Two new ester alkaloids from Portulaca oleracea L. and their bioactivities

Abstract Two new ester alkaloids were isolated from Portulaca oleracea L., identified as (5-aminofuran-2-yl) methyl acetate (1) named oleracone N and 4(S)-ethyl 3-acetamido-3-(dihydroxyamino) propanoate (2) named oleracone O. The structures were elucidated via spectroscopic methods, including 1 D and 2 D NMR, UHPLC-ESI-QTOF/MS and CD spectrometry technique. It was suggested that both oleracone N and oleracone O could significantly inhibit inflammatory factors IL-1β and TNF-α in RAW 264.7 cells induced by LPS. Graphical Abstract


Introduction
Portulaca oleracea L. a well-known traditional Chinese medicine, is an annual succulent herb belonging to the member of the Portulacaceae family (Zheng et al. 2018).The 2020 edition of the Chinese Pharmacopoeia records that P. oleracea has the effect of clearing heat and detoxifying the blood, cooling the blood and stopping dysentery (The Pharmacopoeia Commission of PRC 2020).For thousands of years, P. oleracea L. has played a therapeutic role in traditional Chinese medicine (TCM).while it was popular worldwide due to its abundant nutrition with contents of proteins, carbohydrates, Ca, K, Zn, and Na, and several biological properties (Uddin et al. 2014).Modern pharmacological studies have shown that P. oleracea has anti-inflammatory (Meng et al. 2016;Rahimi et al. 2019), antibacterial (Nayaka et al. 2014), antioxidant (Erkan 2012;Liu et al. 2021;Duan et al. 2022), anticholinesterase (Ma et al. 2021;Xu et al. 2021), and antitumor (Zhao et al. 2013) effects.Trace back to previous chemical research, many active ingredients, such as flavonoids (Yang et al. 2018a, 2018b, Duan et al. 2021), lignans (Ma et al. 2020), furans (Tian et al. 2022), alkaloids (Li et al. 2017;Jiang et al. 2018), esters (Gu et al. 2021) and organic acids (Liu et al. 2022), have been found in P. oleracea.
In the past many years, our laboratory has been devoted to the research of medicinal substances in P. oleracea, and has isolated many bioactive components, many of them have obvious anti-inflammatory activities (Li et al. 2016;Cui et al. 2021;Guo et al. 2022).In this study, we investigated two new ester alkaloids isolated from P. oleracea respectively named oleracone N and oleracone O. Importantly, the experimental results demonstrated that both compounds 1 and 2 can significantly inhibit inflammatory factors including interleukin 1b (IL-1b) and tumor necrosis factor-alpha (TNF-a) in a RAW 264.7 macrophage cell model stimulated with lipopolysaccharide (LPS).Recent studies have shown that IL-1b and TNF-a play significant roles in the treatment of both Alzheimer's disease (AD) (Bona et al. 2008) and ulcerative colitis (UC) (Olsen et al. 2009;Wu et al. 2015), thus suggesting that compounds 1 and 2 have potential for the treatments of AD and UC.

