Jatrophane Diterpenoids from the Seeds of Euphorbia peplus with Potential Bioactivities in Lysosomal-Autophagy Pathway

Abstract Euphopepluanones F − K (1 − 4), four new jatrophane type diterpenoids were isolated from the seeds of Euphorbia peplus, along with eight known diterpenoids (5 − 12). Their structures were established on the basis of extensive spectroscopic analysis and X-ray crystallographic experiments. The new compounds 1 − 4 were assessed for their activities to induce lysosomal biogenesis through LysoTracker Red staining. Compound 2 significantly induced lysosomal biogenesis. In addition, compound 2 could increase the number of LC3 dots, indicating that it could activate the lysosomal-autophagy pathway. Graphic Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-021-00301-4.

alleviation of neuropathic pain and is in phase I human clinical trials in treating severe pain in cancer [5]. In 2012, an ingenane type diterpenoid ingenol 3-angelate from E. peplus was approved by FDA for the treatment of actinic keratosis, a precancerous skin condition [6,7].
E. peplus Linn., a small annual weed native to Mediterranean coast, was introduced into Yunan province of China [8]. The sap from E. peplus has been used in folk medicine for the treatment of asthma, catarrh and internal tumors [9]. Recently, our group discovered ingenane type diterpenoids 20-deoxyingenol and its analogues from E. peplus, possessing activity to promote lysosome biogenesis, limit amyloid plaque formation in APP/SP1 mice's brain, which suggested that the potential of these compounds for the treatment of Alzheimer disease [10]. The subsequent phytochemical studies of the plants of the genus Euphorbia led to isolation of several novel diterpenoids with significant bioactivities [11][12][13][14][15][16]. In our continuing efforts to uncover structurally novel diterpenes capable of inducing lysosomal biogenesis, four new jatrophane type diterpenoids euphopepluanones F − K (1 − 4), along with eight known diterpenoids (5 − 12) were obtained from the seed of E. peplus (Fig. 1). Their structures were elucidated based on extensive NMR, X-ray crystallographic and electronic circular dichroism (ECD) experiments. Furthermore, the activity of compounds 1 − 4 in inducing lysosomal biogenesis were tested, in which only 2 displayed significant activity. Herein, we reported the structural elucidation and biological evaluation of these compounds.
The molecular formula of euphopepluanone I (2)  Comparison of the spectroscopic data of 2 with those of 1 suggested similar structure but with different esterification patterns. The former harbored an angeloxy group (δ H 6.16, 2.01, 1.87) instead of the acetoxy group at C-7. HMBC correlations of the carbonyl carbons (δ C 166.9) and oxymethine protons (δ H 6.09) placed the angeloxy group at C-7. The relative configuration of 2 was the same as that of 1 by their similar ROESY cross-peaks.
The positive HRESIMS data of euphopepluanone J (3) showed an [M + Na] + ion at m/z 693.2512 (calculated for 693.2518), corresponding to the molecular formular C 35 H 42 O 13 . The mass spectrum indicate compound 3 was 42 mass units more than compound 1, suggesting that one of hydroxyl group in 1 was acetylated in compound 3. On comparing its NMR data with those of 1 (Table 1), 3 displayed additional signals responsible for an acetoxy group (δ H 2.19; δ C 169. 8, 22.3), and the absence of the OH-2 signal. Thus, the OH-2 in 1 was inferred as being replaced by an acetoxy group in 3, which was supported by the HMBC correlations from H-2 to the carbonyl carbon. Therefore, the structure of 3 was delineated as shown.
Euphopepluanone K (4) possessed a molecular formula of C 27 H 34 O 8 as deduced from its positive HRESIMS ([M + Na] + , m/z m/z 509.2151, calcd for C 27 H 34 O 8 Na, 509.2146). The spectra data of 4 closely related to those of 5, except for the absence of acetate signals at C-5, C-7, and C-8, suggesting the replacement of them with hydroxyl groups. Indeed, the resonances of C-4, C-6 and C-9 were up-shielded (Δδ C + 2.7, + 6.8, + 6.4 ppm), and C-5 and C-8 were down-shielded (Δδ C -0.7, -1.6) in 4, further supporting the presence of hydroxy groups at C-5, C-7, and C-8, instead of acetoxy groups in compound 5. The relative configuration of 4 was assigned as that of 1 by the nearly identical ROSEY data of these two compounds. Furthermore, the similar electronic circular dichroism (ECD) spectra of compounds 1, 2, 3 and 4 ( Fig. 4) indicated they share the same absolute configurations.

