New Diterpenes from Arenga pinnata (Wurmb.) Merr. Fruits

Three new ent-kauran-type diterpenes (1–3), named arenterpenoids A–C, and five known ones (4–8) were isolated and identified from Arenga pinnata (Wurmb.) Merr. Fruits. The structures of these compounds were established by 1D and 2D NMR spectra and HR-ESI-MS. To the best of our knowledge, this is the first scientific report of diterpenes from Arenga genus.


Introduction
Arenga pinnata (Wurmb.) Merr. are tall evergreen trees belonging to the genus Arenga of the family Palmae. They are widely distributed in Southeast Asian countries, including China. A. pinnata fruits are the fruits of the A. pinnata. [1]. As a kind of folk medicine, it was first recorded in the Song Dynasty's "Kai Bao Ben Cao" and the Ming Dynasty's "Ben Cao Hui Yan" [2,3]. In the folk literature, A. pinnata fruits are made into medicinal liquor, which is rapid and significant in relieving pain [4]. A. pinnata fruits have significant effects on local neuropathic pain, rheumatism, bone pain and traumatic pain [5]. It is abundant and there is a huge development space [6]. At present, the secondary metabolites from A. pinnata fruits have not been reported, and its main effective medicinal ingredients are still not clear. Palm plants contain terpenes, alcohols, alkanes, esters, phenols, quinones, aldehydes and alkaloids, etc [7][8][9]. In recent years, studies have shown that diterpenes have excellent anti-tumor effects in vivo and in vitro [10,11]. The total diterpene of Rabdosia excisa has significant inhibitory effects on P-388, H-22, and Lewis B-16 tumor cells [12]; the diterpenes of Pteris semipinnata L. have significant inhibitory effects on A 549 and CNE-2 tumor cells [13]. This study is the first separation of the Arenga genus by silica gel and ODS column chromatography. We analyzed the chemical constituents of A. pinnata fruits and identified three new diterpenes named arenterpenoids A (1), B (2), and C (3) together with known pseudaminic acids (4) [14], 12α-(β-D-glucopyranosyl)-7β-hydroxy-kaurenolide (5) [15], paniculoside (6) [16], agittarioside b (7) [17], and orychoside B (8) [18]. This report covers the separation and structural analysis of these compounds 1-8 Figure 1.
The relative stereochemistry of 2 was assigned by analysis of the NOESY spectrum. The 1 H-and 13 C-NMR spectra showed similar data for 1 and 2. Thus, it was determined that H-6 was in the β-orientation and that H-7 was in the α-orientation. The correlations of H-6/H-18 showed that they were cofacial and were arbitrarily assigned to be β-oriented.    2). 13 C-NMR and DEPT spectrum (Table 1) of 2 showed an ent-kauran-type diterpene skeleton [22]. The connectivity of these partial structures and the functional groups were investigated by analysis of HMBC of 2. As shown in Figure 2, long range correlations were observed between the following protons and carbon signals: H-18 (CH 3 ) and C-3, C-4, C-5, C-19 (COOH); H-20 (CH 3 ) and C-1, C-5, C-9, C-10; H-14 and C-7, C-8, C-9, C-13; H-15 and C-8, C-15; H-17 and C-13, C-15. Thus, structure 2 was confirmed as shown in Figure 2.
The relative stereochemistry of 2 was assigned by analysis of the NOESY spectrum. The 1 Hand 13 C-NMR spectra showed similar data for 1 and 2. Thus, it was determined that H-6 was in the β-orientation and that H-7 was in the α-orientation. The correlations of H-6/H-18 showed that they were cofacial and were arbitrarily assigned to be β-oriented. H-5, H-9 and H-15 were determined by their correlations with H-18. H-20 and H-11 were determined by their correlations with H-7. Thus, the structure of 2 was determined to be as shown ( Figure 1) and elucidated as, 6α, 7β, 11β, 13β, 17-pentahydroxy-ent-kauran-19-oic acid, named as arenterpenoid B (2).   3). The connectivity of these partial structures and the functional groups were investigated by analysis of the HMBC of 3. As shown in Figure 2, long range correlations were observed between the following protons and carbon signals: H-18 (CH 3 ) and C-3, C-4, C-5, C-19 (COOH); H-20 (CH 3 ) and C-1, C-5, C-9, C-10; H-15 and C-7, C-8, C-9, C-16; H-7 and C-8, C-14, C-15; H-17 and C-13, C-16, C-1 . Thus, the structure of 3 was confirmed as shown in Figure 2.
All these compounds are reported here for the first time in Arenga genus. The kaurane type diterpene is a kind of tetracyclic diterpene with hydrogenated phenanthrene as the mother nucleus [23]. According to the structural rule of the kauri-type diterpene, the compounds 1-8 are all C-20 unoxidized kauri-type. Most of such structures isolated and artificially synthesized in plants have significant biological activities, such as antimicrobial activity and cytotoxicity [24]. This study provides an experimental and scientific basis for drug design and discovery in A. pinnata fruits.

Plant Material
The A. pinnata fruits were collected from Guangxi in China during September 2017, and authenticated by Prof. Weiming Wang of the Heilongjiang Research institute of Chinese Medicine. The fruitage had been deposited at the Heilongjiang Research institute of Chinese Medicine.

Extraction and Isolation
The A. pinnata fresh fruits (30.0 kg) were extracted with 70% EtOH (200 L × 3 h × 3 times). The combined extract was concentrated under vacuum yielding a residue (3.0 kg) which was dissolved in H 2 O (12 L) and extracted sequentially with petroleum, chloroform, ethyl acetate and n-butanol (12 L × 3 h × 5 times). The eluate was separately concentrated in vacuo to give a petroleum syrup (109.0 g), chloroform syrup (123.0 g), ethyl acetate syrup (205.0 g), and an n-butanol syrup (380.0 g). In this study, we only separated the n-butanol layer. The n-butanol (380.0 g) extract was subject to column chromatography on silica gel (4460.0 g) and eluted with CH 2 Cl 2 /MeOH   (Figures 1 and 2).

Acid Hydrolysis and HPLC Analysis
The isolated compounds (1,3) (2.0 mg) were in 1.0 mL HCl and were each heated under reflux for 3 h. After cooling, the two mixtures were separately filtered with Amberlite IRA-400 to give a solution. Assigned with AcOEt to get two layers. The aqueous layer was evaporated to dryness under vacuum, and then subjected to HPLC analysis using an NH 2 column and an optical-rotation detector. D-glucose was confirmed by comparison of the t R with that of an authentic sample (mobile phase: MeCN/H 2 O 85: 15 (v/v); flow rate: 0.8 mL/min; t R = 12.8 min (D-glucose, positive optical rotation))

Conclusions
As a traditional Chinese medicine, A. pinnata fruits were mainly used to treat rheumatism and bone pain. This study obtained eight diterpene compounds from A. pinnata fruits, including three new diterpenes and five known ones. This also reveals some structural characteristics of the chemical constituents in the A. pinnata fruit, which provides some clues for further clarifying the composition of the components and correlations of the relative plant species. In this study we have made this contribution to discover active ingredients and leading compounds and additionally provided an experimental and scientific basis of drug design and drug discovery of the A. pinnata fruits.
Supplementary Materials: The following are available online: Figures S1-S5 and Table S1: The 13 C-NMR data of

Conflicts of Interest:
The authors declare no conflict of interest.