Two new meroterpenoids from the bark of Cinnamomum cassia and their antioxidant activity

Abstract Two new (1 and 2) meroterpenoids were isolated from the bark of Cinnamomum cassia. Their structures were determined by spectroscopic analyses and chemical methods. Antioxidant activities of 1 and 2 were evaluated by the ORAC and DPPH radical scavenging assays, and the results revealed that compound 2 displayed oxygen radical absorbance capacity. The discovery of compounds 1 and 2 added new members of this kind of natural product.


Introduction
Cinnamomum cassia, an evergreen tree in the Lauraceae family, is primarily distributed in tropical and subtropical regions of Asia, including China, Vietnam, Indonesia, and Sri Lanka.In China, Guangxi province is the main growing area of C. cassia [1,2].The bark of C. cassia, named "Rou-Gui" in Chinese, has been utilized as a famous traditional Chinese medicine (TCM) to eliminate cold and relieve pain [1].Recent pharmacological studies showed that it exhibited a series of biological activities, such as anti-inflammatory, anti-bacterial, anti-oxidant, anti-tumour, and neuroprotective properties [3][4][5][6][7][8].Previous studies demonstrated that essential oils, terpenoids, polyphenols, and polysaccharides built a compound library of the bark of C. cassia [9][10][11][12][13].
The present phytochemical study on the bark of C. cassia led to the discovery of two new meroterpenoids (1 and 2) (Figure 1).Considering the biological activities of its bark, antioxidant activities of 1 and 2 were evaluated using the ORAC and DPPH radical scavenging assays.Details of the isolation, structural elucidation, and antioxidant activity of 1 and 2 are reported in this paper.

Results and discussion
Compound 1 was obtained as a brown oil.The quasi-molecular ion at m/z 383.1822 [M þ Na] þ by HRESIMS indicated that the molecular formula of 1 was C 21 H 28 O 5 (8 degrees of unsaturation).The 13 C NMR spectrum of 1 showed 21 carbons.Based on the DEPT-135 data, these carbons could be categorized into two carbonyls, six sp 2 quaternary carbons, four sp 2 methine carbons, four sp 3 methylene carbons, and five methyl carbons.The analysis of the 1 HÀ 1 H COSY experiment and the coupling values of the protons exhibited the presence of 2 subunits (H 2 -1 0 ÀH-2 0 and H 2 -4 0 ÀH 2 -5 0 ÀH-6 0 ) (Figure 2A).Combined with these deduced subunits, molecular formula, and degrees of unsaturation, the key HMBC correlations (Figure 2A) from H-4 to C-2/C-5/C-6/C-1 0 , from H-6 to C-2/C-4/C-5/C-7, from H 2 -7 to C-1/C-2/C-6/C-8, from H 2 -1 0 to C-2/C-3/C-4/C-2 0 /C-3 0 , from H 3 -8 0 to C-6 0 /C-7 0 /C-9 0 , from H 3 -9 0 to C-6 0 /C-7 0 /C-8 0 , from H 3 -10 0 to C-2 0 /C-3 0 /C-4 0 , from OCOCH 3 -2 to OCOCH 3 -2, and from OCH 3 -8 to C-8 and the key ROESY correlations (Figure 2A) between H 2 -7 and H-6/OCOCH 3 -2 deduced the planar structure of compound 1.The assignments of all proton and carbon resonances are provided in Table 1.The key ROESY correlations (Figure 2A) between H-2 0 and H 2 -4 0 and between H 2 -1 0 and H 3 -10 0 indicated that the geometrical configuration of the double bond D 2 0 ,3 0 was assigned as 2 0 E. Since compound 1 is the acetylation product of denudaquinol at C-2, the trivial name of 1 is 2-O-acetyl-denudaquinol.Compound 2 was obtained as a brown oil.The quasi-molecular ion at m/z 523.2144 [M þ Na] þ by HRESIMS indicated that the molecular formula of 2 was C 24 H 36 O 11 (7 degrees of unsaturation).The 13 C NMR spectrum of 2 showed 24 carbons.Based on the DEPT-135 data, these carbons could be categorized into one carbonyl, five sp 2 quaternary carbons, three sp 2 methine carbons, one sp 3 quaternary carbon, six sp 3 methine carbons, five sp 3 methylene carbons, and three methyl carbons.The 1 H and 13 C NMR data of 2 were similar to those of cinnacasside E [14], the difference being the loss of a methyl.Detailed analyses of 1D and 2D NMR data of 2 (Figure 2B; Table S2, Supporting Information) built up its planar structure.The key ROESY correlation (Figure 2B) between H 2 -1 0 and H 3 -10 0 indicated that the geometrical configuration of the double bond D 2 0 ,3 0 was assigned as 2 0 E. The aglycone (2a) of 2 was obtained by acid hydrolysis [15] of compound 2, and the specific rotation of 2a was [a] 25 D À 33.8 (c 0.10, MeOH).The similar specific rotations of the aglycones of 2 and cinnacasside E reported in the reference [14] indicated that the absolute configuration of C-6 0 in 2 was 6 0 S. The planar structure of 2 showed one pyranohexose unit.A precise comparison of 13 C NMR data of the sugar unit with those of glycosides recorded in the literature [16] and the coupling values (J H-1ʺ-H-2ʺ ¼ 7.4 Hz, J H-2ʺ-H-3ʺ ¼ 8.4 Hz, J H-3ʺ-H-4ʺ ¼ 8.8 Hz, J H-4ʺ-H-5ʺ ¼ 8.6 Hz) indicated that the pyranohexose unit was b-Glc.The absolute configuration of the glucopyranosyl was determined by HPLC analysis of products obtained from acid hydrolysis and derivatization reactions by L-cysteine methyl ester hydrochloride and o-tolyl isothiocyanate [15].Analytical HPLC was performed on the Cosmosil Packed C 18 column with an isocratic elution of CH 3 CN À H 2 O À HCOOH (20:80:0.1,v/v/v) for 40 min at a flow rate of 0.8 mL/min, and the peaks of the standard monosaccharide and sample derivatives were recorded at t R 12.2 (L-Glc), 17.2 (D-Glc), and 17.3 (2) min, respectively.This evidence revealed that the glucopyranosyl was D-Glc.Since cinnacasside E is the methyl ester of 2, the trivial name of 2 is 8-demethyl-cinnacasside E.
The antioxidant activities of compounds 1 and 2 were assessed using the ORAC assay with EGCG as the positive control.Figure 3 showed that compound 2 exhibited oxygen radical absorbance capacity.Besides, the antioxidant activities of compounds 1 and 2 were also evaluated using the DPPH radical scavenging assay with vitamin C as the positive control.The scavenging rates of all these compounds (100 lM) were below 20%, and no compounds showed DPPH radical scavenging activity (Table S4, Supporting Information).