Bioactivity Evaluation
To assess the activity to enhance lysosomal biogenesis of the new compounds 1-4, LysoTracker Red staining method was used. All the four new compounds increased the LysoTracker staining intensity. The cells were treated for 3 h with compounds 1 − 4 at 20 μM, and these compounds increased the LysoTracker staining intensity by 141.3%, 151.7%, 136.4% and 130.1%, respectively (Fig. 5a). Hep-14 was used as positive control [10]. It was further tested whether the lysosome biogenesis activities of these compounds are time-and concentration-dependent. As shown in Fig. 5b, HeLa cells were treated for 1, 3 and 6 h with 10, 20 and 40 μM of compound 2 as indicated. Induction of lysosomes was observed in a time-and concentration-dependent manner, with the greatest increase at 40 μM when the cells were treated for 6 h. Many lysosomal genes were upregulated during lysosome biogenesis. To confirm that compound 2 induce lysosomal biogenesis, the expression levels of a set of lysosomal genes were checked, including lysosomal-associated membrane protein 1 (LAMP1), cathepsin B (CTSB), cathepsin A (CTSA), lysosomal sulfatase (ARSB), and ATPase H + transporting V0 subunit E1 (ATP6 V0E1). As shown in Fig. 5c, all these genes were upregulated at mRNA levels 3 h after treatment with compound 2. These data further demonstrated that compound 2 can induce lysosomal biogenesis. Then, we checked the level of LC3 dots, the marker for the activation of autophagy, induced by compound 2. The number of LC3 dots increased with the treatment of compound 2 in a dose-dependent manner (Fig. 5d, e). These results indicate that compound 2 could activate the lysosomal-autophagy pathway.

General Experimental Procedures
Optical rotations were measured with a Jasco P-1020 automatic polarimeter. CD spectra were obtained on the Applied Photophysics circular dichroism spectrometer (Applied Photophysics, Leatherhead, Surrey, UK). High-resolution MS data were measured on an Agilent 1290 UPLC/6540 Q-TOF mass spectrometer in positive mode. IR spectra were determined on a NICOLET iS107 Mid-infrared spectrometer. NMR spectra were measured on Bruker AVANCE III 500 MHz and AV 600 MHz NMR spectrometers with TMS as the internal standard. An Agilent 1260 series instrument equipped with a SunFire-C 18

Plant Material
In August 2018, the seeds of E. peplus were collected from Kunming Botanical Garden, Yunnan Province, People's Republic of China. A voucher specimen (No. kep-09-13) identified by Prof. Hu Shi-Jun (Southwest Forestry University) was deposited in the herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation
The air-dried the seeds of E. peplus (24 kg) were powdered and extracted with methanol thrice at room temperature. The extract was suspended in water and extracted with petroleum ether, and ethyl acetate. The ethyl acetate extract (800 g) was subjected to a silica gel column using petroleum ether/ethyl acetate (100:0 to 0:100, v/v) as eluent to obtain 10 fractions, F1 − F10, in which diterpenes are mainly concentrated in F7 and F8.

X-ray Crystallographic Analyses
Crystallographic Data for Compound 1.

Cell Culture
The activity to enhance lysosomal biogenesis of compounds 1-4 was evaluated using HeLa cell line, which was cultured at 37 °C with 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone), 100,000 U/mL penicillin and 100 mg/mL streptomycin. HeLa cell was purchased from ATCC.

Screening for Compounds That Induce Lysosomal Biogenesis
Briefly, HeLa cells with 85% cell density in 96-well plates were treated with individual compounds at 20 μM in triplicate. Three hours later, cells were grown in fresh medium containing LysoTracker Red DND-99 (0.2 μM) for 30 min. Then, medium was changed to LysoTracker-free medium and images were taken with ArrayScan Infinity (Cellomics, ArrayScan VTI HCS). Positive compounds were subjected to validation by treating HeLa cells with different concentrations (10, 20 and 40 μM) and at 1, 3 and 6 h in triplicate and staining with LysoTracker Red DND-99.

Confocal Microscopy
CFP-LC3 expressing HeLa cells were treated with indicated compounds and images were collected by confocal microscopy. For live-cell imaging, cells grown on glass-bottom dishes were observed directly. All samples were examined with an inverted Olympus FV1000 confocal microscope. Images were analyzed with FV10-ASW 4.0a Viewer.

Quantitative Real-Time PCR with Reverse Transcription (qRT-PCR)
Total RNA was isolated from HeLa cells by using TRIzol Reagent (Invitrogen) according to the manufacturer's recommendation. A reverse-transcription kit (Promega) was used to reverse transcribe RNA (1 µg) in a 20 µL reaction mixture. A real-time PCR system (7900HT Fast; Applied Biosystems) was used to quantify gene expression in triplicate. Amplification of the sequence of interest was normalized with the reference endogenous gene actin.

Statistics and Reproducibility
Data analyses were carried out using Prism 5, and Student's t tests were employed for statistical analyses with a level of significance of p < 0.05.