UPLC-QTOF-MS-guided isolation of anti-COPD ginsenosides from wild ginseng

Four previously undescribed ginsenosides, along with five known analogues were isolated from wild ginseng by a UPLC-QTOF-MS-guided fractionation procedure. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, HR-ESI-MS). The isolated compounds could significantly inhibit the cigarette smoke extract (CSE)-induced inflammatory reaction in A549 cells. The HDAC2 pathway might be involved in the protective effect against the CSE-mediated inflammatory response in A549 cells.


Introduction
Panax ginseng has received much attention not only as a medicinal herb but also as a functional food. 1 Ginseng has a wide range of biological activities such as anti-inammatory, anti-oxidant and immunomodulatory functions. It is the traditional belief that wild ginseng is more medicinally efficacious and more valuable than cultivated ginseng. In the past, natural wild ginseng has been used to refer to the original ecological ginseng. But it is hard to nd the natural wild ginseng because it is nearly extinct now. Currently, according to the "Identication and Grade Quality Standards of Wild Ginseng" (National Standards of People's Republic of China, GB/T 18765-2015), wild ginseng refers to the ginseng cultivated in mountains and grown for more than 15 years in a natural environment without any human intervention. This kind of wild ginseng is a replacement for natural wild ginseng grown within the original ecological environment. It has been reported that there were differences in immunomodulating effects between wild ginseng and cultivated ginseng. 2 In our previous study, it was also found that the wild ginseng (20 year-old mountaincultivated ginseng) was much differentiated from cultivated ginseng. And based on UPLC-QTOF/MS, a total of 126 chemical compositions were tentatively identied or characterized from wild ginseng. 3 Chronic Obstructive Pulmonary Disease (COPD), the fourth leading cause of death in the world currently, is usually caused by signicant exposure to noxious gases or particles. The airow limitation and persistent respiratory symptoms are the typical symptoms. Cigarette smokers have a higher prevalence of respiratory symptoms and higher COPD mortality rate, and the cigarette smoking is the leading environmental risk factor for COPD around the world. 4 Along with the progressive lung inammation, some pro-inammatory mediators such as TNFa, IL-1b and IL-6 participated in the occurrence and development of COPD. 5,6 Ginsenosides are regarded as the main bioactive components in ginseng. 7,8 Ginseng extract 9,10 and monomer saponins such as Rg 1 (ref. 11), Rh 2 (ref. 12) and CK 13 had been reported to signicantly inhibit COPD-related inammation in vivo and in vitro. Reduced HDAC2 activity and expression was found in COPD, resulting in amplication of the inammatory response. Meanwhile, HDAC2 dysfunction is thought to play an important role in the development of corticosteroid resistance in COPD. [14][15][16] Thus, increasing HDAC2 activity may be a promising strategy to overcome corticosteroid resistance in COPD. Existing treatments such as theophylline, nortriptyline, macrolides and selective phosphatidylinositol-3kinase-d inhibitors have been reported to increase HDAC2 activity effectively. 17 As part of our continuing efforts to elucidate the chemical composition of wild ginseng furtherly and search for anti-COPD compounds from wild ginseng, a UPLC-QTOF-MS-guided fractionation procedure was performed to target ginsenosides from the anti-COPD fractions of wild ginseng, leading to the isolation of 4 previously unreported ginsenosides (1)(2)(3)(4) and ve known analogues (5)(6)(7)(8)(9). Herein, the isolation and structural elucidation of the isolated compounds as well as their anti-COPD activity on the CSE-stimulated A549 cells were discussed. The potential mechanism against the CSE stimulation was also preliminarily investigated in the paper.

Plant materials
The 20 years-old mountain-cultivated ginsengs were collected from cultivation area in Fusong, Jilin Province, the main source of ginseng in China. The ginseng was identied by our group according to Chinese Pharmacopoeia (2015 version). The voucher specimen (no. MCG170931) was deposited at the Research Center of Natural Drug, Jilin University, Changchun, China.