General experimental procedures
The specific details about instruments used see "General experimental procedures" section in Supporting Information.
The oxygen radical absorbance capacity of compounds 1 and 2 (12.5 mM)

Plant materials
The bark of Cinnamomum cassia was purchased from Guangzhou Qingping Traditional Chinese Medicine Market, Guangzhou, China in April, 2021.The plant materials were identified by one of the authors (Hong-Xia Fan).A voucher specimen (CC-202104) was deposited in the School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, China.

Extraction and isolation
The dried bark of Cinnamomum cassia (20.0 kg) was thumped into small lumps.Then, the lumps were refluxed three times with 200 L of 60%

Acid hydrolysis
Acid hydrolysis was performed according to the previously described method with minor modifications [15].The compound (1.0 mg) was hydrolyzed with 2 M of HCl for 1 h at 90 C.After extracted with EtOAc two times, the H 2 O layer was evaporated in vacuo to furnish a monosaccharide residue.The residue was dissolved in pyridine (1.0 ml) containing L-cysteine methyl ester hydrochloride (2.5 mg) and heated at 60 C.After 1 h, 5 ll of o-tolyl isothiocyanate was added to the reaction mixture and further reacted at 60 C for 1 h.Then, the reaction mixture was directly analyzed by the Shimadzu HPLC system and detected by an UV detector (at 254 nm).Analytical HPLC was performed with an isocratic elution of CH 3 CN À H 2 O À HCOOH (20:80:0.1,v/v/v, 0.8 ml/min) for 40 min.The standard monosaccharides of D-Glc and L-Glc were subjected to the same method.

Oxygen radical absorbance capacity (ORAC) assay
The ORAC assay was performed in accordance with the previously described method [17].The automated ORAC assay was performed on a Synergy H1 microplate reader (Bio-Tek Instruments Inc., Vermont, USA) with an excitation/emission filter pair of 485/527 nm.Sodium fluorescein was used as a fluorescence probe, and the reaction was started with the addition of AAPH.EGCG was used as the positive control.The results were calculated according to the difference in the area under the fluorescence decay curve between the AAPH control and each sample.The ORAC values of tested samples were calculated as the relative values of the area under the fluorescence decay curve using Trolox as a standard, and expressed as micromoles of Trolox equivalents (TE) per micromoles of sample (lmol TE/lmol).All of the samples were performed in quadruplicate.

DPPH radical scavenging assay
The DPPH radical scavenging assay was performed on the basis of the method of Xie et al. with slight modifications [18].Briefly, 100 ll of 0.2 mM DPPH radical solution in ethanol was put into 100 ll of sample solutions in ethanol.The mixture was wobbled for 30 min in the dark.Vitamin C was used as the positive control.The absorbance values were measured at 517 nm using a Thermo Varioskan LUX microplate reader (Thermo Fisher Scientific Inc., Waltham, USA).All of the samples were performed in triplicate.The DPPH radical scavenging rate (S %) was calculated as follows: S % ¼ [(A 0 -A 1 )/A 0 ] Â 100 (A 1 and A 0 are the absorbance of the incubation DPPH radical solution with and without the tested sample, respectively).

Statistical analysis
The data were plotted by GraphPad Prism 6.0 software, and expressed as means ± SD.

FluorescenceFigure 3 .
Figure 3. Antioxidant capacity of compounds 1 and 2 evaluated by the ORAC assay.

Table 1 .
1H (400 MHz) and13C (100 MHz) NMR spectral data of 1 and 2 in DMSO-d 6 (d in ppm, J in Hz).Assignment may be interchanged.a The indiscernible signals due to overlap or having a complex multiplicity are reported without designating the multiplicity.