood formation. Up to date, there is no phytochemistry study of Taiwania sapwood. In this study, three new sesquiterpenoids, Taiwania A (1), Taiwania B (2), and Taiwania C (3), together with 75 known compounds in the Taiwania sapwood. The structures of extractives were determined by analysis of spectroscopic data and comparison with the literatures. This study supported secondary reaction lignans could be found in sapwood that confirmed our previous research on the Taiwania-type of heartwood formation.


Introduction
Heartwood is the large part of the wood, and the extractives contained in it are closely related to the properties of the wood, such as strength, durability, color, and odor. The formation mechanism of heartwood has always been of interest and an important research topic for researchers. The heartwood formation could be classified into three types based on distribution patterns of extractives in stem wood of various trees species [1,2]. Type I heartwood formation, i.e., Robinia-type heartwood formation, where the accumulation of phenolic extractives starts in the transition zone (TZ). In this case, no phenolic precursors were found in the aging sapwood. Type II (Juglans-type) heartwood formation, where the phenolic precursors gradual accumulated centripetally with progressive aging of the sapwood tissues. The extractives that characterize the Type II heartwood were formed in the TZ either by de novo biosynthesis or secondary reactions (oxidation or hydrolysis) of precursor substances. Type III (Taiwania-type) heartwood formation, which most of phenolic compounds are synthesized in sapwood [2].
Taiwania (Taiwania cryptomerioides Hayata) is a native tree species growth in Taiwan. Taiwania also is the highest conifer in East Asia, it can reach 80 m. From 1960s to date, Taiwania is the important plantation species in Taiwan. Due to its excellent durability and processing property, Taiwania is the popular wood material for building and furniture. With regard to phytochemical study of Taiwania, more than 300 compounds, including terpenoids, lignans, isoflavones, and other compounds have been isolated from Taiwania during the past 90 years . The putative bioactivities compounds of Taiwania, and evaluated the potential usages of the phytochemicals isolated from Taiwania for pharmacological applications were carried on by our research group. We demonstrated that sesquiterpenoids isolated from the heartwood of Taiwania against bacteria, fungi, mite, and termite [24][25][26][27][28]. The diterpenoids also exhibited the antioxidant and anti-inflammatory activities [29,30]. In the meantime, the lignans of Taiwania presented the potent anti-inflammatory, antiviral, and anticancer activities [26,[31][32][33][34][35][36][37].
The mechanism of heartwood formation in Taiwania is very unique. We found that most of the skeletons of the compounds are already synthesized in the sapwood. Although there have been numerous research reports on the chemical composition of Taiwan cedar, so far, no discussion has been made on its sapwood composition. For understanding the heartwood formation in Taiwania, it is important to clarify the difference of composition between the heartwood and sapwood. This study accurately distinguished sapwood from heartwood in Taiwania, focusing the composition investigation of Taiwania sapwood. Totally, 78 compounds from sapwood of Taiwania, including 3 new skeleton sesquiterpenoids. The results obtained in this study provide a valuable reference for further heartwood formation and metabolites biosynthesis investments.

Materials and methods
General experimental procedures 1 H, 13 C, and 2D NMR spectra were recorded on a Bruker AVANCE III NMR spectrometer (Bruker, Billerica, Massachusetts, US), acquiring 1 H data at 400 MHz and 13 C data at 100 MHz, using standard experiments from Bruker pulse programs library. High-resolution mass spectrometry (HR-MS) was determined using an LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.). The compositions of the essential oil were analyzed by an ITQ 900 mass spectrometer coupled to a TRACE GC Ultra gas chromatography (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.). Methanol (MeOH) extracts were fractionated on silica gel 60 (230-400 mesh ASTM, Merck) and then purified with semi-preparative normal-phase column (luna silica (2), 250 × 10 mm, 5 μm, Phenomenex) on an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, California, U.S.).

Plant materials
A 30-year-old Taiwania used in this study was collected from the Huisun Experimental Forest Station of National Chung-Hsing University in August 2014; and was identified by Prof Yen-Hsueh Tseng, Department of Forestry, National Chung Hsing University. The voucher specimen was deposited in the herbarium of the same university. The sapwood chips (excluding the heartwood and knots) were prepared from a green cut tree and stored in room temperature with avoiding light irradition.

Essential oil analysis
The air-dried sapwood chips (350 g) were subjected to hydrodistillation for 8 h using a Clevenger type apparatus. The moisture-free oil which yield 0.01% was obtained by treating with anhydrous Na 2 SO 4 . The compositions of the essential oils were analyzed by an ITQ Series GC mass system, equipped with a DB-5MS capillary column (30 m length × 0.25 mm inside diameter × 0.25 μm film thickness, J & W Scientific) and helium as a carrier gas with a flow rate of 1 ml min −1 . The injector temperature was 240 ℃ and spilt ratio was 1:200. The oven temperature was start at 40 ℃, and increased by 5 ℃ min −1 to 130 ℃, then rose to 160 ℃ at a rate of 2 ℃ min −1 , finally increased to 280 ℃ by 10 ℃ min −1 and held for 10 min. The EI source was 70 eV and 250 ℃. Quantification was obtained from percentage peak areas from the gas chromatogram. A Wiley/ NBS Registry of Mass Spectral Data search and authentic reference compounds were used for substance identification. The Kovats retention index (KI), which is a parameter calculated in reference to n-alkanes that converts retention times into system-independent constants, was also confirmed [38]. Chromatography results expressed as area percentages were calculated with a response factor of 1.0.
(1) Type I (Robinia-type) heartwood formation, where the accumulation of phenolic extractives starts in the transition zoon, while no phenolic precursors were found in the aging sapwood. (2) Type II (Juglans-type) heartwood formation, where the phenolic precursors gradual accumulated centripetally with progressive aging of the sapwood tissues. Our previously study proposed the type III, Taiwania-type of heartwood formation, which was found the secondary reaction for lignans in sapwood [2]. This study further confirms that secondary reaction lignans occurred in sapwood of Taiwania. The biosynthesis of lignans, e.g., matairesinol, hinokinin, savinin, helioxanthin, and Taiwanin E has been synthesized in the sapwood.

