Screening of traditional Chinese medicines with therapeutic potential on chronic obstructive pulmonary disease through inhibiting oxidative stress and inflammatory response

Background Chronic obstructive pulmonary disease (COPD) is a major public health problem and gives arise to severe chronic morbidity and mortality in the world. Inflammatory response and oxidative stress play dominant roles in the pathological mechanism of COPD, and have been regarded to be two important targets for the COPD therapy. Traditional Chinese medicines (TCMs) possess satisfying curative effects on COPD under guidance of the TCM theory in China, and merit in-depth investigations as a resource of lead compounds. Methods One hundred ninety-six of TCMs were collected, and extracted to establish a TCM extract library, and then further evaluated for their potency on inhibitions of oxidative stress and inflammatory response using NADP(H):quinone oxidoreductase (QR) assay and nitric oxide (NO) production assay, respectively. Results Our investigation observed that 38 of the tested TCM extracts induced QR activity in hepa 1c1c7 murine hepatoma cells, and 55 of them inhibited NO production in RAW 264.7 murine macrophages at the tested concentrations. Noteworthily, 20 of TCM extracts simultaneously inhibited oxidative stress and inflammatory responses. Conclusion The observed bioactive TCMs, particularly these 20 TCMs with dual inhibitory effects, might be useful for the treatment of COPD. More importantly, the results of the present research afford us an opportunity to discover new lead molecules as COPD therapeutic agents from these active TCMs. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1347-y) contains supplementary material, which is available to authorized users.


Background
Chronic obstructive pulmonary disease (COPD) is a disease characterized by progressive and not fully reversible airflow limitation, which is associated with abnormal inflammatory response of the lung to noxious particles and gases [1]. Tobacco smoke, indoor and outdoor air pollutions, as well as exposure to occupational dust and chemicals are the three dominant risk factors for COPD. It is the fourth leading cause of chronic morbidity and mortality in the United States. On the basis of investigation by the World Bank/World Health Organization, COPD is predicted to rank fifth in 2020 as a worldwide burden of disease. A horrifying fact is that half of global deaths from COPD occur in the Western Pacific Region, with the majority of these existing in China, which might be contributing to high incidence of smoking and severe air pollution in the industrialization advancement [2].
Cumulative evidences indicate that inflammatory response, oxidative stress, and protease imbalance play dominant roles in the pathological mechanism of COPD [3,4]. Briefly, exogenous irritants and reactive oxygen species (ROS) activate inflammatory cells (e.g. macrophages, neutrophils) and epithelial cells in the respiratory tract that release ROS, inflammatory mediators [e.g. leukotriene B4 (LTB4), interleukin-8 (IL-8), tumor necrosis factor α (TNFα), transforming growth factor-β (TGF-β)], proteases (e.g. cathepsins, matrix metalloproteinases) [3,5,6]. ROS stimulates nuclear factor kB (NF-kB) and increase the release of inflammatory cytokines, inflammatory mediators promote the production of endogenous ROS, while proteases cause alveolar destruction and mucus secretion. Hence, the synergistic reactions of inflammation, oxidative stress, and protease imbalance amplify pathophysiology of COPD, and inhibitions of these three processes are regarded to be effective strategies for the treatment, as well as drug research and development of COPD [7].
Plenty of traditional Chinese medicines (TCMs) have been used clinically to treat COPD in the form of single or compound prescription under guidance of the TCM theory in China, and demonstrated satisfying curative effects [8,9]. Their clinical effectiveness implies that TCM is an important resource of new drugs and/or lead compounds with COPD therapeutic potential. Based on this rationale, we have launched a systemic research on discovering new drugs and lead molecules for COPD treatment from TCM targeting inhibitions of oxidative stress and inflammatory response. We firstly collected and extracted TCM materials to establish a TCM extract library, and then carried out a biological screening of these TCMs using NADP(H):quinone oxidoreductase (QR) assay and nitric oxide (NO) production assay to find the TCMs with potential therapeutic effect on COPD.

