Two novel amide alkaloids from Portulaca oleracea L. and their anti-inflammatory activities

Abstract In this article, two novel amide alkaloids were identified as (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(5-hydroxy-6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-1-yl)prop-2-en-1-one (1) and (E)-1-(5-hydroxy-6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-1-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (2), the two compounds were named oleraindole E and oleraindole F, respectively. The structures were elucidated using 1D and 2D NMR and HR-ESI-TOF-MS spectra. Additionally, the anti-inflammatory activities were evaluated on RAW264.7 cells induced by LPS, compounds 1 and 2 exhibited anti-inflammatory activities at 20 μM. Graphical Abstract


Introduction
Portulaca oleracea L. is an annual grassy plant belonging to the Portulacaceae family, which is the homology of medicine and food (Tian et al. 2015). It is listed by the World Health Organization (WHO) as one of the most widely used medicinal plants, and it has been given the term 'Global Panacea' (Xu et al. 2006). P. oleracea is distributed widely in the world and especially the tropical and subtropical areas (Zhou et al. 2015). It has a reputation for 'vegetable of long life' in China, and it has been used as traditional Chinese Medicine for thousands of years (Chen et al. 2009;Jin et al. 2013). P. oleracea is sour in taste and cold in nature, and it is used to clear heat, resolve toxins, cool the blood and stanch bleeding. The constituents of P. oleracea have been isolated, including alkaloids (Xu et al. 2020;Liu et al. 2019), flavonoids (Yan et al. 2012;Duan et al. 2020), organic acids (Uddin et al. 2014), terpenoids (Xin et al. 2008), and so forth. Additionally, modern pharmacology studies showed that P. oleracea has many biological activities, such as anti-inflammatory (Lee et al. 2012), antiulcerogenic (Karimi et al. 2004), antimicrobial (Zhu et al. 2006), antioxidant (Karimi et al. 2011;Duan et al. 2020), anticancer (Yan et al. 2012;Zheng et al. 2014), antidiabetic (Lan and Lu 2003;El-Sayed 2011), neuroprotective (Yang et al. 2012;Abdel Moneim 2013) and hepatoprotective (Elkhayat et al. 2008) activity. Particularly in terms of anti-inflammatory and antimicrobial activity, P. oleracea is known as 'natural antibiotic', which is used for the treatment of carbuncle, dysentery, diarrhoea and hematochezia. In a previous study, alkaloids of the plant exhibited marked anti-inflammatory activities, such as Oleracimine (Li et al. 2016) and Oleracone . Therefore, it is of significance to further study alkaloids and bioactivities of P. oleracea. In this study, two novel amide alkaloids, oleraindole E (1) and oleraindole F (2) (Figure 1) were isolated from P. oleracea and their anti-inflammatory activities were further evaluated on RAW264.7 cells stimulated by lipopolysaccharide (LPS).

Anti-inflammatory activity
P. oleracea has various kinds of biological activity, and it is well known for its antiinflammatory effect, so P. oleracea is used for the treatment of carbuncle, dysentery and ulcerative colitis. In addition, it is well known that IL-1b plays a key role in inflammatory diseases (Bauer et al. 2010). Thereby, the anti-inflammatory activity could be evaluated by testing the concentration of IL-1b. In this article, cytotoxicity test of compounds was measured by the method of CCK-8 at first, then the inflammation model of RAW264.7 cells was constructed using LPS (Zhong et al. 2018), and the activity was evaluated by testing the concentration of IL-1b. The results showed that the two compounds are non-cytotoxic when the concentration was 50 lM ( Figure S32, in supplementary material), compound 1 and compound 2 displayed anti-inflammatory activities at 20 lM ( Figure S33, supplementary material).

General instruments
The structures of the compounds were identified by 1 H and 13 C NMR spectroscopy (Bruker Avance 600 MHz spectrometer, Switzerland), with methanol-d 4 used as the solvent. The molecular weights of the compounds were measured by a 6520 quadrupole-time-of-flight mass spectrometer (Agilent, Palo Alto, CA). In the separation process, silica-gel (100-200 and 200-300 mesh, Qingdao Marine Chemical Co., Qingdao, China), polyamide resin (80-100 mesh, Taizhou Luqiao Sijia Biochemical Plastic Factory, Zhejiang, China) and ODS (20-40 lm, GE Healthcare, Marlborough, MA) were used for the separation of compounds. A Nexera X2 UHPLC LC-30A system (Shimadzu, Kyoto, Japan) with a Kromasil C18 column (150 mm Â 4.6 mm, 5 lm, Dalian Jiangshen Separation Science and Technology Corporation) was used for the purification of compounds.

Cell culture and cytotoxicity test
The RAW264.7 cells were cultured in DMEM, and the medium consists of 10% heatinactivated fetal bovine serum and antibiotics (100 U/mL penicillin and 100 lg/mL streptomycin), the 96-well microplates incubated in a humidified incubator at 37 C for 24 hours.
The WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium) assay was used for evaluating cell viability. The cells were added to the 96-well plates and cultured for 24 h, the initial density is 10 4 cells/mL. Then, compounds were dispensed into the wells of the microwell plates and incubated for 1 h, the concentrations of compounds were 0, 5, 10, 25, 50 lM. Next, 1 lg/mL LPS was added to the 96-well plates and incubated for 24 h. After that, the CCK-8 solution (10 lL) was added to the wells of the microwell plates, and the absorbances were detected using a microplate reader at 450 nm.

Anti-inflammatory activity
As described above, the inflammation model was constructed by LPS, and the RAW 264.7 cells were cultured in DMEM. First, the cells were added to the 48-well plates and cultured for 24 h, the initial density is 10 5 cells/mL. Then, compounds were dispensed into the wells of the microwell plates and incubated for 1 h, the concentrations of compounds were 0, 1, 5, 10, 20 lM. Next, 1 lg/mL LPS was added to the plates and incubated for 24 h. After that, the activity was evaluated by testing the concentrations of IL-1b in the supernatant according to the IL-1b ELISA kit.

Conclusions
Two novel amide alkaloids, oleraindole E (1) and oleraindole F (2), were isolated from P. oleracea. What's more, the results of anti-inflammatory experiments showed that both oleraindole E and oleraindole F could inhibit the production of IL-1b at 20 lM, and there was a dose-dependent effect on IL-1b inhibition. We could find that the activities of two compounds were similar, and the results may due to their same core structure. These results could provide some valuable information for further investigation on P. oleracea.

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