Bioactivities of Compounds from Elephantopus scaber, an Ethnomedicinal Plant from Southwest China

Elephantopus scaber is an ethnomedicinal plant used by the Zhuang people in Southwest China to treat headaches, colds, diarrhea, hepatitis, and bronchitis. A new δ-truxinate derivative, ethyl, methyl 3,4,3′,4′-tetrahydroxy-δ-truxinate (1), was isolated from the ethyl acetate extract of the entire plant, along with 4 known compounds. The antioxidant activity of these 5 compounds was determined by ABTS radical scavenging assay. Compound 1 was also tested for its cytotoxicity effect against HepG2 by MTT assay (IC50 = 60 μM), and its potential anti-inflammatory, antibiotic, and antitumor bioactivities were predicted using target fishing method software.


Introduction
Elephantopus is a genus comprised of about 30 species worldwide, mainly distributed in South America, with only 2 species E. scaber and E. tomentosus found in Southwest China [1]. From 2008 to 2012, our ethnobotanical investigation in the traditional medicinal market, held during the Dragon-Boat Festival in the fifth month of the Chinese lunar calendar with a history of over 700 years, found that Elephantopus scaber L. (Asteraceae) is a common medicinal plant used by the Zhuang people in Jingxi County of Southwest China. The local Zhuang people use E. scaber commonly as a traditional herbal medicine to treat many ailments including headaches, colds, diarrhea, hepatitis, and bronchitis.
To date, 30 compounds have been reported from E. scaber, including 4 sesquiterpene lactones, 9 triterpenes, and 5 flavones. Previous bioactivity studies on E. scaber demonstrated that the extracts or compounds from this species have antibiosis, antivirus, and cytotoxicity activities [2]. The sesquiterpene lactones in particular have been explored for their anti-inflammatory and hepatoprotective activities [3], which partially proved the traditional knowledge of E. scaber.

Antioxidant Assay.
The antioxidant activity of compounds 1-5 was evaluated with ABTS radical scavenging assay as described previously [8]. The IC 50 was expressed as millimoles per liter (mM).

Cytotoxicity
Assay. Compound 1 was tested for cytotoxicity using a slightly modified MTT method [9]. Briefly 150 L (10 M, 20 M, 30 M, and 40 M) of samples was added to 96-well plate containing a confluent HepG2 cell monolayer in sextuplicate; 10 g/mL of norcantharidin (NCTD) and blank medium were used as the positive and control group, respectively. After a 72 h incubation at 37 ∘ C, 100 L MTT solution (5 mg/mL phosphate buffered saline) was added to each well, which was further incubated for 4 h for the formation of the formazan product. After removing the medium, 150 L DMSO was added to dissolve the formazan crystals. The optical density (OD) was measured at 550 nm with a microplate reader. The rate of inhibition was calculated by the following formula: rate of inhibition = (1 − sample OD)/control OD. The concentration causing inhibition of viable cells by 50% (IC 50 ) was determined from a doseresponse curve, which was based on triplicate measurements.

Virtual
Screening. The potential activity of compound 1 was predicted by the "Target Fishing" functional model software (Discovery Studio). The target fishing process was conducted as follows. The DockScore energy function was utilized to minimize the energy of compound 1 conformation. Setting full minimization as minimization gave the smart conformation of compound 1. Then, pharmacophore search Evidence-Based Complementary and Alternative Medicine 3 was set to be screened and profiled. Screen and profile was set to be ligand profiler. PharmaDB pharmacophores were set to be all. Conformation generation was set to be the best. Maximum conformation was set to be 200. Energy threshold was set to be 10. Saved conformations were set to be true, and other parameters were set to be default.
Top 14 candidate receptors were ranked according to the fit value (as shown in Table 2), which is based on force field approximation and specifically examined the compound internal energy and the compound-receptor interaction energy, which is taken as the sum of van der Waal force and electrostatic energy [10].      . Meanwhile, the HMBC spectrum of compound 1 presented the correlations from H-10 to C-9, H-8 to C-9 and C-8 , H-7 to C-2 and C-6, H-11 to C-10 , from C-10 to H-9 and H-11 , from C-8 to C-9 , and from C-7 to H-2 and H-6 , respectively ( Figure 2). Consequently, the structure of compound 1 was deduced to be ethyl, methyl 3,4,3 ,4 -tetrahydroxy--truxinate, which was further confirmed by HMQC, COSY, and NOESY spectra. This paper reports a new -truxinate derivative in Elephantopus genus for the first time. Compounds 2-5 were identified, respectively, as 5-O-caffeoylquinic acid (2), chlorogenic acid methyl ester (3), deoxyelephantopin (4), and isoscarbertopin (5) by comparing their NMR and MS data with reported literature values.

