Bioactive Constituents from the Aerial Parts of Pluchea indica Less

Four new thiophenes, (3′′R)-pluthiophenol (1), (3′′R)-pluthiophenol-4′′-acetate (2), 3′′-ethoxy-(3′′S)-pluthiophenol (3), 3′′-ethoxy-(3′′S)-pluthiophenol-4′′-acetate (4), together with twenty-five known compounds were obtained from the 70% ethanol-water extract of the aerial parts of Pluchea indica Less. Their structures were elucidated by spectroscopic methods. Among the known isolates, compounds 7, 8, 11, 14, 15, 18, 20, 23, 25–27 were isolated from Asteraceae family firstly, while compounds 6, 9, 10, 12, 13, 16, 19, 21, 28 were isolated from Pluchea genus for the first time. Meanwhile, compounds 1, 2, 10, 13, 18, 23 displayed significant inhibitory activities on LPS-induced NO production at 40 µM from RAW 264.7 macrophages, while compounds 3, 4, 26–29 possessed moderate inhibitory effects.


Introduction
As one of the largest families, the Asteraceae (Compositae) family contains about 1600-1700 genera and 24,000-30,000 species. Most of the Asteraceae family plants are herbs and shrubs, and have been widely used as herbal medicines since ancient times all over the world [1]. Pluchea indica Less., belongs to Pluchea genus, Asteraceae family, is a 1 to 2 meters high shrub. It mainly distributes in the tropical and subtropical regions of Africa, Asia, America, Australia, and China's southern provinces. As an amphibious woody semi-mangrove plant, it plays an important role in maintaining the ecological balance in the coastal areas of Southeast Asia in China [2]. As a folk medicine in Guangxi, it exhibits the function of softening hardness and dissolving lump [3]. As a type of food, it possesses the activity of warming the stomach [4]. Its main chemical compositions are thiophenes, quinic acids, sesquiterpenes, lignans, flavonoids, and sterols [2]. Pharmacological studies have shown that the plant exhibits many pharmacological functions such as anti-inflammatory [5], anti-cancer [6], anti-oxidant [7], anti-microbial [8], and insecticidal activities [9].
Through the summary of relevant literature, it is found that the pharmacodynamic material basis is not yet clear for the lack of systematic research on the plant. In the course of studying the anti-inflammatory activity of various medicinal plants, 70% EtOH extract of P. indica was found to possess significant in vitro anti-inflammatory bioactivity. Based on the anti-inflammatory activity on LPS-induced NO production from RAW 264.7 macrophages, a systematic chemical component study of P. indica aerial parts was carried out. In this paper, the isolation and identification of constituents were described as well as their inhibitory effect on the production of NO in RAW 264.7 cells induced by LPS.
Molecules 2018, xx, x 2 of 11 study of P. indica aerial parts was carried out. In this paper, the isolation and identification of constituents were described as well as their inhibitory effect on the production of NO in RAW 264.7 cells induced by LPS.
The potential in vitro anti-inflammatory effects of 70% EtOH extract (PI) and 95% EtOH eluent (PIE) and compounds 1-29 obtained from the aerial parts of P. indica on LPS-stimulated NO production were accomplished by pretreating RAW 264.7 macrophages cells with them for 1 h before stimulating with LPS (500 ng/mL) for 24 h, respectively. Griess reagent (St. Louise, MO, USA) was used to measure NO concentrations in the culture medium. Comparing to unstimulated normal (negative control), NO production in LPS-stimulated RAW 264.7 macrophages was markedly induced (Table 5). PI and PIE displayed potential inhibitory activities on LPS-induced NO production at 100 µg/mL. Further, using the same activity screening assay, the compounds isolated from active fractions were tested at a final concentration of 40 µM. Under this concentration, cells showed no significant influence on cell viability by dimethyl thiazolyl diphenyl tetrazolium (MTT) assay. Compared with untreated cells, the changes in cell viability were less than 10% (data not shown). As results, compounds 1, 2, 10, 13, 18, 23 showed significant inhibitory effects at 40 µM, while 3, 4, 26-29 possessed moderate in vitro anti-inflammatory activity. These results suggested that compounds 1, 2, 10, 13, 18, 23 may exhibit potent anti-inflammatory activity. Table 5. Inhibitory effects of positive control, PI, PIE and compounds 1-29 obtained from the aerial parts of P. indica on NO production in RAW 264.7 macrophages. 103.9 ± 6.7

Plant Material
The aerial parts of Pluchea indica Less. were collected from Hepu city, Guangxi province, China and identified by Dr. Wei Songji (Zhuang Medical College, Guanxi University of Chinese Medicine). The voucher specimen was deposited at the Academy of traditional Chinese Medicine of Tianjin University of TCM.