Structure clarification
The structure of compounds 1 and 2 were confirmed by comprehensive 1 D and 2 D NMR analyses.According to the analysis data, there were no previous reports about them.
Compound 1 was obtained as yellowish-brown powder, which turned orange with Dragendorff's reagent.The 1 H NMR (600 MHz, CD 3 OD-d 4 ) and 13 C NMR (150 MHz, CD 3 OD-d 4 ) data have been listed.(Table S1, in supplementary material) combining 1 H-NMR, 13 C-NMR, and DEPT spectroscopic data, it is speculated that the molecular formula of the compound is C 7 H 9 NO 3 , and the degree of unsaturation is 4. UHPLC-QTOF/ MS provided molecular ion peak at m/z:154.0511 [M-H] -(calcd 154.0509) (Figure S3, in supplementary material).
The 13 C-NMR, DEPT and HMBC spectra (Figures S5, S6, and S9, in supplementary material) displayed seven carbon signals, specifically one methyl (d C 20.7), one methylene (d C 59.0), two olefinic methines (d C 113.4, 119.8), and three quaternary carbons, including one carbonyl carbon (d C 172.2) and two olefinic carbons (d C 146.9, 155.3).The 1 H-NMR spectrum (Figure S4, in supplementary material) showed a methyl signal at d H 2.07 (3H, s) and a methylene signal at d H 5.10 (2H, s), moreover, it contained two furan proton signals at d H 7.16 (1H, d, J ¼ 3.6 Hz, H-3) and d H 6.59 (1H, d, J ¼ 3.6 Hz, H-4).The correlation of 1 H-1 H COSY (Figure S7, in supplementary material) from H-3 to H-4 indicated that C-3 (d C 119.8) connected to C-4 (d C 113.4).In the 13 C-NMR spectrum (Figure S5), C-2 (d C 146.9) and C-5 (d C 155.3), clearly displayed the low field chemical shift, furthermore, HMBC correlations (Figure S9) from H-3 (d H 7.16) to C-2 and C-5  and from H-4 (d H 6.59) to C-2 and C-5 Compound 2 was obtained as yellowish-brown powder, which turned orange with Dragendorff's reagent.The 1 H NMR (600 MHz, CD 3 OD-d 4 ) and 13 C NMR (150 MHz, CD 3 OD-d 4 ) data have been listed (Table S2, in supplementary material).Its molecular formula was identified as C 7 H 14 N 2 O 5 based on a molecular ion peak at m/z 207.0986 3).According to the correlations from H-4, H-5, H-8 to the C-6 (d C 171.9) in HMBC spectra (Figure S17) indicated that C-5, C-8 connected to the ester-based carbon (d C 171.9).Finally, due to the low-field chemical shift of C-4 and the molecular formula, it can be determined that the dihydroxyamino group is attached to C-4.The absolute configuration of the C-4 stereogenic center of the compound was established by the circular dichroism (CD) spectrum (Figure S19) and compared with the similar compounds reported previously (Huang et al. 2013).This indicated that the absolute configuration of the compound 2 is 4S.Based on the discussion above, compound 2 was identified as ethyl 3-acetamido-3-(dihydroxyamino) propanoate and was given the trivial name oleracone N.

Anti-inflammatory activity
AD is characterized by progressive loss of memory and other cognitive functions.Typically, a decade or so passes before the illness has taken its course and patients die in a completely helpless state (Coelho and Moreno 2019).The major neuropathological hallmarks of the disease are extracellular senile plaques mainly composed of amyloid-b (Ab) peptides, which are often surrounded by reactive glia and dystrophic neurites.A growing number of studies further implicated neuroinflammation in the pathogenesis of AD (Akiyama et al. 2000;Zotova et al. 2010;Mrak 2012).Since neuroinflammation seems to play a role in neurodegeneration, several studies have suggested that cytokines might enhance this inflammatory process contributing to synaptic dysfunction and subsequent neuronal death.It has been reported that inflammatory factors, such as IL-1b, TNF-a can be detected in reactive astrocytes surrounding Ab deposits (Griffin et al. 1995;Mehlhorn et al. 2000) and Ab was shown to induce the production of IL-1b and TNF-a (Gitter et al. 1995).
UC is an immunerelated inflammatory disease with unknown etiology.The incidence of UC is continuously increasing all over the world.Although the exact pathogenic mechanism of the disease remains unclear, many studies indicate that IL-1b is a major inflammatory factor leading to UC and increased after the NLRP3 inflammatory signaling pathway was activated, also the activation of caspase-1 by the NLRP3 inflammasome will cause pro-IL-1b to cleavage and release IL-1b (Zhong et al. 2018;Saber et al. 2021).Studies have shown that IL-1b plays a pivotal role during the pathogenesis of UC.On the other hand, Anti TNF-a agents have become a significant advance in the management of ulcerative colitis (Pugliese et al. 2017).
Consequently, the anti-inflammatory activities of the compounds 1 and 2 were tested via IL-1b and TNF-a production and ELISA assays after lipopolysaccharide-stimulated macrophages.Using dexamethasone (DEX) as a positive control.The results showed that compound 1 could inhibit the production of inflammatory factors IL-1b and TNF-a at a concentration of 10 lM, compound 2 could inhibit the production of two inflammatory factors at 10 lM and 20 lM (Figure S20, S21, in supplementary material).It follows that compounds 1 and 2 have therapeutic potential for AD and UC.

