Terpenoids with anti-influenza activity from the leaves of Euphorbia leucocephala

Abstract Two rearranged terpenoids with a rare 3,4,5-trimethyl-cyclohexa-2,5-dien-1-one moiety, namely leucocephins A (1) and B (2), and a megastigmane, namely leucocephin C (3), as well as three known compounds, hollongdione (4), 3-acetoxy-lup-12,20(29)-diene (5), and β-amyrin acetate (6) were isolated from the leaves of Euphorbia leucocephala. Their structures and absolute configurations were determined by spectroscopic methods and comparing with literature data. Compounds 4–6 exhibited potent anti-influenza A virus activity comparable to the positive control, betulinic acid. Graphical Abstract


Introduction
The genus Euphorbia comprises more than 2000 species, some of which are traditionally used as medicinal plants (Jassbi 2006). The chemical constituents have been well known for the diverse chemical structure (Frezza et al. 2018;Lahmadi et al. 2020;Chen et al. 2021;Deng et al. 2021;Hassan et al. 2022;Tran et al. 2022;Yan et al. 2022;Zhang et al. 2022) and wide spectrum of bioactivities (Shi et al. 2008). Some of the terpenoids in Euphorbia plant were found to have significant bioactivities for the treatment of actinic keratosis (Gupta and Paquet 2013), detrusor hyperreflexia, diabetic peripheral neuropathy, cancer pain (Kissin and Szallasi 2011), and HIV infection (Miana et al. 2015).
Euphorbia leucocephala Lotsy, a densely branching shrub, has many common names, such as white Christmas bush, snow flake, and little Christmas flower. It is endemic to Mesoamerica and Mexico, and has been widely commercially available as a garden potted and ornamental plant. Its phytochemical ingredients have not been examined to date.
Influenza A virus (IAV), a highly infectious respiratory illness, which causes 3-5 million population to be affected annually worldwide and usually accompany with high mortality and morbidity. Our preparatory assay for the extracts from the leaves of E. leucocephala revealed that both hexane and nonalkaloid layers, at 25 lg/mL, exhibited significant anti-IVA activity. In the course of our persisting efforts to investigate antivirus natural products from medicinal plants (Chao et al. 2014(Chao et al. , 2016(Chao et al. , 2018, the active layers of E. leucocephala were studied, which resulted in the characterisation of three new terpenoids (1-3), and three known compounds (4-6). Herein, we described the details of isolation, structural elucidation, and anti-IVA activity of the isolated terpenoids.

