Two new monoterpene esters from Illigera paviflora Dunn roots

Abstract Two new monoterpene esters, illigerates H and I (1 and 2), and six known compounds actinodaphine (3), bulbocupnine (4), stephanine (5), hypserpanine B (6), betulinic acid (7) and gallic acid (8) were obtained from the root of Illigera paviflora Dunn. Their structures were elucidated by spectroscopic analysis. Anti-inflammatory and α-glucosidase inhibitory activity of some isolated compounds were assessed. Two monoterpenes 1 and 2 exhibited weak in vitro anti-inflammatory activity (IC50 64.5 ± 5.3 and 79.2 ± 7.5 μM) while compounds 3–6 showed inhibition of α-glucosidase with IC50 values ranged from 87.17 to 118.74 μM. Graphical Abstract


Introduction
The Illigera is one genus of the family Hernandiaceae (Flora of China Editorial Committee 1982).Some species were used to treat rheumatic arthralgia in traditional Chinese medicine.Alkaloids (Chen et al. 2011;Ge et al. 2018;Li et al. 2021), sesquiterpenoids (Dong et al. 2018) and monoterpenes (Dong et al. 2017;Zhou et al. 2019aZhou et al. , 2019b;;Pu et al. 2021) were reported as the main bioactive components corresponding to the pharmacological activity.
Illigera paviflora Dunn is one of the 14 species distributed in China.The root is traditionally used to treat rheumatoid arthritis in Chinese (Flora of China Editorial Committee 1982).Previous phytochemical investigations on this plant afforded six alkaloids (Zhang et al. 1991).In our search for bioactive metabolites, two monoterpene benzoic acid esters, illigerates H and I (1 and 2), four known aporphine alkaloids actinodaphine (3), bulbocupnine (4), stephanine (5), hypserpanine B ( 6), together with a pentacyclic triterpene betulinic acid (7) and gallic acid (8) were obtained from the roots of I. paviflora (Figure 1).We herein report the structure elucidation of two new metabolites as well as the anti-inflammatory and a-glucosidase inhibition activity of the isolates.
As I. paviflora was used to treat inflammation in traditional Chinese medicine, we evaluated the anti-inflammatory activity of compounds 1-6.Compounds 1 and 2 showed weak anti-inflammatory activity with IC 50 of 64.5 ± 5.3 and 79.2 ± 7.5 lM, respectively.Compounds 3-6 exhibited no activity at 100 lM.Guo reported that it was the -OH at C-8 rather than the -OCH 2 O-at C-1 and C-2, or the hybridization type of the nitrogen at C-6 was responsible for anti-inflammatory activity in aporphine alkaloids (Guo et al. 2018).
The ethyl acetate extract portion of the plant displayed potent a-glucosidase inhibition activity (IC 50 83.9± 11.5 mg/ml) in our terms of a-glucosidase inhibitory assay.The a-glucosidase is an enzyme with the function to hydrolysis carbohydrates into sugars.A a-glucosidase inhibitor will resulted to lower the glucose level after meal in patients with type 2 diabetes.(Ajebli et al. 2021;Proenc¸a et al. 2022) Compounds 1-6 were evaluated for a-glucosidase inhibition effects.Compounds 3-6 exhibited potent hypoglycemic activity with IC 50 values of 101.7 ± 10.9, 87.2 ± 10.2, 118.7 ± 12.4 and 117.8 ± 13.0 lM, respectively, while acarbose has an IC 50 value of (246.8 ± 10.1 lM).Compounds 1 and 2 exhibited no obvious a-glucosidase inhibition activity at the dose of 100 lg/ml.Alkaloids were distributed in a large variety plants and can be used to prevent and treat endocrine diseases including diabetes (Ajebli et al. 2021).

General experimental procedures
UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer.IR spectra were measured using a Bio-Rad Wininfmred spectrophotometer (KBr disc).Optical rotations were measured on a Horiba SEPA-300 polarimeter.NMR spectra were obtained on a Bruker AV-600 spectrometer with solvent peaks as reference.chemical shifts (d) are expressed in ppm.HRESIMS was acquired with a VG Autospec-3000 spectrometer.Column chromatography was done on silica gel (200-300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China).

Plant material
The roots of I. paviflora were collected from Nanning, Guangxi province in China, in October 2018 and verified by Prof. Qing-song Yang.A voucher specimen (YMU-J-20181011) has been deposited in the Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan Minzu University.

Anti-inflammatory assay
The method of anti-inflammatory activity assay was performed as described in the previous report (Pu et al. 2021) with L-NMMA (Biomedical, Inc.) as a positive control.Briefly, murine macrophage RAW264.7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 lg/mL penicillin and 100 lg/mL streptomycin at 37 C in a 5% CO 2 atmosphere.The samples were dissolved in DMSO and then diluted into different concentrations using DMEM [3.13,6.25,12.5,25,50and 100 lM, final DMSO concentration: 0.01% (v/v)].Cells were pretreated with the samples at the different concentrations and coincubated with LPS (2 lg/mL) for 24 h.Then, 70 lL of cell culture medium were mixed with 50 lL of Griess reagent I and 50 lL of Griess reagent II and incubated at room temperature for 5 min.
Then the absorbance at 540 nm was measured in a microplate reader.L-NMMA was used as a positive control and DMSO were used as a negative control.The percentage inhibition of NO was calculated by the following formula: 3.5.Alpha-glucosidase inhibitory assay The experiment was carried out as our previous report (Jiang et al. 2020) with Acarbose as a positive control.Firtsly, samples were dissolved in MeOH-phosphate buffer solution (PBS, pH 6.8) (1:1).A reaction mixture containing 50 lL PBS (100 mM), 10 lL reduced glutathione (1.0 mg/mL), and 10 lL of the sample (test concentration at 0.1 mg/mL, in triplicates), was added to each well of a 96-well plate, followed by addition of 20 lL a-glucosidase solution (0.5 U/mL).Then 20 lL of p-nitrophenyl a-D-glucopyranoside substrate was added to the mixture to start the reaction.The reaction mixture was incubated at 37 C for 15 min, and afterwards, 50 lL Na 2 CO 3 (0.1 M) solution was added to stop the reaction.The absorbance (A) was immediately recorded at 400 nm using a microplate spectrophotometer to estimate the enzymatic activity.The inhibition percentage was calculated as: The IC 50 value was defined as the concentration of a a-glucosidase inhibitor that inhibited 50% of a-glucosidase activity.