Two new anthraquinone derivatives from Saprosma crassipes H. S. Lo

Abstract Two new anthraquinone derivatives sapranquinones A and B (1 and 2) together with two known biogenetically related anthraquinone derivatives (3 and 4) were isolated from the stems of Saprosma crassipes H. S. Lo. The structures of these compounds were elucidated using comprehensive spectroscopic methods. Compounds 1-4 were evaluated for their antibacterial activities and compounds 1 and 3 had a broad spectrum antibacterial activity against Staphylococcus albus, Escherichia coli, Bacillus cereus, Micrococcus tetragenus, and Micrococcus luteus with MIC values ranging from 1.25 to 5 μg/mL. Graphical Abstract


Introduction
The genus Saprosma Blume (Rubiaceae) is represented by about 50 species of small trees or shrubs, which are mainly distributed in the tropical Asian region, and has been used as a traditional medicine for the treatment of fever (Ling et al. 2002;Singh et al. 2006;Lu et al. 2010).Anthraquinones, iridoids, alkaloids, phenolics, and triterpenoids were reported as the constituents of this genus (Ling et al. 2002;Wang et al. 2011Wang et al. , 2014;;Zhang et al. 2014;Zhou et al. 2016).S. crassipes is an endemic plant in Hainan, P. R. China, mainly distributed in Dongfang, Qiongzhong city of Hainan Province.S. crassipes is a shrub plant belonging to Rubiaceae family.The tree is 1 to 2 meters high, and the flower, white in color, grows at the top of the branch.A literature survey showed that there are no phytochemical and biological studies previously reported with S. crassipes.In this study, S. crassipes attracted our attention since its ethanol extract exhibited antibacterial activities.Bioassay-guided fractionation of the bioactive extract led to the isolation two new anthraquinone derivatives sapranquinones A and B (1 and 2) together with two known biogenetically related anthraquinone derivatives (3 and 4) (Figure 1).The structures of these compounds were elucidated using comprehensive spectroscopic methods.The inhibitory activities of all compounds against S. albus (ATCC 8799), E. coli (ATCC 25922), B. cereus (ATCC 14579), M. tetragenus (ATCC 13623), and M. luteus (ATCC 9341) were evaluated.
Compound 2 was also obtained as yellow amorphous powder, with the molecular formula C 17 H 14 O 4 from HRESIMS data (m/z 305.0781 for 2 [M þ Na] þ ; calc'd 305.0786) combined with 1 H and 13 C NMR spectroscopic data.The first preliminary investigation of its 1 H and 13 C NMR showed that 2 was closely related to 3-hydroxy-2-hydroxymethylanthraquinone (Chokchaisiri et al. 2021), except for the presence of two methoxy group signals (d H 3.50/59.1 and d C 3.89/55.9)in 2. The location of the two methoxy groups at C-2 and C-11 were confirmed by the HMBC correlations from 2-OMe to C-2, from 11-OMe to C-11.Detailed analysis of 2 D NMR (HSQC, 1 H-1 H COSY and HMBC) spectra confirmed that the other part of the molecule were the same as those of 3hydroxy-2-hydroxymethylanthraquinone.Thus, the structure of 2 was established as 2methoxy-3-(methoxymethyl)anthraquinone, and named sapranquinone B.
The inhibitory activities of all compounds against S. albus, E. coli, B. cereus, M. tetragenus, and M. luteus were evaluated.Compounds 1 and 3 exhibited a broad spectrum antibacterial activity against five terrestrial pathogenic bacteria (Table S2).The MIC values of 1 and 3 were further tested by microplate assay method.The results (Table S2) showed that 3 had potent antibacterial activity against E. coli and B. cereus with the MIC values of 1.25 lg mL À1 for each.Compound 1 showed significant activity against E. coli with the MIC values of 1.25 lg mL À1 .Compounds 1 and 3 had the similar antibacterial activity, but 1 (MIC values: 5.0 lg mL À1 ) showed weaker activity than 3 (MIC values: 5.0 lg mL À1 ) against S. albus.The MIC values of compounds 2 and 4 higher than 20 lg mL À1 were defined as inactive.These results suggest that -OH groups of anthraquinone can be important for antibacterial activity (Wang et al. 2010;Lee et al. 2016).

General
IR spectra were recorded on a Nicolet 6700 spectrophotometer. 1 D and 2 D NMR spectra were recorded on a Bruker AV spectrometer (400 MHz for 1 H and 100 MHz for 13 C) and a JEOL JEM-ECP NMR spectrometer (600 MHz for 1 H and 150 MHz for 13 C) with residual solvent peaks as references (d H 7.260 and d C 77.160 for CDCl 3 ).TMS was used as an internal standard.CDCl 3 was used as solvents.HRESIMS spectra were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer.The mass spectra were acquired using a TripleTOF TM 5600þ system with a Duo Spray source (AB SCIEX, Foster City, CA, USA) in negative and positive ESI mode.Semi-Preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector (detection wavelength: 210, 230, 254, and 280 nm) using an Agilent Eclipse XDB-C18 column (9.4 Â 250 mm, 5 mm) (flow phase: MeCN and H 2 O, velocity of flow: 2.5 mL/min).Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200-300 mesh), octadecylsilyl silica gel (YMC; 12 nm-50 lm) were used for column chromatography (CC).Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin layer chromatography (TLC).

Plant material
The stems of S. crassipes were collected from Bawangling National Nature Reserve, Changjiang County, Hainan Province (Northern latitude 18 582539 0 , East longitude 108 876352), China in June 2018, and identified by Prof. Xue-Ming Zhou, College of Chemistry and Chemical Engineering, Hainan Normal University.The plant was collected under the guidance of the local forestry bureau and we identified it as S. crassipes by comparing with the description of this plant in the literature.A voucher specimen (No.RMS20180622) was deposited at the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University.

Biological assays
Antibacterial activity was determined against five terrestrial pathogenic bacteria, including S. albus, E.coli, B. subtilis, M. tetragenus and M. luteus (These strains were purchased from Ningbo taisituo Biotechnology Co., Ltd, China) by microplate assay method (Pierce et al. 2008).The inocula of the bacterial strain were prepared from 24h-old cultures and suspensions were adjusted to 0.5 McFarland standard turbidity.The 96-well plates were prepared by dispensing into each well 100 mL of Muller-Hinton broth/Luria-Bertani.A volume of 100 mL aliquot from the stock solutions of the samples initially prepared was added into the first wells.Then, 100 mL from their twofold serial dilutions were transferred into consecutive wells.100 mL of the inocula were added to achieve a final inoculum concentration of 5 Â 10 5 CFU/mL.The final volume in each well is 200 mL.After incubation at 37 C for 24 h, growth was monitored by Microplate Reader at 625 nm.The broth medium containing pathogenic bacteria was used as the blank group and DMSO as the negative control, ciprofloxacin was used as the positive control.MIC (50% minimum inhibitory concentration) was calculated by Reed & Muench method (Reed and Muench 1938).

Conclusions
In this study, two new anthraquinone derivatives sapranquinones A and B (1 and 2) together with two known biogenetically related (3 and 4) were isolated from the stems of S. crassipes.Compounds 1 and 3 exhibited a broad spectrum antibacterial activity against S. albus, E. coli, B. cereus, M. tetragenus, and M. luteus with MIC values ranging from 1.25 to 5 lg/mL.Thus, we suggest that anthraquinone derivatives may play an important role in the antibacterial activity of S. crassipes and may deliver benefits in the treatment of bacteria-related infectious diseases.Further work is here warranted.