Results and discussion
Three new terpenoids, leucocephins A-C (1-3), and three known compounds (4-6), were isolated from the methanolic extract of the leaves of Euphorbia leucocephala. By comparison of experimental and literature data, the known compounds were characterised as hollongdione (4) (Phongmaykin et al. 2008), 3-acetoxy-lup-12,20(29)-diene (5) (Bohlmann et al. 1982), and b-amyrin acetate (6) (Bibi et al. 2010) (Figure 1). Leucocephin A (1), [a] 25 D À36, was found to have a molecular formula of C 13 H 18 O 2 by the analysis of its HRAPCIMS ( Figure S6, Supporting Information) and 13 C NMR data (Table S1 and Figure S2, Supporting Information). The IR absorption bands denoted the presence of hydroxyl (3406 cm À1 ) and conjugated carbonyl (1667 cm À1 ) groups. The latter was confirmed by a diagnostic UV absorption band at k max 232 nm.  Figure S1 and Table S1, Supporting Information). Analysis of the 13 C NMR data, with the assistance of DEPT and HSQC spectra ( Figures S2 and S5, Supporting Information), indicated 13 carbon resonances suggestive of a carbonyl group (d C 186.1), three double bonds (d C 163.1, 163.0, 136.7, 131.3, 127.1, and 127.0), four methyls (d C 23.6, 20.7, 20.0, and 19.9), a quaternary sp 3 carbon (d C 48.3), and an oxymethine carbon (d C 68.3). The olefinic H-8 show a large coupling constant (15.6 Hz) with H-7 and COSY correlations with both H-7 and H-9, while the latter showed a strong correlation with a methyl doublet H 3 -10 ( Figure S3, Supporting Information), suggesting the presence of a trans-but-3-en-2-ol moiety. On the other hand, a rare 3,4,5-trimethyl-cyclohexa-2,5-dien-1-one moiety was constructed based on the HMBC correlations from H 3 -11 to C-1, C-2, and C-6, from H 3 -13 to C-4, C-5, and C-6, and from H 3 -12 to C-1, C-5, and C-6 ( Figure S5, Supporting Information). The trimethylcyclohexdienone fragment attached to the but-3-en-2-ol moiety was assigned by the crucial HMBC correlation from H 3 -12 to C-7. Accordingly, a rare structure with a rearranged 12(1!6)-abeo-megastigmane scaffold was established (Jiang et al. 2020). Due to the limited amount of compound 1, the absolute configuration at C-9 could not be assigned by the application of Mosher's method. Thus, the absolute configuration of 1 was determined by comparison of its specific optical rotation with those of known analogues. Compound 1 ([a] 25 D À36) and the aglycone of isodonmegastigmane I ([a] 25 D À46) shared the same sign of specific optical rotation, indicating that compound 1 have R-configuration at C-9 (Matsumoto et al. 2017). Based on the above data, the chemical structure of leucocephin A (1) was ascertained as shown.
(þ)-HRAPCIMS of leucocephin B (2) gave a protonated adduct ion at m/z at 221.1537 [M þ H] þ , indicative of a molecular formula of C 14 H 20 O 2 with 14 mass unit higher than that of 1 (Figure S12, Supporting Information). As compared to 1, the IR spectrum of 2 showed the same conjugated carbonyl (1667 cm À1 ) group, but there is no OH peak visible around 3200-3400 cm À1 . Inspection of the 1 H and 13 C NMR data of 1 and 2 (Table S1, Supporting Information), the structure of 2 was suggestive to be similar to 1, except that the methoxy group was observed at C-9 position [d H 3.27 (3H, s, OMe), d H 3.75 (1H, m, H-9); d C 56.2 (OMe), d C 77.6 (CH, C-9)]. This was confirmed by the HMBC correlations from OMe to C-9 and from H 3 -10 to C-8 and C-9 ( Figure S11, Supporting Information). The 9 R absolute configuration was again established by comparing its specific optical rotation ([a] 25 D À14) with those of 1 and similar analogues (Matsumoto et al. 2017).
The molecular formula of 3 was assigned as C 18 H 22 O 2 according to the (-)-HRAPCIMS and 13 C NMR data (Table S1 and Figure S19, Supporting Information). The IR absorption band at 1714 cm À1 implied the presence of carbonyl group. The 13 C and DEPT NMR data of 3 showed downfield signals characteristic of two ketone groups (d C 208.3 and 210.7) and upfield signals attributable to aliphatic carbons comprising four methyls, four methylenes, two methines, and one quaternary carbon. The NMR data of 3 were quite similar to sarmentol I (Ninomiya et al. 2007); however, the difference was the absence of hydroymethyl group attached at C-9 and the replacement by a methyl group in 3, which was confirmed by the HMBC correlations from H 3 -10 to C-8 and C-9 (Figure S17, Supporting Information). By a further confirmation of 2 D NMR experiments, the planar structure of 3 was established (Figure 1). The NOE correlations were observed for the following protons: H 3 -11/H 2 -7, H 3 -11/H-5, H 3 -11/H-2a, H-6/H-2b, H-6/H-4a ( Figure S18, Supporting Information). These correlations suggested that the methyl group at C-5 and the side chain at C-6 occupied the equatorial position, revealing a trans relationship of these two substituents. The CD spectra of 3 showed a negative Cotton effect [294 nm (De À0.01)] due to the n!p Ã transition, which is opposite to (5 R,6S)-sarmentol I [288 nm (De þ 0.12)] (Ninomiya et al. 2007), suggesting a 5S,6R-configuration for 3. Also, analysis of the specific optical rotation of 3 ([a] 25 D À3), with opposite sign to sarmentol I ([a] 24 D þ 19.9) (Ninomiya et al. 2007), also confirmed its absolute configuration to be 5S,6R.
Compounds 3-6 were subjected for anti-IAV activity using plaque reduction assay, while the cytotoxicity evaluation towards Madin-Darby canine kidney (MDCK) cells were performed using MTS assay. The tested compounds were found to be non-toxic towards the host cells, MDCK cells, at 100 lM ( Figure S27a

General experimental procedures
Measurement of optical rotations were performed on a JASCO P2000 digital polarimeter. Spectra data for UV and IR were collected a PerkinElmer Lambda-265 UV/Vis spectrophotometer and a PerkinElmer Spectrum Two FT-IR spectrometer, respectively. CD spectrum was measured on a JASCO J-715 spectropolarimeter. The NMR spectra were recorded on a Bruker Avance-400 and a BRUKER AVIIIHD700X spectrometers at 400 and 700 MHz, respectively. The high concentrated CDCl 3 (99.96%) was used for NMR measurements of compounds 1 and 2. The chemical shifts were calibrated to residue signals of CDCl 3 (d H 7.26; d C 77.0). APCIMS and HRAPCIMS were measured with a Finnigan LCQ ion-trap mass spectrometer and a LTQ Orbitrap XL mass spectrometer, respectively. Normal phase and reverse phase column chromatography was performed using Silica gel 60 (230 À 400 mesh, Merck) and SiliaBond C18 silica gel (40-63 mm, 60 Å, 17% carbon loading, Silicycle), respectively. TLC analysis was performed using precoated silica gel plates (Kieselgel 60 F 254 , 0.25 mm, Merck) and silica gel based RP-18 plates (Kieselgel 60 F 254 S, Merck).

