Two new amides from the seeds of Coix lacryma-jobi var. lacryma-jobi

Abstract Phytochemical investigation of the seeds of Coix lacryma-jobi var. lacryma-jobi resulted in the isolation of eight compounds, including two new structures, coixdines A-B (1-2) and six known compounds (3-8). The structures of these compounds were determined by 1 D and 2 D NMR spectra referring to the literatures, together with HR-MS analysis. Coixdine A and B are first examples of spermidine skeleton in genus Coix. In the present paper, all compounds were evaluated for the cytotoxicity against two cancer cell lines (CT-26 and BxPC-3) in vitro, and none of the compounds exhibited obvious cytotoxic activity. The present investigation suggests that these amides seem to be of great chemotaxonomic value for C. lacryma-jobi.


Introduction
Coix lacryma-jobi var. lacryma-jobi (Poaceae) is native to Southeast Asia and has been cultivated in East and Southeast Asia for thousands of years (Zhu 2017). The ripe seed of C. lacryma-jobi is an important food in some parts of Asia and is a staple food item in some Asian countries before the rise of maize (Roder 2006). Apart from being a food item, the seeds of C. lacryma-jobi have been used in traditional folk medicine for thousands of years (Devaraj et al. 2020). As a traditional Chinese medicine, the seed of C. lacryma-jobi recorded in 'ZhonghuaBencao' was used to treat edema, rheumatism, pulmonary abscess and so on (State Administration of Traditional Chinese Medicine Editorial Board 1999). A series of biological activities, such as anti-cancer, anti-inflammatory, anti-oxidant, anti-diabetic, anti-obesity and melanogenesis inhibitory activity have been reported for pure compounds and crude extracts from the seed of C. lacryma-jobi (Wang et al. 2013;Byun et al. 2015;Choi et al. 2015;Amen et al. 2017;Qu et al. 2017). In previous phytochemical investigations, the chemical constituents of C. lacryma-jobi seeds have been reported to be essential oil, polysaccharides, lignans, lactams and spiroenone (Kuo et al. 2002;Chung et al. 2011;Liu et al. 2015;Yao et al. 2015). As part of a continued search to discover structurally unique natural products with interesting biological activities from medicinal and edible homologous plant, two new amides with spermidine skeleton, coixdine A and B, along with six known compounds ( Figure 1) were isolated from the seeds of C. lacryma-jobi. The isolation and structural determination of the new compounds are described here.
Compound 2 was obtained as white amorphous powder. Its molecular formula was deduced as C 26 H 31 N 3 O 5 Na þ by HR-ESI-MS at m/z 488.2153 [M þ Na] þ (calcd. for C 26 H 31 N 3 O 5 Na þ , 488.2155). The 1 H and 13 C NMR data of 2 (Table S1) were similar to those of 1, with the main difference being the presence of a formyl group in 2, as shown by the proton signal at d H 8.02 (1H, s, H-1 0 ) and the carbon signal at d C 163.9/ 163.8 (C-1 0 ). The acetyl group in 1 was substituted in 2 based on the replacement of the formyl group [d C 163.9/163.8 (C-1 0 )] in 2 by an acetyl carbon [d C 173.3/173.2 and 22.5 (C-1 0 and C-2 0 )] in 1. The attachment of the formyl group to N-1 of 2 was confirmed by the HMBC correlation observed between d H 8.02 (1H, s, H-1 0 ) and d C 38.6/ 38.4 (C-2); 3.26/3.25 (2H, t, J ¼ 6.1 Hz, H-2) and d C 163.9/163.8 (C-1 0 ). Assignments for all function groups of 2 were achieved through 1 H-1 H COSY, HSQC and HMBC experiments ( Figure S17). Consequently, the structure of 2 was established as N 1 -formyl-N 6 ,N 10 -(E)-di-p-coumaroylspermidine and named coixdine B.

General experimental procedures
The 1 D and 2 D NMR spectra were recorded on either a Bruker DPX 400 instrument. Optical rotations were measured in MeOH on a JASCO P-2000 polarimeter. The Semipreparative HPLC procedure was performed with a Shimadzu LC-16D instrument with an RID-20A (reflective index detector) detector and a reversed-phase C 18 column (250 Â 10 mm, 5 lm). The purifications by column chromatography (CC) were conducted using silica gel (200-300 mesh, Qingdao Marine Chemical Corporation, Qingdao, China), octadecyl silica (ODS) (50 lm, Merck, Germany).

Plant material
The seeds of C. lacryma-jobi were collected from Xingren of Guizhou Province (China), and identified by Prof. Sheng-Hua Wei from Guizhou University of Traditional Chinese Medicine. The voucher specimen (No. 20200301) was deposited at Guizhou University of Traditional Chinese Medicine.

Extraction and isolation
The air-dried C. lacryma-jobi seeds (98.0 kg) were extracted under reflux with methanol (MeOH). The MeOH extracts were concentrated in vacuum (40 C) by rotary evaporators to obtain residue (1480 g). The residue was suspended in H 2 O and then sequentially partitioned with petroleum (PE), ethyl acetate (EtOAc), n-butyl alcohol (n-BuOH).

Cytotoxic activity
The cytotoxic activity of compounds (1-8) against two human cancer cell lines, namely CT-26 (colon cancer cells) and BxPC-3 (pancreatic cancer cells) cells were evaluated by the MTT colorimetric assay described in previous papers (Yin et al. 2019;2022).

Conclusions
In conclusion, our chemical investigation of the seeds of C. lacryma-jobi resulted in the isolation and identification of eight compounds, including two new ones, coixdines A-B (1-2) and six known compounds (3-8). All compounds were firstly isolated from C. lacryma-jobi except 5 and 7. Therefore, we believe that this plant could serve as an additional source for the isolation of amide derivatives with diverse structures and potential for further biological investigation.

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