New sesquiterpenoids identification
Three new sesquiterpenoids were identified in this study, the structure's elucidation was reported in the following. Compound 1 was obtained as a colorless oil. The 1 H NMR spectrum of 1 (Table 2)  Ascribing to cnjugated double bond, H-5 exhibited very low field at δ H 4.95, and the carbon signals at δ C 122.6 (CH), δ C 132.8 (C), δ C 134.0 (C), δ C 157.8 (C), and δ C 173.2 (C) indicated the existence of a C = CH, a C = C, and a O = C-OH systems. The remaining two degrees of unsaturation identified 1 as a bicyclic compound. The HMBC (Fig. 3) data showed correlations H-12/C-1, C-11, C-13; H-13/C-1, C-11, C-12, and the COSY (Fig. 4)   H-14/C-8. Taking the above evidences together, 1 identified as a 5-7 ring compound and pinpointed the location of 4-carboxyl group, OH-5, OH-6, and methyl group (Me)-7. The NOESY (Fig. 5) signals showed correlations of H-5/H-14; H-6/H-14; H-7/H-11 as well as the coupling constant confirmed that H-5, H-6, and Me-7 were β and isopropyl-1 and H-7 were α configuration. Based on these data confirmed the proposed structure of 1 and named Taiwania A, and it is a new skeleton sesquiterpene to the best of our understanding.
Compound 2, a colorless oil, was assigned a molecular formula of C 15 H 22 O 5 on the basis of HR-APCI-MS and 13 C NMR. The 1 H NMR and 13 C NMR data of 2 ( Table 2) were similar to those of 1, indicated that compound 2 was also the same type sesquiterpenoid derivative. Analysis of NMR data revealed that OH-7 of 2 replaced H-7 of 1. This supported by the COSY (Fig. 4)   The molecular formula of compound 3 was C 16 H 24 O 6 by electrospray ionization mass spectrometry (ESI-MS) and NMR data, indicated five degree of unsaturation. Sixteen carbon signals were observed in the 13 C NMR spectrum of 3 and were assigned by the DEPT experiments displayed four aliphatic methyl, two aliphatic methylene, two aliphatic methine, two oxygenated methine, three oxygenated quaternary, one olefinic methine, one olefinic quaternary, and one carbonyl carbons. The carbon signals at δ C 132.8 (CH), δ C 134.1 (C), and δ C 153.5 (C) indicated the existence of a C=CH and a C=O systems. The carbonyl carbon (C-1′) exhibited very high field at δ C 153.5 supported that was carbonate group [-O-C(= O)-O-] [86]. The remaining three degrees of unsaturation identified 3 as a tricyclic compound. Its 1 H NMR spectrum of 3 (Table 2) (Fig. 3) correlations showed H-12/C-1, C-11, C-13; H-13/C-1, C-11, C-12, and the COSY (Fig. 4) correlations showed H-1/H-11; H-11/H-12 and H-13 confirmed that the isopropyl group attached to C-1. The carbonyl carbon (C-1′) was attached on C-4 and C-5 by the HMBC correlations showed H-5/C-4, C-6, C-2′; H-15/C-3, C-4, C-10. The double bond was assigned to located C-8 and C-9, basing on the HMBC correlation showed H-8/C-1, C-6, C-10. The three hydroxyl groups were located at C-6, C-7, and C-10, respectively, which were assured by the HMBC correlations showed H-6/C-5, C-7, C-14; H-14/C-6, C-7, C-8; H-15/C-3, C-4, C-10. The NOESY

Conclusion
The biosynthesis and accumulation of the extractives is an important process for the formation of heartwood, and the content and types of the extractives in the heartwood also influence the special properties of wood. Previously, we proposed a new type of Taiwania-type heartwood formation mechanism, i.e., phenolic compounds have completed the secondary reaction in the sapwood, forming a complete structure (Tsao et al. [2]). Although the current research on the phytochemistry of Taiwania is quite complete, there is no research on the sapwood extractives. This study isolated and identified 78 compounds from sapwood of Taiwania, including 1 fatty acid, 6 monoaromatics, 1 monoterpenoid, 34 sesquiterpenoids, 6 diterpenoids, 9 steroids, and 21 lignans and norlignans. Among these, 3 new skeleton sesquiterpenoids, which were Taiwania A, Taiwana B, and Taiwania C were first time identified. During the past decades, a number of studies have reported the metabolites of Taiwania's wood. However, to our best of knowledge, this is the only study focusing on the elucidation of sapwood compounds. Interestingly, besides 3 new skeleton compounds, all of the 75 known compounds had been reported previously. This study confirmed again that the secondary reaction of lignans occurred in the sapwood of Taiwania. It provided the evidence for type III, Taiwania-type of heartwood formation (Tsao et al. [2]). However, the unique and dominant lignan, Taiwanin A, was not found in the sapwood. The result once again confirmed that all lignans of Taiwania are synthesized in sapwood, except Taiwanin A.