Three new secolignans from Peperomia blanda (Jacq.) Kunth

Abstract Three new secolignans were found in the detailed chemical study of Peperomia blanda (Jacq.) Kunth collected from China. Detailed NMR data analysis, especially 1H NMR, 13C NMR and 2 D NMR, elucidates the structures of the three new secolignans. Graphical Abstract


Introduction
Peperomia blanda (Jacq.) Kunth (Piperaceae), mainly grows in Yunnan, Guangdong, Guangxi and Fujian provinces in China, has been used in folk remedies to treat stomach, liver, mammary and esophageal cancers (Wang et al. 2013). At present, there are relatively few studies on the chemical constituents of Peperomia blanda (Jacq.) Kunth. Secolignans are a typical secondary metabolite with similar structural characteristics in Peperomia blanda (Jacq.) Kunth. The secolignans found from this plant were mainly peperomin A (Wang et al. 2014), peperomin B (Wang et al. 2014), peperomin C (Wang et al. 2014), peperomin D (Chen et al. 2008), peperomin E (Govindachari et al. 1998), peperomin F (Govindachari et al. 1998), peperomin G (Lin et al. 2011), peperomin H (Lin et al. 2011). These secolignans have extensive biological activity. In vitro studies, peperomin A and peperomin B showed moderate inhibitory effects on HIV-1 IIIB growth in C8166 cells (Zhang et al. 2007). Peperomin E and peperomin B have significant inhibitory effect on the formation of human umbilical vein colorectal cells tube (Lin et al. 2011). Peperomin E showed significant antiproliferative activity on cultured prostate cancer cells in a dose-dependent manner . Peperomin E can inhibit the invasion and migration of gastric cancer cells, and has low toxicity to normal cells, so it has the potential to treat gastric cancer (Wanga et al. 2018). Peperomin E is characterised by the presence of an a-methylene-c-butyrolactone group structure, which has a strong anticancer activity (Wang et al. 2017).
In this paper, we introduce the isolation, purification and structural identification of three new secolignans from Peperomia blanda (Jacq.) Kunth.

Results and discussions
The extraction of ethyl acetate layer was separated and purified by a series of chromatographic techniques, and three new secolignans were obtained, namely peperomin I (1 mg), peperomin J (1.4 mg) and peperomin K (1.4 mg).

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.2(C-3) signal; 4.90(1H, brs, H-1)correlates with 45.8(C-2), 42.2(C-3), 71.0(C-4), 60.9(C-6), 53.5(C-9). Comparing with the literature data (Feng et al. 2010), a furan ring was deduced from the key HMBC cross peaks between C-2, C-3, C-4 and the H-1. In summary, suggesting the existence of part A ( Figure S21). Because in the HMBC experiment, 3.71(1H, d, J ¼ 11.5 Hz, H-5)correlates with 42.2(C-3), the plane structure of the compound is inferred by connecting part A to part B. In order to determine the relative configuration of the compound, NOESY experiment was measured. In NOESY experiment, the signal of 3.71(1H, d, J ¼ 11.5 Hz, H-5) is correlated with the signal of 3.93(1H, t, J ¼ 11.0 Hz, H-6b) and 4.14(1H, dd, J ¼ 11.0, 4.5 Hz, H-6a). Signals of 3.93(1H, t, J ¼ 11.0 Hz, H-6b) and 4.14(1H, dd, J ¼ 11.0, 4.5 Hz, H-6a) correlate with 4.90(1H, brs, H-1), suggesting the relative configuration of this compound (Figure 1). The compound was identified as an unreported new compound and named as peperomin J. Peperomin K (Figure 1) (Table S2), peperomin K has the same plane structure as peperomin J. In order to determine the relative configuration of the compound, the NOESY experiment was measured. In NOESY experiment, the signal of 2.62(1H, m, H-2) is correlated with the signal of 4.98(1H, d, J ¼ 4.0 Hz, H-1); and the signal of 3.36(3H, s, H-9) is correlated with the signal of 6.63(1H, d, J ¼ 1.8 Hz, H-6 0 ), 6.59(1H, d, J ¼ 1.8 Hz, H-6 0 '), and 6.48(1H, d, J ¼ 1.8 Hz, H-2 0 '), so the relative configuration of the compound was speculated (Figure 1). Compared with the NMR data of peperomin J, the main difference is that the peak type of 4.98(1H, d, J ¼ 4.0 Hz, Table S2, Position H-1) is significantly changed, while the 4.90(1H, brs, Table S1, Position H-1) of peperomin J is brs peak type. And, the compound was identified as an unreported new compound and named as peperomin K. Peperomin I (Figure 1) is a white powdered solid(chloroform, methanol, acetone). The molecular formula was determined to be C 23 H 26 O 9 by HR-TOF-MS at m/z 469.1478 [M þ Na] þ , the calculated value is 469.1475. First, its 1 H NMR(500 MHz, methanol-d 4 ) and 13 C NMR(125 MHz, methanol-d 4 ) (Table S3) data were obtained. Then the HMBC and HSQC experiments were measured. The NMR data of this compound were compared with peperomin J, and it was found that this compound lacked the signal value of the acetyl group. The plane structure of the peperomin I can be deduced ( Figure  1). In NOESY experiment, the signal of 2.22(1H, m, H-2) is correlated with the signal of 3.48 (1H, m, H-3), both of which are on the same side. The H signal at position 1 of peperomin J is brs peak type (Table S1, Position H-1), and the H signal at position 1 of peperomin K is d peak type (Table S2, Position H-1). In contrast, the H signal at position 1 of this compound is also a brs peak without splitting peak (Table S3, Position H-1). So we can infer the relative configuration of this compound (Figure 1 ). Since this compound is missing an acetyl group, the main difference compared with the NMR data of Peperomin J and K is that the position H-6 (Table S3, Position H-6) signal value moves significantly towards the high field. And, the compound was identified as an unreported new compound, named peperomin I.

