A New 1,3-Benzodioxole Compound from Hypecoum erectum and Its Antioxidant Activity

The purpose of this study was to identify the chemical components in aerial parts of Hypecoum erectum. A new 1,3-benzodioxole derivative, identified as Hypecoumic acid (1), was isolated, together with the three known compounds: protopine (2), coptisine (3), and cryptopine (4). Their structures were identified based on extensive spectroscopic experiments, including nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectra (HR-ESI-MS), as well as comparison with those reported in the literature. Meanwhile, the in vitro antioxidative activity of all compounds was determined using a DPPH-scavenging assay, and compound 1 (IC50 = 86.3 ± 0.2 μM) was shown to have moderate antioxidative activity.


Introduction
The oxidative-antioxidant balance affects the proper functioning of homeostasis. Overproduction of reactive oxygen species (ROS) causes oxidative stress, and is one of the most common reasons for homeostasis disorders. The antioxidative activities of herbal medicines have received a great amount of attention as being primary preventive ingredients against various diseases, such as cancer [1], diabetes [2], and cardiovascular diseases [3]. H. erectum, belonging to the Papaveraceae family, is widely distributed in North, Northeast, and Northwest China [4]. H. erectum has traditionally been used to treat inflammation, fever, and pain in folk medicine in China. The chemical constituents of H. erectum L. are mainly isoquinoline alkaloids, such as hypecorine and hypecorinine [5], hyperectine [6], protopine, coptyine and allocryptopine [7], isohyperectine [8], 2,3-Dimethoxy-N-formylcorydamine and 2,3-Dimethoxyhypecorinine [9], and leptocarpinine [10]. Recent pharmacological studies involving in vitro experiments have been performed on the anti-inflammatory [9], antimicrobial [11], antibacterial and analgesic activity [12] of H. erectum. However, few comprehensive studies have investigated its in vivo antioxidative abilities and identified the bioactive metabolites. In preliminary experiments, we found that the extract of aerial parts of H. erectum showed antioxidative activity, and therefore set out to identify the bioactive compounds. Herein, we report on the isolation and structural elucidation of a new 1,3-benzodioxole derivative (1), along with three known compounds (2-4) ( Figure 1).

DPPH-Scavenging Assay
The DPPH is a stable free radical, which accepts the hydrogen radical or an electron, forming a stable diamagnetic molecule [13]. This antioxidant ability can be assessed by the determination of IC 50 values related to the amount of the sample required to reduce 50% of free radicals. The four compounds were further investigated using the DPPH-scavenging assay to evaluate antioxidant activity ( Table 2). The most effective antioxidant was found to be compound 1, with an IC50 of 86.3 µM, followed by coptisine (3), protopine (2), and cryptopine (4) with values of IC 50 252.6, 345.2, and 430.1 ± 1.6 µM, respectively.

Plant Material
H. erectum specimens were purchased from the Affiliated Hospital, Inner Mongolia Minzu University, Tongliao, China, in May 2017 and identified by Prof. Xi Quan of the Inner Mongolia University for Nationalities. A sample (No. 20170613) was deposited at the Inner Mongolia Minzu University.

DPPH-Scavenging Assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent (at a concentration of 0.1 µM, dissolved in MeOH) was used for assaying the reduction of free radicals by the various compounds, as measured by UV spectrometry [16,17]. Compounds 1-4 were dissolved in MeOH to obtain a suitable concentration (100-150 µg/mL), and were dispensed into a cuvette containing 3.0 mL of DPPH solution. The resultant mixture was incubated for 30 min at ambient temperature in the dark and then monitored at 517 nm. The DPPHscavenging activity was calculated according to the following equation: where A C is the initial concentration of the stable DPPH free radical without the test compound, and A S is the absorbance of the remaining concentration of DPPH in the presence of MeOH. The IC 50 values (µM) were determined from a plotted graph of DPPHscavenging activity against the concentrations of the respective compounds, where IC 50 is defined as the total amount of antioxidant needed to decrease the initial DPPH free-radical concentration by 50% (mean ± SD, n = 3).

Conclusions
In this study, the crude extract of H. erectum revealed the isolation and purification of one new and three known compounds. Compound 1 was clarified to be 4-(butoxycarbonyl)benzo[d] [1,3]dioxole-5-carboxylic acid, named Hypecoumic acid. This is the first report of the isolation of a 1,3-Benzodioxole compound from the Papaveraceae family. All the isolates were evaluated for their antioxidative activity. Among them, Hypecoumic acid (1) exhibited moderate capabilities against ·OH. These findings enrich the knowledge of the chemical diversity and biological potential of H. erectum.