Three new triterpenoid glycosides from Aronia melanocarpa (Michx.) Elliott

Abstract Three new triterpenoid glycosides, 2α,3α,23,24-tetrahydroxyurs-12,19- dien-oic acid 28-O-β- D -glucopyranoside (1), 2α,3β,23,24-tetrahydroxyurs-12, 19(29) -dien-28-oic acid 28-O-β- D -glucopyranoside (2), and 2α,3β,23,24-tetrahydroxyurs-12, 18-dien-28-oic acid 28-O-β- D -glucopyranoside (3) were isolated from Aronia melanocarpa (Michx.) Elliott. Their structures were elucidated by extensive spectroscopic methods. All the isolated compounds displayed moderate inhibitory activity against nitric oxide production in macrophages. Graphical Abstract


Introduction
Aronia melanocarpa originated in the east of North America and introduced to Liaoning Province of China (Wei et al. 2017) is a new small berry fruit tree.The species was used for food industry, for example, the production of juice, fruit wine, jam, fruit powder, food-grade colorants and so on (Guo et al. 2020).Its pharmacological studies displayed that Aronia melanocarpa contained anti-inflammatory (Zapolska-Downar et al. 2012;Ho et al. 2014;Badescu et al. 2015), antioxidant (Teleszko and Wojdyło 2015a;Appel et al. 2015b), anti-cancer (Skarpanska-Stejnborn et al. 2014;Gao et al. 2018), antihypertensive (Valcheva-Kuzmanova et al. 2007) activities.Besides, it also used as medicine for the treatment of dyslipidemia (Kardum et al. 2014), obesity (Lim et al. 2019).At present, there are many researches on the antioxidant activity but less on the anti-inflammatory activity of Aronia melanocarpa.
The isolated compounds 1-3 were evaluated for their antioxidant activity using DPPH method and anti-inflammatory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7.The experiment displayed that none of the compounds showed antioxidant activity with IC 50 > 100 lM, while compounds 1-3 showed moderate anti-inflammatory inhibitory activities with IC 50 values of 32.5, 41.6, 35.7 lM, respectively, compared with the positive control Aminoguanidine (IC 50 11.2).The experiment was repeated three times, the average value as the experimental results.

General experimental procedures
Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter.UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers, respectively.NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as d values with TMS as the internal standard).HRESIMS spectra were performed on a LTQ-Obitrap XL spectrometer.Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and an YMC Pack C 18 column (250 mm Â 10 mm, i.d., 5 lM, YMC Co. Ltd., Japan) eluted with MeOH-H 2 O at a flow rate of 2 mL/min.C 18 reversed-phase silica gel (40～ 63lM, Merk, Darmstadt, Germany), MCI gel (CHP 20P, 75～ 150lM, Mitsubishi Chemical Corporation, Tokyo, Japan) was used for column chromatography.The gas chromatograph system GC-2010 plus (Shimadzu, Japan) equipped with a FID and an AOC-20i automatic sample injector.The Rtx-1701 capillary column (length ¼ 30 m, inner diameter ¼ 0.25 mm with thickness of stationary phase ¼ 0.25 lM, Restek, USA) was used for separation.And pre-coated silica gel GF 254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic of China) were used for TLC.D-Glucose and L-Glucose are Standards (Sinopharm Chemical Reagent Co.,Ltd).All solvents used were of analytical grade (Beijing Chemical Works).

Plant material
Aronia melanocarpa (Michx.)Elliottwere were provided by Guozhen Health Technology (Beijing) Co., Ltd., Beijing, People's Republic of China, and authenticated by Prof. Rong-Tao Li.A voucher specimen (CS200516) has been deposited at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

Hydrolysis and derivatization
Weigh 1.5 mg of Compound 1, add 0.5 mL of 2.0 mol/L trifluoroacetic acid, seal the tube with N 2 , react at 110 C for 6 h, then blow to dry with N 2 at 70 C. Weigh 2.0 mg of D-Glucose, 2.0 mg L-Glucose and the above hydrolyzed sugar respectively, add 1.0 mg of hydroxylamine hydrochloride, 2.0 mL pyridine, shak for 30 min at 90 C water bath temperature, take out and cool to room temperature, add 0.5 mL of acetic anhydride, continue to react 30 min at 90 C for acetylation, the reaction products were analyzed by gas chromatography after filtering through the membrane.

Antioxidant activity
Compounds were tested for their antioxidant activities using the DPPH method, with the vitamin E as a positive control.The assay was conducted in a 96-well format using serial dilutions of 100 lL aliquots of tested compounds (ranging from 100 to 10.0 lM) and vitamin E, respectively.Then, the absorbance was measured at 515 nm after 30 min in the dark.Three replicate wells were set in parallel for every group.Methanol was used as a negative control.The DPPH scavenging capacity (SC) was calculated according to the following formula: SC% ¼ [(A0-A1/A0)] Â 100% (Ma et al. 2021).A0 is the absorbance value of the control group; A1 is the absorbance value of the sample group.

Bioassay for anti-inflammatory activity
The inhibitory ability against LPS-induced NO production was evaluated using RAW 264.7 macrophages (Kamada et al. 2018;Chen et al. 2022).The cells were seeded in 96-well microtiter plate(10 4 cells/well), Then, the cells were coincubated with drugs and LPS 2 lg/ml for 20 h.The amount of NO was assessed by measuring the concentration of nitrite in the culture medium using Griess reagent.The absorbance was recorded using a microplate reader at a wavelength of 570 nm.

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