Apparatus and chemicals
The HR-ESI-MS spectra were performed on Waters Xevo G2-XS QTOF mass spectrometer (Waters, Milford, MA, USA). NMR spectra were measured on a Bruker Avance-600 spectrometer (Bruker, Karlsruhe, Germany) in DMSO-D 6 operating at 600 MHz for 1 H NMR, HMBC and HMQC, 150 MHz for 13 C NMR, respectively. Tetramethylsilane (TMS) was used as internal standard. Column chromatography was performed with silica gel (200-300 mesh) purchased from Qingdao Ocean Chemical Group Co. Ltd (Qingdao, China). Thin-layer chromatography was conducted on silica gel G plates (Qingdao Marine Chemical Inc.). Semi-preparative HPLC with 1525 binary pump combined Waters 2998 UV detector (Waters Co., Milford, MA, USA.) and YMC C 18 column (5 mm, 20 mm Â 10 mm; YMC Co., Ltd., Japan) were also used to isolate the compounds. Methanol (MeOH) and acetonitrile (ACN) were HPLC grade (Fisher, USA). Puried water was purchased from YiBao Co, Ltd (Shenzhen, China). Other solvents used were analytical grade (Beijing Chemical Works, Beijing, China).

Spectroscopic data
White amorphous powder; Libermann-Burchard reaction was positive; Molisch reaction was positive; suggesting the existence of triterpenoid structure; 1 H and 13 C NMR: see Table 1 In the present study, the cigarettes used were Xiongshi cigarette (China Tobacco Zhejiang Industrial Co., Ltd, Hangzhou, China) containing 11 mg of tar, 0.7 mg of nicotine and 13 mg of carbon monoxide per cigarette. Cigarette smoke extract (CSE) was prepared essentially as reported previously. [18][19][20] Smoke from one cigarette was bubbled into 20 mL of culture medium (300 s per cigarette). The CSE solution was ltered through a 0.22 mm sterile lter aer being incubated at 37 C for 30 min. The CSE solution was prepared freshly and was used within half an hour. This CSE solution was regarded as the highest concentration (100%).
2.5.2 Cell viability assay. Human lung carcinoma A549 cells were obtained from the Department of Pathogen Biology, Basic Medical College, Jilin University. Each ginsenoside was dissolved in DMSO to obtain the stock solution stored in 4 C. The  For all group, A549 cells were cultured for 18 h in 96-well plates at a density of 5 Â 10 5 cells per mL. In control group, A549 cells were cultured normally without the CSE stimulation. In CSE group, the cells were stimulated with a certain amount of CSE without any drug intervened. In ginsenoside groups, the cells were treated simultaneously with CSE and 10, 20, 40, and 80 mM of each ginsenoside.
2.5.4 Enzyme-linked immunosorbent assay. IL-1b, IL-6 and TNF-a contents in the cell culture supernatant were determined with ELISA kits (Nanjing Jiancheng Bioengineering Institute) aer 18 h of incubation. All procedures were performed following the manufacturer's instructions.
2.5.5 Western blotting. Western blotting was performed as previous described 22 to discuss the effects of ginsenosides on HDAC2 expression in CSE-stimulated A549 cells. Both anti-HDAC2 (#ab32117) and anti-b-actin (#ab137550) were purchased from Abcam Company (Cambridge, UK). The HDAC2 band intensities were compared with reference to b-actin control.
2.5.6 Statistical analysis. Graphpad Prism 6.0 soware (CA, USA) was used for all statistical analysis. The results are expressed as x AESD. Statistical signicance was calculated with two tailed test or a one-way analysis of variance (ANOVA), and pvalue < 0.05 was considered as statistically signicant.
2.5.7 Molecular docking assay. The GLIDE 6.7 soware and Maestro Elements 2.2 soware (Schrödinger, New York, NY, USA) were applied to perform molecular docking. Firstly, the 3D structures of ginsenosides were acquired respectively; secondly, the ginsenosides' bond angles and orders were then assigned using LigPrep module; thirdly, the 3D X-ray crystal structure of HDAC2 (PDB ID: 5IWG) was retrieved from Protein Data Bank (PDB) and was optimized according to the processes previous reported. 23 Fourthly, the docking were performed with the following main parameters: the energy of conjugate of ligand and receptor was minimized to a root-mean-square deviation of 0.30 A in optimized potentials for liquid simulations 3 force eld, and the grid box at active site was set at the size of 20 A. Finally, the highest XPG score pose was chosen as the dominant binding form and the visual analysis was performed by PyMol (Schrödinger) soware.