Experimental materials
The whole plant(Peperomia blanda (Jacq.) Kunth) was purchased in 2017 from Bozhou City, Anhui Province, China. A voucher specimen, identified by Prof. Chen (Liaoning Normal University), was deposited with registration No.04026 in the College of Life Sciences and Biotechnology, Dalian University.

NMR measurements
NMR spectra were measured on Bruker AM-500 with TMS (d ¼ 0.00 ppm) as the internal standard and CD 3 OD(dH ¼ 3.40 ppm, dC ¼ 49.00 ppm) as the solvent for NMR measurement. For peperomin J, peperomin K and peperomin I, 1 H NMR, 13 C NMR, HMBC, HSQC, NOESY experiment were measured.

Extraction and isolation
The 9.6 kg of experimental material was dried and crushed. Using 95%(V/V) EtOH(15L) reflux extraction method, the extraction was carried out at 100 C for 3 times, 4 h each time. The alcohol extract was concentrated to dry state under reduced pressure, and the extraction amount was 1.064 kg. After the ethanol extract was fully suspended with deionized water, the same volume of petroleum ether was used to extract the colorless liquid. The petroleum ether layer was compressed and concentrated to obtain 635 g of petroleum ether extract. The same volume of ethyl acetate was added into the water layer for extraction until the extract was colorless, and 136.2 g of ethyl acetate extract was obtained. The remaining water layer is 168.6 g.

Conclusions
In this study, we isolated three new secolignans from Peperomia blanda (Jacq.) Kunth, and determined the plane structures and relative configurations of them by NMR experiments. These three new secolignans have been named peperomin I/J/K because they share a similar skeleton structure with the previously isolated peperomins compound from Peperomia blanda (Jacq.) Kunth. It can be inferred that these compounds may share a common biosynthetic pathway in this plant.
In our study, three other compounds were isolated from Peperomia blanda (Jacq.) Kunth: N-trans-feruloyldopamine, isoscutellarin-4 0 -methylether-8-O-a-L-arabinoyl (1!4)b-D-glucoside and 5,7,8-trihydroxy-4 0 -methoxyflavone-8-O-b-D-glucoside, these three compounds were tested for neuraminidase activity according to the method in the literature (Lu et al. 2019).The obtained IC50 values are 6.31 lg/mL, 2.48 lg/mL, 0.93 lg/mL (Table S4), which have the activity of inhibiting neuraminidase, which explains the mechanism of Chinese medicine against influenza virus to a certain extent. However, peperomin I/J/K did not inhibit neuraminidase activity. Unfortunately, the bioactivity of the three new secolignans could not be determined due to the limited number of isolates. And, a comparison with the peperomin compounds that have been found provides three new peperomins.

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

Funding
This work was supported by Excellent Youth Team for Scientific Research, Innovation and Entrepreneurship of Dalian University(XQN202004).