Acronyols A and B, new anti-inflammatory prenylated phloroglucinols from the fruits of Acronychia crassipetala

Abstract Two new phloroglucinols, acronyols A (1) and B (2) along with the four known (3–6) pholoroglucinols were identified following anti-inflammatory activity guided fractionation from the fruits of Acronychia crassipetala (family Rutaceae). The pholoroglucinols (1–6) were evaluated for their inhibitory effects on NO production and downregulation of TNF-α in RAW 264.7 macrophage cell lines. Graphical Abstract


Introduction
The genus Acronychia (family Rutaceae) comprises approximately 50 species with a geographical distribution extending from South East Asia to Australia and islands of the western Pacific Ocean (Epifano et al. 2013). Members of the genus have been used as traditional medicines throughout this region and a wide range of biologicallyactive phytochemicals has been isolated from Acronychia spp. including acridone alkaloids, flavonoids, acetophenones, coumarins, cinnamic acids, lignans, steroids and triterpenes (Epifano et al. 2013) Phloroglucinols have been identified as common metabolites in different plant parts of the genus Acronychia. For example, structurally similar prenylated pholorogulcinols, acronyculatins A-E were isolated from the stem and root bark of Acronychia pedunculata (Su et al. 2003). While recently a series of acetophenone monomers with one of the oxidized prenyl rings cyclizing to form either a furan or pyran ring were isolated from the bark of Acronychia trifoliolata (Miyake et al. 2016). Mildly cytotoxic prenylated acetophenone derivatives were isolated from the leaves of A. oligophlebia (Yang et al. 2015;Niu et al. 2018). A more recent investigation into the leaves of A. pedunculata afforded anti-proliferative isoprenylated acetophenone-quinolone hybrids (Panyasawat et al. 2021).
Herein we report the isolation, structural elucidation and the anti-inflammatory activity of two new (1-2) and four known (3-6) pholoroglucinols from Acronychia crassipetala, an understory tree endemic to upland and lower montane rainforests in northeastern Queensland, Australia.
Compound 3 was identified as 6-demethylacronylin a minor constituent previously isolated from the bark of Acronychia laurifolia (Banerji et al. 1973). Compound 4, identified as 2 0 ,6 0 -dihydroxy-5 0 -prenyl-4 0 -prenyloxyacetophenone, was only reported as perhaps one of the constituents having anti-inflammatory and anti-allergy extracts from nettle reported in a patent (Alberte et al. 2010) and as a synthetic analogue (Masao et al. 1994). There were no records of this compound as a natural product isolated from a plant source to the best of our knowledge. Compound 5, a methylated derivative of acronylin was only ever reported as a synthetic intermediate (Jain and Zutshi 1972) and as an alkaline degradation product of a derivative of villosinol (Jayaraman et al. 1980). The presence of compounds 4 and 5 as new natural products from Acronychia crassipetala deserves a noteworthy attention, highlighting the chemical diversity that may be present in other Acronychia sp. Finally, the known compound 6 was identified as the recently reported acetophenone, crassipetalonol A from A. crassipetala (Tran et al. 2020).
The anti-inflammatory activity (Table S3) of the isolated compounds (1-6) was evaluated by observing the LPS and IFN-c induced NO (nitric oxide) inhibition and TNF-a downregulation in RAW 264.7 macrophage cells.
2 0 ,6 0 -dihydroxy-5 0 -prenyl-4 0 -prenyloxyacetophenone (4) exhibited the highest level of potency in terms of NO inhibition, while at the same time being moderately cytotoxic. Interestingly, crassipetalonol A (6) had similar level of potency as (4) but exhibited a much lower level of cytotoxicity, structurally they were very similar with the only difference being the change from the methyl ketone (4) to a methylene hydroxy ketone (6) (Figure 1, Table S3).
In comparison, the new pholoroglucinols, acronyol A (1) which lacked the second isoprene unit and an increased saturation of oxy-methyls on the phloroglucinol and isoprene backbone, displayed moderate anti-inflammatory activity with low levels of cytotoxicity. Acronyol B (2) on the other hand with an altered oxygenated isoprene unit displayed weak anti-inflammatory activity (Figure 1, Table S3). The discovery of these small subset pholoroglucinols from the fruits of A. crassipetala lead to an observed structure activity relationship which could be utilized for a broader series of similar prenylated pholoroglucinols.