Results
The antioxidant activity of 5 compounds isolated from E. scaber was evaluated by the ABTS radical scavenging assay, and the results are presented as IC 50 in Table 1. Antitumor and obesity [39,40] 2brc-01 ATP-dependent molecular chaperone HSP90 Protein 3.26648 Antitumor and antivirus [41,42] 1c1b-01-s HIV-1 reverse transcriptase (A-chain) Protein 3.25549 Anti-HIV [43][44][45] The most active radical scavengers were the new compound ethyl, methyl 3,4,3 ,4 -tetrahydroxy--truxinate (IC 50 = 0.44 ± 0.039 mM). The other 2 quinic acid derivatives 5-Ocaffeoylquinic acid and chlorogenic acid methyl ester also showed radical scavenging potential (IC 50 = 0.96 ± 0.096 and 0.89 ± 0.140 mM, resp.), while the antioxidant activity of the other 2 sesquiterpene lactone compounds deoxyelephantopin and isoscabertopin was not detected. Comparing the structures of these 5 compounds, the different antioxidant activities were attributed to the existence of phenolic hydroxyl groups in compounds, which were supported by the previous reports [12]. Compound 1 was also tested for in vitro cytotoxicity against HepG2 cell line with norcantharidin (NCTD, 60 M) as positive control at 72 h incubation ( Figure 3). Compound 1 exhibited a dose-response inhibition curve from 27% growth inhibition at 10 g/mL to 81% at 40 g/mL, demonstrating that it has significant and dose-dependent inhibition on the growth of HepG2 (IC 50 = 60 M). Further work will be conducted on the mechanism by which compound 1 induces apoptosis.
With the rapid development of computer-aided drug design (CADD), virtual screening technique has been used more and more widely in drug design and bioactivity screening of compounds [13]. The potential bioactivities of compound 1 have been predicted by the target fishing method which was based on the Discovery Studio software and Protein Date Bank (PDB) including over twelve thousand 3D macromolecular structure data determined experimentally by X-ray crystallography and NMR. The top 14 biological molecular targets ranked as the fit value (FV) are reported (Table 2).
Theoretically, FV > 3 means this target should be explored experimentally. The strongest activity of compound 1 was predicted to be anti-inflammatory (FV = 4.05271) and anti-AIDS (FV = 3.25549), respectively ( Table 2). Further experiments on the biological functions of 1 should be directed towards its potential anti-inflammatory, antibiotic, antivirus, and anticancer activities.

Discussion and Conclusion
With more and more present modern drugs discovered from traditional medical knowledge, the traditional knowledge is getting more extensive attention, which also led to the development of important drugs such as reserpine (a treatment for hypertension) podophyllotoxin (the base of an important anticancer drug), and vinblastine (used in the treatment of certain cancers) [46]. Previous studies showed that pulmonary oxidant stress can cause some disease conditions, such as acute lung injury, Evidence-Based Complementary and Alternative Medicine 5 radiation injury, COPD (chronic obstructive pulmonary disease), and inflammation [47]. Meanwhile, previous clinical and experimental studies described that antioxidant supplementation including flavonoids and vitamins may inverse the oxidant-mediated cough depression by modulating the inflammatory process in lung disease [48,49]. Interestingly, our work using ABTS assay demonstrated that compounds 1-3 showed strong antioxidant activity, especially compound 1 (IC 50 = 0.44 ± 0.039 mM). Moreover, E. scaber was also reported as the source of a number of sesquiterpene lactones, such as compounds 4 and 5, which have shown significant contribution to the anti-inflammatory activity of plants [50]. Some of the sesquiterpenes from the genus Elephantopus have demonstrated significant anti-inflammatory as well as hepatoprotective activities and are being considered as drug lead compounds [3]. Based on the above analysis, we hypothesize that Zhuang people use this plant to treat headaches, bronchitis, and hepatitis,due to its anti-inflammatory and antioxidant effects.
According to the in vitro cytotoxicity assay with NCTD (60 M) as control group and activity virtual screening, compound 1 exhibited good (IC 50 = 60 M) and doseresponse inhibition on HepG2 cell line and potential antiinflammatory, antibiotic, antivirus, and anticancer activities, which indicated that the further research of E. scaber could be focused on anticancer and anti-inflammatory activity. The present work further developed the usage of this traditional medicine plant.