Determination of a new chromone from Aurantii Fructus Immaturus by DFT/GIAO method

Investigation of EtOAc fraction from the 95% ethanol extract of Aurantii Fructus Immaturus led to the isolation of one new chromone (1) and seven known flavonoids (2–8). Their structures were elucidated mainly by NMR and HR-ESI-MS, as well as on comparison with the reported NMR data. The final substituent pattern of 1 was defined by comparison of 13C NMR data calculated by DFT/GIAO with those of experimental NMR data.


Introduction
Aurantii Fructus Immaturus (Zhi Shi), being used as a traditional Chinese medicine, was the dried young fruit of Citrus aurantium L. or Citrus sinensis Osbeck. It is widely distributed in the provinces of Sichuan, Jiangxi, Fujian, Jiangsu, etc. Some of the chemical constituents are antioxidant, antiviral, antisepsis, anti-inflammatory, etc, in nature. It has been reported that Aurantii Fructus Immaturus had antitumour (Satoh et al. 1996) and protective activities on gastric mucosal lesions (Takase et al. 1994). It has also been shown to reduce the damage to the livers and kidneys in mice caused by the oral medication of Magnolia officinalis rehd (Chen et al. 2013), which aroused our interests in studying its chemical constituents. Thus, a systematic research on its chemical components was implemented and as a result, one new chromone, along with seven known flavonoids, was isolated. Herein, we report the isolation and structural elucidation of the new compound 1.
Compound 1 was obtained as a light yellow crystal with a molecular formula of C 10 H 8 O 5 as determined by data of the HRESI-MS (m/z 209.0442 [M þ H] 2 ). The 1 H NMR spectrum of compound 1 showed six proton signals, including two phenolic hydroxyl proton signals at d H 10.85 (1H, s) and 12.39 (1H, s), two meta-coupled phenyl proton signals at d H 6.28 (1H, d, J ¼ 6.0 Hz) and 8.27 (1H, d, J ¼ 6.0 Hz), one methoxyl proton signal at d H 3.73 (3H, s) and one phenyl proton signal at 6.29 (1H, s). The 13 C NMR spectrum gave nine sp 2 carbon signals at d C 99. 6, 105.1, 110.8, 128.1, 150.4, 156.7, 157.6, 157.8 and 181.8, along with one methoxyl signal at d C 61.1. All the proton signals were assigned to their directly attached cartons by HSQC spectral data. As a result, NMR signals at d C 110.8(d H 6.29), 157.7(d H 8.27) and 181.8, being identical to those of 5,7-dihydroxychromone (Zhang et al. 2010), led to the elucidation of a a,bunsaturated pyrone ring. Compound 1 was also considered to be a chromone due to the resemblance of the NMR data of compound 1 and 5,7-dihydroxychromone.
There were two hydroxyl groups and one methoxyl group that were not assigned, according to the analysis described earlier. Being able to interact with the carbonyl group of C-4 to form a hydrogen bond, which enabled the proton chemical shift to be over twelve, the phenolic proton at d H 12.39 revealed the presence of a hydroxyl group at C-5. In the NOE spectrum, the correlation between -OH(C-5, d H 12.39) and H-6 (d H 6.29) was observed, leading to the determination of the location of the unassigned hydroxyl and methoxyl groups at C-7 and C-8. Thus, two possible structures of compound 1 can be derived as 1a and 1b as shown in Figure 2.
In order to reveal the final structure of compound 1, the DFT/GIAO method was adopted to calculate the 13 C NMR data of 1a and 1b, respectively. The calculated and experimental carbon data were compared by a liner fit method. The R-square value of 1a was 0.998 (Figure 3) was better than those of the 1b (0.993, Figure 3). Thus, the structure of 1 was established to be 5,7-dihydroxy-8-methoxychromone.

Plant material
The dried young fruits of Citrus aurantium L. were purchased in An-guo medicine market, Hebei province, China. The plant was identified by Prof. Guanghai Shen of Yanbian University College of Pharmacy and a voucher specimen was deposited in the central experiment laboratory of Yanbian University Hospital (No. 20140123zh).

13 C NMR data calculation
The structures of 1a and 1b were optimised by the DFT method with b3lyp/6-311g(d). The optimised structures were subjected to calculation of 13 C data by Gauss 09 program using the DFT/GIAO Method by b3lyp/6-311 þ g (2d,p).

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported partially by the National Natural Science Foundation of China [grant number 81460563].