Triterpene constituents from the fruits of Cyclocarya paliurus and their anti-HIV-1IIIB activity

Abstract Four new norceanothane-type triterpenes, cyclopalin A-D (1-4), and sixteen known compounds (5-20) were obtained from the fruits of Cyclocarya paliurus. Their structures were determined by spectroscopic data, experimental electronic circular dichroism (ECD) and X-ray single crystal analyses. All isolated compounds were assayed for their anti-HIV-1IIIB activity. Compound 18 exhibited potent anti-HIV-1IIIB activity with an EC50 value of 1.32 μM (SI = 151.52). Graphical Abstract


Introduction
Cyclocarya paliurus, belonging to the Juglandaceae family, is an endemic plant growing in China (Editorial Committee of Flora of China, 1979). Due to the sweet taste of its leaves, it is often used as herbal tea and commonly known as the 'sweet tea tree' (Kennelly et al. 1995). In addition, its leaves have been used as Chinese folk medicine to prevent dyslipidemia, hypertension, and diabetes (Xie and Li 2001;Wright et al. 2014). The extracts of this plant have exhibited antioxidant, hypoglycemic and hypolipidemic effects in type 2 diabetic rats (Kurihara et al. 2003;Wang et al. 2013;Ma et al. 2015).
2．Results and discussion
The molecular formula of cyclopalin D (4) (Table S1), categorised into six methyls, nine methylenes (one olefinic carbon at d C 110.1), seven methines (two oxygenated carbons at d C 92.1 and 84.6), and seven nonprotonated carbons (one olefinic carbon at d C 152.1, and one carbonyl at d C 180.1). The aforementioned spectroscopic analysis suggested that 4 is a norlupane-type or norceanothane-type triterpenoid derivative. The 1 H and 13 C NMR data of 4 (Table S1) were similar to those of ceanothic acid (5) (Li et al. 1997), except for the absence of a carboxyl group of C-2 and the presence of two oxygenated methine at C-1 (d C 86.4) and C-3 (d C 92.1) in 4. This assumption was verified by the HMBC correlations of H-1 (d H 3.45 s), H 3 -23 (d H 0.82, s) and H 3 -24 (d H 1.03, s) with C-3, H-1 (d H 3.45 s) and H 3 -25 (d H 0.87, s) with C-1. 1-OH was assigned to be a-oriented by the ROESY correlation of H-1/H-11b and H-1/H 3 -25. The relative configurations of the remaining stereogenic centers were identical to those of 2 by analysis of the 13 C NMR and ROESY data. Ultimately, the structure of 4 was identified as 17b-carboxy-(1a, 3b)-dihydroxy-28-norceanoth-20(29)-en-28-oic acid.
According to the structural features, the hypothetical biosynthetic pathway of 1, 2, and 4 was proposed on the base of pinacol rearrangement and oxidation of the coexisting compound 10 ( Figure S5).

Anti-HIV-1 IIIB activity
Compounds 1-20 were tested for their potencies in preventing the cytopathic effects of HIV-1 IIIB in TZM-bl cells. Cytotoxicity was measured in parallel to determine antiviral activity with TDF as a positive control (EC 50 ＝1.21 ± 0.56 nM and CC 50 >200lM). Some compounds showed anti-HIV IIIB activity, and the range of EC 50 was 1.32 ± 0.44-159.05 ± 23.71 lM. Among them, compound 18 exhibited the best anti-HIV-1 IIIB activity with EC 50 value of 1.32 ± 0.44 lM and a selectivity index (SI) more than 151.52 (Table S2).
Among the ceanothane-type triterpenes (1-7) tested, compound 3 generated the strongest anti-HIV-1 IIIB activity, which may be attributed to its b-H configuration at C-5. Compounds 8-12 are lupine-type triterpenes, while 8 and 9 did not exhibit anti-HIV-1 IIIB activity, possibly because of the absence hydroxyl groups at both C-2 and C-30. Compared to compound 18, the isolated ursane-type triterpenoids (13-17, 19 and 20) showed relatively weak activity (EC 50 >20lM). However, compound 18 exhibited potent anti-HIV-1 IIIB activity with high SI (Selectivity index). These results suggested that the hydroxy group at C-2, C-3, and C-19 in 18 might contribute to its anti-HIV-1 IIIB activity.