Plant material
The leaves of Euphorbia leucocephala were collected in April 2017 from Taichung City, Taiwan and were identified by Dr. C.-H. Chao. A voucher specimen (specimen no. EL-Chao006) was deposited at School of Pharmacy, China Medical University, Taichung, Taiwan.

Extraction and isolation
Fresh leaves were dried in air for weeks, and crushed by hands before extraction. The dried material (4.57 kg) was extracted exhaustively with MeOH (3 Â 25 L) and concentrated to give a residue (545 g), which was partitioned between hexane and 90% methanolic solution. The hexane layer was concentrated under reduced pressure to obtain a residue (EH, 109 g). The aqueous MeOH layer was passed through a cationexchange resin (Dowex 50WX4) and the collected eluent was again concentrated in vacuum to give the non-alkaloid residue (410 g), which was subjected for silica gel vacuum liquid chromatography (VLC) and eluted gradiently with a solvent mixture comprising EtOAc and hexane to obtain seven fractions (EN1-EN7). Fraction EN2 (2.87 g), eluted with EtOAc-hexane (40%), was further subjected to a silica gel column chromatography (CC) with a gradient elution from 0% to 50% EtOAc in hexane, and a total of 18 fractions (EN2A-EN2R) were pooled based on TLC analysis. Fraction EN2J (255 mg) was fractionated using RP-18 CC to give 16 fractions (EN2J1-EN2J16). Compound 3 (6.9 mg) was obtained from fraction EN2J6 using RP-18 HPLC (MeOH-H 2 O, 50%). Fraction EN2J11 was subjected for purification by HPLC (MeOH-H 2 O, 60%) to obtain compounds 1 (0.4 mg) and 2 (0.2 mg). Fraction EN2J15 was purified by RP-18 CC with a gradient solvent system (MeOH-H 2 O, gradient, 50% to 100%) to afford compound 4 (7.2 mg). The hexane layer (EH) was subjected for gradient chromatography on silica gel (EtOAc-hexane, 0% to 18%) to give 18 fractions, of which the fourth fraction was further separated by silica gel CC (EtOAc-hexane, 0% to 5%) to afford 17 fractions (EH4A-EH4Q). Fraction EH4E was further purified by RP-18 CC to obtain compounds 5 (35 mg) and 6 (48 mg).

Anti-influenza virus assay
The anti-influenza virus assay was performed by plaque reduction assay with A/WSN/ 33 (H1N1) virus as described previously (Li et al. 2019). In brief, Madin-Darby canine kidney (MDCK) cells at density of 10 4 cells/well were seeded in the 96 well plate were cultured with Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS for 24 h at 37 C. For cytotoxicity detection of each compound, up to 100 mM was added to the cells for 48 h and was measured by MTS assay (CellTiter96V R AQueous One Solution Assay (Promega, Madison WI)) and the non-cytotoxic compounds were subsequently applied to plaque reduction assay as following: Influenza virus (A/WSN/1933 (H1N1), 100 PFU/well) mixing with 25 mM of each compound was used to infect the MDCK cells in a six-well plate (2 Â 10 6 cells/well). The DMSO was used as a negative control and betulinic acid was used as a positive control. After 1 h adsorption, the viral suspension was removed and the cells were washed three times with PBS, and then overlaid with 0.3% agarose in medium with or without the indicated compound for further 48 h. The cells were then fixed and stained with 1% crystal violet. The number of plaques was counted and the antiviral activity of the indicated compound was calculated by comparing with that of the virus infected control. The data are presented as mean-± standard error of at least three individual experiments. Statistical significance was determined using the 2-tailed Student's t-test (GraphPad Prism version 5.0; GraphPad Software, Inc., La Jolla, CA).

Conclusions
In this paper, three terpenoids, leucocephins A-C (1-3), as well as three known compounds, hollongdione (4), 3-acetoxy-lup-12,20(29)-diene (5), and b-amyrin acetate (6), were isolated and characterised from the leaves of Euphorbia leucocephala. Among them, the isolation of 1 and 2 enrich the members of the rare 12(1!6)-abeo-megastigmane group. The chemical constituent from title plant and its anti-IAV activity were reported for the first time. The structure of 4-6 and betulinic acid are triterpenoids, which implied that the triterpenoids might play a major role in preventing IAV infection. The present study showed that this ornamental plant could also be potentially used as anti-IAV agent.