Acridone alkaloids and flavones from the leaves of Citrus reticulata

Abstract A new acridone alkaloid, reticarcidone A (1), decorated with an oxygenated isopentenyl group between C-1 and C-2, was isolated from the leaves of Citrus reticulata Blanco, together with nine known acridone alkaloids (2–10) and fifteen flavones compounds (11–25). The structure of those compounds were confirmed by analysis of comprehensive 1D and 2D NMR, and MS data. Reticarcidone A (1) was the first pyrano[2,3-a]acridone isolated from the genus Citrus. Some of these compounds showed moderated cytotoxicity against the five human tumor cell lines MCF-7, SMMC-7721, HL-60, A549 and SW480. Graphical Abstract


Introduction
The genus Citrus is an important economic crops and belongs to the Rutaceae family, of which are widespread throughout the tropical and subtropical regions (Rendeiro et al. 2016). Various species of the Citrus genus are used as food, beverages, folk medicine and perfumes, which have important medical and economic value (Mar ın et al. 2007;Han et al. 2010). Mounts of diverse secondary metabolites have been identified from this genus, including flavones, coumarins, limonoids, acridone alkaloids and essential oil (Wu et al. 1983a;Gonzalez et al. 1988;Akiyoshi et al. 1990;Panthong et al. 2013). The flavonoids were the largest number of components in Citrus genus, which exhibited extensive biological activities, such as anti-depression, anti-anxiety, anti-bacteria, and anti-inflammatory (Lichius et al. 1994;Benavente-Garcia and Castillo 2008;Cuong et al. 2015).
Citrus reticulata Blanco, is widely distributed in Sichuan, Zhejiang, Jiangxi, Hunan, Guangdong provinces of China. Arcidone alkaloids, a group of important bioactive compounds, are mainly founded in the roots of Cirtus genus (Dzierzbicka et al. 2001;Samuel et al. 2019). In this study, a new arcidone alkaloid (1), nine known acridone alkaloids (2-10) and fifteen flavones (11-25) compounds ( Figure 1) were isolated from the leaves of C. reticulata. Some compounds displayed moderate cytotoxic activities. Herein, the details of the separation, structure elucidation and cytotoxic activity assessment of these compounds are described.

Results and discussion
The molecular formula C 19 H 17 NO 4 of reticarcidone A (1) was assigned by the 13 C NMR and HRESIMS data (m/z 322.1091 [M -H] -, calcd for C 19 H 16 NO 4 , 322.1085), indicating 12 indices of hydrogen deficiency (IOHD). The 1 H NMR data indicated the presence of one hydroxyl proton at d H 10.52, three conterminal aromatic ring protons at d H 7.15 (dd, J ¼ 7.9 Hz), 7.23 (dd, J ¼ 7.9, 1.0 Hz), and 7.73 (dd, J ¼ 7.9, 1.0 Hz), one singlet aromatic ring protons at d H 5.70, and three singlet methyls at d H 3.96, 1.42, and 1.42. The 13 C and DEPT NMR data (Supplementary material Table S1) revealed the existence of 19 carbon resonances that were attributed as ten quaternary carbons, six methines, and three methyls. Analysis of these data manifested the characteristic resonances of one aromatic ring with three substituents, one aromatic ring with five substituents, one carbonyl, and a double bond with Z-configuration. Further study of the NMR data of 1 suggested that the structure of 1 was similar to those of the known compound 5-hydroxynoracronycin (2), an acridone alkaloid derivative decorated with an isopentenyl group. This conclusion was verified via the 1 H-1 H COSY correlations of H-6/H-7/ H-8 and H-1 0 /H-2 0 , as well as the HMBC correlations of OH-5 with C-5/C-5a/C-6; H-8 with C-5a/C-8a/C-9; H-4 with C-2/C-3/C-4a/C-9a, and N-methyl to C-4a/C-5a; Me-4 0 and Me-5 0 with C-2 0 /C-3 0 (Supplementary material Figure S1). The distinct difference between 1 and 2 was the appearance of the hydroxyl proton at d H 14.45 in 1, the replacement of a methine (d C 97.0) and N-methyl (d C 48.6) in 2 by a methine (d C 92.1) and N-methyl (d C 40.9) in 1, respectively. The HMBC correlations between H-1 0 /C-1, C-2, and C-3, and H-2 0 /C-2 indicated the presence of the linear pyrano[2,3-a]arcidone in 1, instead of the linear pyrano[2,3-c]arcidone in 2. Therefore, the structure of reticarcidone A (1) was determined as shown in Figure 1, which was known as the first pyrano[2,3-a]acridone isolated from the genus Citrus.
These isolated compounds were tested for their in vitro cytotoxic activities against five human tumor cell lines SMMC-7721, MCF-7, HL-60, A549, and SW480 by using MTT method (Cory et al. 1991

Plant material
The leaves of C. reticulata were collected from Pengshan, Meishan, Sichuan, People's Republic of China, in March 2018. The specific name was authenticated by Dr. En-De Liu, and the voucher specimen (201803-L02) was deposited at the Kunming Institute of Botany.

Cytotoxicity assay
The cytotoxic assay was evaluated by using the MTT method according to the previous procedure (Ye et al. 2018). The detailed process was described in the Supplementary Information.

Conclusions
The chemical study of the leaves of C. reticulata has resulted in the isolation of a new acridone alkaloid, reticarcidone A (1), which was decorated with the linear pyrano[2,3a]acridone, as well as nine known acridone alkaloids and fifteen known flavones compounds. Ten of the isolated twenty-five compounds showed cytotoxic activity against the SMMC-7721 cell lines.

Disclosure statement
There are no conflicts to declare.

Funding
This research was funded by the Technical Program of China Tobacco Sichuan Industrial Co., Ltd. [hx201907].