Two new bibenzyls from Pleione grandiflora (Rolfe) Rolfe and their antioxidant activity

Abstract Two new bibenzyls (1 and 2) were isolated from the pseudobulbs of Pleione grandiflora (Rolfe) Rolfe along with six known compounds, including isoarundinin I (3), isoarundinin II (4), bulbocodin D (5), batatasin III (6), 5,3'-dihydroxy- 4-(p-hydroxybenzyl)-3-methoxybibenzyl (7) and shancigusin F (8). Their structures were established on the basis of spectroscopic methods. These compounds showed potent DPPH free radical scavenging effects with IC50 values ranging from 49.72 ± 0.35 μM to 65.41 ± 0.49 μM. Graphical Abstract

is cultivated as a medicinal plant and circulated in the traditional Chinese medicine market (Wu et al. 2020). Previous phytochemical studies led to the isolation of plentiful phenanthrenes, bibenzyls and a few glucosyloxybenzyl succinate derivatives from P. bulbocodioides, P. yunnanensis and P. grandiflora (Wu et al. 2019;Han et al. 2020).
Oxidative stress and its injuries have been reported to play a key role in disease and damage of animals or plants (Gutteridge and Halliwell 1994). DPPH radical scavenging activity is often used as an important index to evaluate the antioxidant activity of compounds (Abubakr et al. 2012). Some compounds from the genus of Pleione such as pleionosides K and pleionosides L were reported for antioxidant activity (Han et al. 2019). In our previous works, we found that the crude extract of P. grandiflora showed potent antioxidant activity. Motivated by a search for potentially bioactive secondary metabolites from this valuable natural source, the chemical constituents of P. grandiflora pseudobulbs were investigated resulting into the isolation of two new bibenzyls (1, 2) with six known compounds (3-8) ( Figure 1). Herein, the isolation, structural elucidation and antioxidant activities of 1-8 were described in this paper.

Result and discussion
Compound 1 was obtained as a light-yellow amorphous powder (CHCl 3   d 4 ) of 1 combined with DEPT spectra exhibited 22 carbons in the structure, including eighteen aromatic carbons, three methylene carbons and a methoxyl. Based on these data, the structure of compound 1 was implied to be a bibenzyl derivative.
All the above compounds were tested for their in vitro antioxidant activity by 1,1diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay, and vitamin C was used as a positive control. Compounds 1-6 showed potent DPPH free radical scavenging effects with IC 50 values range of 49.72 ± 0.35 lM to 65.41 ± 0.49 lM, the IC 50 value of vitamin C was 53.36 ± 0.68 lM (Table S1).
The hydroxyl groups in these compounds are helpful for DPPH radical scavenging, which is consistent with the features of some reported antioxidant compounds (Olszewska and Kwapisz 2011). Generally, the number of hydroxyl groups in the chemical structure is positively correlated with the antioxidant capacity. However, the antioxidant activity of the above compounds did not increase with the increase of hydroxyl groups. The structure-activity relationship of antioxidant activity of compounds needs further experiments.

General experimental procedures
UV spectra were acquired on a JASCO V650 spectrophotometer. IR spectra were measured with a Fourier Transform Infrared Spectroscopy. The Thermo Scientific LTQ Orbitrap XL was used to collect HRESIMS spectra. NMR spectra (1D and 2D) were acquired on a Bruker AV400 nuclear magnetic resonance instrument from German Bruker in deuterated (Methanol-d 4 ), using the residual nondeuterated solvent peak as an internal standard (Methanol-d 4 with d H 4.87 and d C 49.00). Waters W2695-QDA High Performance Liquid Chromatograph-Mass Spectrometer (Waters Corporation, America) with a YMC-Pack ODS-A reversed phase chromatography column (4.6 Â 150 mm, 5 mm, YMC, Japan). Preparative HPLC was conducted on a Shimadzu LC-20AP instrument with an SPD-20A detector, using a YMC-Pack ODS-A column (20 Â 250 mm, 5 mm, YMC, Japan). Semipreparative HPLC was conducted on a Shimadzu LC-16P instrument with an SPD-M20A detector, using a YMC-Pack ODS-A column (10 Â 250 mm, 5 mm, YMC, Japan). Absorbance was detected by microplate Reader (Thermo Scientific, America). Column chromatography was run with Macroporous adsorption resin (Hecheng new material technology Co. Ltd, Zhengzhou, P. R. China), SiO 2 (Fuchen chemical reagents Co. Ltd, Tianjin, P. R. China).

Plant material
The pseudobulbs of P. grandiflora were collected in Nujiang Lisu Autonomous Prefecture, Yunnan Province of P. R. China (Longitude: 98

DPPH radical scavenging assay
Compounds 1-8 were carried on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay (Kim et al. 2014;Talla et al. 2017). 8 mg DPPH was prepared as a solution and stored away from light at 4 C. Compounds 1-8 and vitamin C were dissolved in ethanol to prepare 0.2, 0.3, 0.4, 0.5 and 0.6 gÁL À1 solution. 100 lL samples and 100 lL DPPH (0.2 mmolÁL À1 ) were added to the 96-well plate in triplicate. After the reaction of 30 min in the dark, the absorbance of each well was measured by the microplate reader at 517 nm, and the DPPH radical scavenging rate of the sample was calculated. Vitamin C was used as the positive control, and the test was repeated for three times.

Conclusions
In this study, two new bibenzyls (1-2) and six known (3-8) compounds were isolated from the pseudobulbs of P. grandiflora. Antioxidant activity investigation revealed that compounds 1-8 showed potent antioxidant activity. Their DPPH radical scavenging activity is equivalent to that of vitamin C.