Experimental section
3.1. General experimental procedure UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained by a Tensor 27 spectrophotometer with KBr pellets. ECD spectra were measured on a Chirascan Circular Dichroism Spectrometer. Optical rotations were measured with a Jasco P-1020 polarimeter. One-dimensional (1 D) and two-dimensional (2 D) NMR spectra were recorded on Bruker AV-400 and AV-500 spectrometers with tetramethylsilane as the internal standard. HREIMS was performed on an Agilent UPLC/Q-TOF, 1290, and LC/MSD TOF liquid-mass spectrometer. Semipreparative HPLC was performed on an Agilent 1260 liquid chromatograph system with a Zorbax SB-C18 (9.4 mm Â250 mm, 5 lm) column at a flow rate of 3 mL/min. Column chromatography (CC) was performed on silica gel (100-200 and 200-300 mesh; Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), MCI gel (75-150 lm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH-20 (Pharmacia). All fractions were monitored by TLC, and spots were visualised by UV light (254 nm) and sprayed with 10% H 2 SO 4 in ethanol, followed by heating. All solvents were distilled prior to use.

Extraction and isolation
Air-dried fruits of C. paliurus (100 kg) were extracted three times with 80% ethanol under reflux and evaporated to afford a crude extract (11.0 kg). The extract was suspended in water and partitioned with ethyl acetate (EtOAc) to yield a EtOAc-soluble extract (3.8 kg). The EtOAc solution was then concentrated under reduced pressure, and the residue was chromatographed on a silica gel column (6 Kg, 100-200 mesh) and eluted stepwise with a petroleum ether (PE)-EtOAc gradient system (1:0 À 0:1) to afford the major fractions A À F. Fraction A (1.5 Kg) was a mixture of fatty substances, as indicated by the 1 H NMR spectrum.

Anti-HIV-1 IIIB activity assay
The inhibitory effect on acute HIV-1 IIIB infection was measured by luciferase activity assay. In the presence or absence of various concentrations of samples, exponentially growing TZM-bl cells were infected with HIV-1 IIIB at an appropriate multiplicity of infection (MOI) and cultured in 96-well plates at 37 C in 5% CO 2 cell culture incubators. 48 h later, and cells were lysed with Glo Lysis Buffer (Promega) and luciferase activity of lysate was measured by D-Luciferin potassium. The relative luciferase activity was measured by using Molecular Devices microplate reader FlexStation 3.
The cellular toxicity of compounds on TZM-bl cells was also assessed by MTT colorimetric assay, which is based on the reduction of yellow colored MTT by mitochondrial dehydrogenase of metabolically active cells to a blue-purple formazan that can be dissolved in 50% DMF-15% SDS. After the formazan was dissolved completely, the plates were analysed by Bio-Tek 800TS microplate reader at 570 nm/630 nm.

Conclusion
Chemical constituents and anti-HIV activity of the fruits of C. paliurus were reported in our work. Twenty triterpenoids, including ceanothane-type (1-7), lupine-type (8-12), and ursane-type (13-20), were obtained from the fruits of the plant. Among them, four new norceanothane-type triterpenes, cyclopalin A-D (1-4), were isolated for the first time. The structures of these four previously undescribed compounds were elucidated by spectroscopic analysis, ECD, and single crystal X-ray diffraction.
Moreover, eighteen compounds showed anti-HIV activity, and the range of EC 50 was 1.32 ± 0.44-159.05 ± 23.71 lM; compound 18 possesses the most significant anti-HIV-1 IIIB activity. The hydroxy group at C-2, C-3, and C-19 in 18 highly contributes to the anti-HIV-1 IIIB activity.

Disclosure statement
No potential conflict of interest was reported by the authors.