General experimental procedures, chemical and reagents
Dried P. oleracea was extracted and concentrated by using a multifunctional extraction and concentration machine (Shanghai Shunyi Tech. Co. Ltd., Shanghai).The 1 D and 2 D NMR experiments were performed by an AVANCE 600 MHz instrument with cryogenic probe (Bruker Corporation, Switzerland), while the compounds were dissolved in CD 3 OD-d 4 .UHPLC-ESI-QTOF/MS were acquired in the positive or negative ion mode on a 6520 quadrupole-time-of-flight mass spectrometer (Agilent, Palo Alto, CA).A Shimadzu Nexera X2 UHPLC LC-30A system (Shimadzu, Kyoto, Japan), which was equipped with a solvent delivery pump (LC-30 AD), a vacuum degasser (DGU-20A), a UV spectrophotometric detector (SPD-20A) and LabStation software (Shimadzu), was used for separation and analysis.The column of analysis was Kromasil C18 column (150 mm Â 4.6 mm, 5 lm, Dalian Jiangshen Separation Science and Technology Co., flow rate 1.0 mL/min).In the separation process, column chromatography including silica gel Qingdao Marine Chemical Co.,Qingdao,China), ODS (40-70 lm) and Sephadex LH-20 (GE Healthcare, Marlborough, MA).TLC was performed on TLC plates precoated with silica gel GF254 (Qingdao Marine Chemical Co., Qingdao, China).

Plant material
P. oleracea was collected from Shijiazhuang (Hebei, China) in September 2020, and were identified by Prof. Xixiang Ying.The voucher specimen (No. 20200901) was exhibited at the School of Pharmacy, Liaoning University of Traditional Chinese Medicine.

Cell culture, treatment and MTT assay
The macrophage cell line RAW 264.7 was maintained in DMEM supplemented with 10% heat-inactivated FBS and antibiotics (100 units/mL penicillin and 100 lg/mL streptomycin) and incubated at 37 C in a humidified incubator with 5% CO 2 .The cytotoxicity compounds 1 and 2 were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay.Then the RAW 264.7 cells were seeded in 96-well plates at a density of 1 Â 10 4 cells/well, subsequently pre-incubated with or without various concentrations (5, 10, 25, and 50) of compounds 1 and 2 for 1 h in the incubator, then stimulated with 1 lg/mL LPS for 24 h, followed by 1 lg/mL LPS for 24 h, then removed the media, and the cells were incubated with 5 mg/mL MTT solution for 4 h at 37 C. Formazan was dissolved in 150 lL of dimethyl sulfoxide (DMSO), and the absorbance was detected at 570 nm by BIO-TEK Microplate Reader; the untreated group was considered as 100% viable.The cytotoxic effects of compounds 1 and 2 on RAW 264.7 cells were non-observed at a concentration of 25 lM by MTT assays.Therefore, concentrations between 1 and 25 lM were considered non-cytotoxic and were used for subsequent experiments.

Determination of IL-1b and TNF-a production
LPS (1 lg/mL) induced RAW 264.7 cells pre-incubated with or without the indicated concentrations (1, 5, 10, and 20 lM) of compounds 1 or 2 were collected and applied to quantitate the production of IL-1b and TNF-a using ELISA kits dexamethasone (Dex, 10 lM) was used as the positive IL-1b and TNF-a release inhibitor.

Conclusion
In this study, two new ester alkaloids were isolated from P. oleracea for the first time.The bioactivities assay showed that compounds 1 and 2 exhibited significant antiinflammatory activity in LPS-induced RAW 264.7 macrophages, while compound 1 exhibited better anti-inflammatory activity, being active at 10 lM than compound 2 that showed anti-inflammatory activity at 10 lM and 20 lM respectively.

Disclosure statement
No potential conflict of interest was reported by the authors.
, typically two carbons attached to O, indicated that the compound owns a furan ring.Additionally, The HMBC correlations from H-6 (d H 5.10) to C-4 (d C 113.4) and C-5 (d C 155.3) showed that the methylene group was attached to the furan ring through C-5.Moreover, there was an obvious ester group signal at C-8 (d C 172.2), and in combination with the key HMBC correlations of H-6 and H-9 (d H 2.07) to C-8, indicated that C-6 (d C 59.0) and C-9 (d C 20.7) are connected to C-8.Finally, based on the low-field chemical shift at C-2, the evidence of the molecular formula, and the color reaction of the compound, an amino group should attach at C-2. Accordingly, compound 1 was identified as (5-aminofuran-2-yl) methyl acetate and was given the trivial name oleracone N (Figure 1).