Bioactivity evaluation
Ginsenoside Rg 1 (ref. 11) and Rh 2 (ref. 12) had been reported to signicantly inhibit COPD-related inammation. As part of an ongoing effort to search for anti-COPD agents from wild ginseng, 23 compounds 1-7 were evaluated for their anti-inammatory effects on CSE-induced A549 cells. Dexamethasone was selected as positive drug (Pos). 32 3.2.1 Cytotoxicity of cigarette smoke extract (CSE) and compounds 1-7 on the viability of A549 cells. The results of MTT showed that the cell viability of A549 cells was signicantly affected (p < 0.01) by CSE (S30%) (Fig. 3A). Therefore, in subsequent experiments, 20% CSE was chosen as stimulation. As shown in Fig. 3B, the cell viabilities of A549 cells were not signicantly affected by 1-7 at 10 mM-80 mM. Then we evaluated the anti-inammatory effects of 1-7 at 10 mM-80 mM on CSEstimulated A549 cells.
3.2.3 Effect of compounds 1-7 on CSE-Mediated protein expression of HDAC2 in vitro. It has been demonstrated that histone deacetylase 2 (HDAC2) activity and egg self-expression in alveolar macrophages, peripheral lung tissues and bronchial tissues of COPD patients are signicantly decreased. 33,34 CSE can reduce HDAC2 activity and protein expression in rats, 35 which can activate rat lung tissue NF-kB pathway. 36 Other studies have shown that CSE exposure can cause the decrease of HDAC2 protein and activity in A549 cell nucleus 37 and activate the NF-kB pathway in vivo. 36 In order to explore the underlying mechanism of anti-inammatory property of compounds 1-7, we investigated the effect of them on the protein expression of HDAC2 in CSEexposed A549 cells. In the present study, aer 18 hours of CSE exposure in A549 cells, the expression of HDAC2 protein in each group of 40 mM concentration was measured as shown in Fig. 5. The results showed that the level of HDAC2 protein in A549 cells decreased signicantly (p < 0.01) aer CSE 18 hours exposure. Interestingly, treatment with each ginsenoside could signicantly activated the expression of HDAC2 (p < 0.01, p < 0.05) as compared to the CSE group (Fig. 5) in detail, which suggested that these compounds may up-regulated the expression of HDAC2 in CSE-exposed A549 cells.

Conclusions
In the present study, four minor previously undescribed ginsenosides, named as ginsenoside Rm1 (1), Rm2 (2), Rm3 (3) and Rm4 (4), along with ve known ginsenosides, Rb 2 (5), Rd (6), Rg 3 (7), Rg 1 (8) and Rh 2 (9) were isolated from wild ginseng by a UPLC-QTOF-MS-guided fractionation procedure. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HR-ESI-MS). The anti-COPD activities were investigated using CSE-stimulated A549 cells. Compounds 1-7 could ameliorate inammatory reaction in vitro. Furthermore, the protective effect against CSE could be related to the HDAC2 pathway. The study provided some evidences to further elucidate the chemical composition of wild ginseng, and revealed that the compounds isolated from wild ginseng had the protective effect on the injury of cigarette smoke in vitro. The study provides a theoretical basis for the further utilization and development of the wild ginseng. It reminds us that it is meaningful to explore the protective effect of the wild ginseng against the COPD in vivo.

Conflicts of interest
The authors declare no conicts of interest.