General experimental procedures
UV spectrum was recorded on a Shimadzu spectrophotometer model UV-2550. Chiroptical measurements ([a] D ) were obtained on a Polax-D, ATAGO system polarimeter in a 100 Â 2 mm cell at 25 C. NMR spectra were recorded on a Bruker Ascend 400 and 600 MHz spectrometer (Bruker Biospin GmbH, Germany), in the solvents indicated and referenced to residual 1 H signals in deuterated solvents. HRMS was carried out using a Waters Xevo Q-TOF mass spectrometer operating in the positive ESI mode. HPLC was carried out using the Agilent 1290 series.

Pro-inflammatory activation of cells
For assays, 90 lL of each concentration of samples (8 concentrations made by serial dilution in DMEM) were added an hour prior to addition of 10 mL of activator. A combination of 10 lg ml À1 LPS and 10 U ml À1 (1 unit ¼ 0.1 ng/mL) IFN-c, were used for activation. After activation, the cells were incubated for 24 h at 37 C and then NO, TNF-a and cell viability was determined. Unactivated cells (exposed to media alone) were used as negative control and activated cells as positive control.
3.7. Determination of nitrite (as a measure of nitric oxide production) by the griess assay Nitric oxide was determined by Griess reagent quantification of nitrite. Griess reagent was freshly made up of equal volumes of 1% sulfanilamide and 0.1% napthyethylenediamine in 5% HCl. From each well, 50 ml of supernatant was transferred to a fresh 96well plate and mixed with 50 ml of Griess reagent and measured at 540 nm in a POLARstar Omega microplate reader (BMG Labtech, Mornington, Australia). The concentration of nitrite was calculated using a standard curve with sodium nitrate (0-500 lM), and linear regression analysis.

Determination of TNF-a by ELISA
The diluted supernatants were used for determination of TNF-a using a commercial sandwich ELISA (Peprotech) according to the manufacturer's protocol with small modifications. In brief, the capture antibody was used at a concentration of 1.5 lg ml À1 in PBS (1.9 mM NaH 2 PO 4 , 8.1 mM Na 2 HPO 4 ,154 mM NaCl) (pH 7.4). Serial dilutions of TNFa standard from 0 to 10,000 pg mL À1 in diluent (0.05% Tween-20, 0.1% BSA in PBS) were used as internal standard. TNF-a was detected with a biotinylated second antibody and an Avidin peroxidase conjugate with TMB as detection reagent. The colour development was monitored at 655 nm, taking readings every 5 min. After about 30 min the reaction was stopped using 0.5 M sulphuric acid and the absorbance was measured at 450 nm using a POLARstar Omega microplate reader (BMG Labtech, Mornington, Australia) and expressed as a percentage of that in control cells after conversion of the concentrations by using a standard curve constructed with defined concentrations of TNF-a. Curve fitting of this standard curve and extrapolation of experimental data were performed using non-linear regression analysis.
3.9. Determination of cell viability by the alamar blue assay 100 ml of Alamar Blue solution (10% Alamar Blue (Resazurin) in DMEM media) was added to each well, incubated at 37 C for 2 h. After incubation, fluorescence intensity was measured with the microplate reader (excitation at 530 nm and emission at 590 nm) and results were expressed as a percentage of the intensity of that in control cells, after background fluorescence was subtracted.

Statistical analysis
Data calculations were performed using MS-Excel 2010 software. IC 50 values were obtained by using the sigmoidal dose-response function in GraphPad Prism. The results were expressed as mean ± standard deviation (SD).

Conclusion
We isolated six phloroglucinols (1-6) from the fruits of A. crassipetala. Acronyol A (1) and B (2) are previously undescribed while this is the first report of the occurrence of phloroglucinols 4 and 5 in this species. All compounds displayed weak to moderate anti-inflammatory activity (IC 50 values as low as 14.7 and 37.8 lM) in terms of downregulation of NO and TNF-a production respectively.