Sacraoxides A–G, Bioactive Cembranoids from Gum Resin of Boswellia sacra

Seven undescribed cembranoids, sacraoxides A–G (1, 3–8) were isolated from the gum resin of Boswellia sacra. Their structures were elucidated by extensive physicochemical and spectroscopic analysis, as well as ECD calculation, modified Mosher’s method and X-ray diffraction crystallography. Compounds 6 and 7 exhibited inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in RAW264.7 cells with IC50 values of 24.9 ± 1.7 and 36.4 ± 2.9 μM.


INTRODUCTION
Cembranoids are a class of diterpenes biosynthesized from the cyclization of geranylgeranyl diphosphate to generate a 14-membered ring backbone decorated with a variety of oxidation patterns (Li and Pattenden, 2011). Natural occurrence of cembranoids has been found from both terrestrial and marine organisms, which showed not only a large structural diversity but also a wide range of biological activities (Yang et al., 2012). Plant-derived cembranoids have a relatively limited distribution, and have been reported mainly from tobacco (Yan et al., 2016), as well as the genera of Croton, Euphorbia, Macaranga (Euphorbiaceae), Pinus (Pinaceae), Echinodorus (Alismataceae), and Boswellia (Burseraceae), etc (Wahlberg et al., 1992;Yang et al., 2012).
Olibanum is an aromatic oleogum resin that exudes from incisions in the bark of Boswellia trees and has been used as incense and perfumes since antiquity. Olibanum has also been used in traditional medicines for the purpose of relieving pain and removing blood stasis. Cembranoids and triterpenes were reported as the bioactive constituents responsible for these effects (Banno et al., 2006;Takada et al., 2006;Al-Harrasi et al., 2019). Boswellia sacra, known as Olibanum-tree or Frankincense, is a small deciduous tree that is native to the Arabian Peninsula and northeastern Africa, and is one of the plants known to produce olibanum. Previous chemical investigations on the gum resin from B. sacra have reported the isolation of a number of cembranoids with neuroprotective, hepatoprotective, anti-inflammatory and anti-depression activities (Moussaieff et al., 2012;Pollastro et al., 2016;Wang et al., 2020).
As part of a continuing research for the discovery of bioactive natural products from medicinal plants (Xia et al., 2017;Zhang et al., 2020), a chemical investigation was carried out on the gum resin of B. sacra. Herein, we report the isolation and structural elucidation of nine cembranoids (1-9) as well as their inhibitory activities against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse monocyte-macrophages.

Plant Material
The gum resin of Boswellia sacra Flueck. syn. Boswellia bhawdajiana Birdw. originated in Ethiopia, was furnished by Tianjin Tongrentang Group Co., Ltd. The resin was authenticated by Professor Lin Ma (Tianjin University of Traditional Chinese Medicine). The voucher specimen (accession number: 11037Q) was deposited in the School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, P. R. China.

Extraction and Isolation
The gum resin of B. sacra (6.8 kg) was powdered and extracted with 95% EtOH, then the extracts was evaporated to yield a residue (2.5 kg). The residue was separated by silica gel CC, and eluted sequentially with PE (petroleum ether), CH 2 Cl 2 and MeOH. The CH 2 Cl 2 fraction (513 g) was subjected to silica gel CC and eluted with a gradient of PE-EtOAc-MeOH to afford ten fractions (A-I).

ECD Calculations
The details of the quantum chemical ECD calculations for compounds 4, 6-8 are provided in Supplementary data.

NO Inhibitory Assay
The RAW 264.7 (ATCCTIB-71) mouse monocyte-macrophages were cultured in RPMI 1640 medium supplemented with penicillin G (100 units/mL), streptomycin (100 mg/ml) and 10% FBS. The cells were seeded in 96-well plastic plates with 1 × 10 5 cells/well and allowed to adhere for 24 h at 37°C in a humidified atmosphere containing 5% CO 2 . Then the medium was replaced with fresh medium, containing LPS (1 μg/ml) together with test compounds at various concentrations and then incubated for 24 h. NO production was determined by measuring the accumulation of nitrite in the culture supernatant using Griess reagent. Briefly, 100 μL of the supernatant from incubates were mixed with equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylene-diamide dihydrochloride in 2.5% H 3 PO 4 ) and were allowed to stand for 10 min at 37°C in a humidified atmosphere containing 5% CO 2 . Absorbance at 540 nm was measured using microplate reader. The nitrite concentrations were calculated according to the literature (Jin et al., 2016).

Cell Viability
Cell viability was determined using the mitochondrial respiration-dependent MTT reduction method. After transferring the required supernatant to another plate for the Griess assay, the remaining supernatant was aspirated from the 96-well plates, 100 μL of fresh medium and 10 μL of MTT (5 mg/ ml PBS) were added to each well. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . After incubating for 4 h, the medium was removed and the violet crystals of formazan in viable cells were dissolved in DMSO. Absorbance at 570 nm was measured using a microplate reader.
Nitric oxide (NO) plays significant roles in immune and inflammatory responses. The inhibition of NO release may be considered therapeutic in the treatment of inflammatory diseases (Strowig et al., 2012). Taking account of the traditional usage of the olibanum, cembranoids (1-9) were evaluated for their inhibitory activities against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse monocyte-macrophages. Compounds 6 and 7 showed the most potent inhibitory activities with the IC 50 values of 36.4 and 24.9 μM respectively, while the others were less active or inactive ( Table 4). In addition, the MTT assay indicated that none of these compounds showed cytotoxicity in RAW264.7 cells at a FIGURE 8 | Experimental and calculated ECD spectra of 7 and its enantiomer (red and blue calculated at the B3LYP/6-311++G (2d,p)//B3LYP/6-31+G (d,p) level in CH 3 OH; black, experimental in CH 3 OH).
Frontiers in Chemistry | www.frontiersin.org March 2021 | Volume 9 | Article 649287 concentration of 50 μM. The presence of an α,β-unsaturated carbonyl functionality seems essential for the inhibitory activity.

CONCLUSION
A phytochemical investigation on the gum resin of B. sacra resulted in the isolation and structural elucidation of seven undescribed and two known cembranoids. These cembranoids possess a common tertrahydrofuran ring structure through an ether bond between C-1 and C-12. The structures including absolute configurations were determined by extensive physicochemical and spectroscopic analysis, as well as ECD calculation, modified Mosher's method and X-ray diffraction crystallography. Compounds 4, 6, and 7 displayed inhibitory activities against LPS-induced NO production in RAW 264.7 cells with IC 50 values ranging from 24.9 to 72.1 μM. These findings will be of particular value for further studies of structurally interesting cembranoids with biological activities from the genus of Boswellia.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
BZ, DL, and WJ were responsible for the isolation of compounds. BZ and KO elucidated the structures. FZ and KH tested NO inhibitory effects of the compounds. BZ and WL interpreted the data, and wrote the paper. WL, KK, and FQ revised the manuscript. FQ was the project leaders organizing and guiding the experiment. All authors read and approved the final manuscript.