Collection and Identification of tested TCMs
Traditional Chinese medicine (TCM) materials were purchased from the Jinan Jianlian TCM Co. Ltd in Shandong province, Anguo TCM market in Hebei province, and Bozhou TCM market in Anhui Province. These TCMs were identified by Prof. Lan Xiang, School of Pharmaceutical Sciences, Shandong University, through comparing their characteristics in plant morphology and taxonomy with that described in Chinese Pharmacopoeia. Voucher specimens (Voucher ID see Table 1) of TCMs have been deposited at the Laboratory of Pharmacognosy, School of Pharmaceutical Sciences, Shandong University.

Preparations of TCM extractions
Crushed aerial parts or leaves of plant materials (50 g) were extracted under reflux for 2 h with 75 % ethanol (EtOH, 2 × 500 mL), and then EtOH was removed under reduced pressure. The yield of each extract was presented as a percentage of weight of dried plant material, and has been summarized in Table 1.
Nitric oxide (NO) production assay Inhibition of NO production by LPS-stimulated RAW 264.7 murine macrophages was applied to evaluate antiinflammatory functions of TCM extracts. RAW 264.7 cells (8.0 × 10 4 cells/well) were seeded in 96-well plates and treated with 1 μg/mL LPS, in the absence or presence of tested TCM extractions for 24 h. Then, 100 μL of supernatant was removed to a new 96-well plate and added with 100 μL of Griess reagent (0.1 %      naphthylethylenediamine and 1 % sulfanilamide in 5 % H 3 PO 4 solution) at room temperature for 15 min. Absorbance was measured at 570 nm on the Model 680 plate reader (Bio-rad). Nitrite concentration was calculated from a NaNO 2 standard curve. Didox (100 μM) was used as a positive control.

Cell viability assay
The anti-proliferative effect of TCM extracts on RAW 264.7 cells were simultaneously determined using a 3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. Briefly, 100 μL of DMEM media containing 0.4 % MTT were added to each wells, after removing 100 μL of supernatant as described in NO production assay. Then, the cells were incubated at 37°C for 3 h, and absorbance was measured at 570 nm on the Model 680 plate reader (Bio-rad).

Statistical analysis
One way analysis of variance (ANOVA) and post hoc multiple comparison Bonferroni test were applied to compare the significant difference between two groups.
Results are presented as the mean ± SD. P < 0.05 was considered to be significant.

Results and Discussion
To establish a TCM extract library for biological screening, we firstly selected 196 TCMs based on Chinese Pharmacopoeia (Edition 2015) and TCM literatures, and collected TCMs from Jinan local TCM drugstore, as well as the two biggest Chinese TCM markets, Anguo and Bozhou TCM markets. After plant material authentication, TCMs were extracted with 75 % EtOH to prepare their EtOH extracts, and then the concentrations of 200, 100, 50, 25, 12.5, 6.25 μg/mL were selected as tested doses for bioassays. Names, origin, extract yields and biological activities of investigated TCMs were summarized in Table 1. We adopted a bioassay measuring QR activity in hepa 1c1c7 murine hepatoma cells to evaluate the ability of TCM extracts on inhibiting of oxidative stress [10]. Although QR is a phase II detoxification enzyme, it possesses same regulating mechanism with antioxidant enzymes [e.g. glutamate-cysteine ligase, modifier subunit (GCLM) and heme oxygenase-1 (HO-1)], since these  enzymes are antioxidant response element (ARE)-containing target genes and are mediated by ARE located in their promoter region [11]. Specially, upon the exposure of cells to oxidative stress and/or toxicants, nuclear factor E2-related factor 2 (Nrf2) translocates into the nucleus, binds to the ARE sequence, and activates the transcription of these ARE-target genes [12]. Therefore, QR and antioxidant enzymes (e.g. GCLM and HO-1) possess same responses against endogenous and exogenous insults, which have also been verified by our recent researches [13,14]. Considering above mentioned inducing mechanism of QR and antioxidant enzymes, determination of QR activity is a rational and effective method for analyzing the potency of oxidative stress inhibition. In the current study, we normalized the data by setting the untreated control group as 1, and then the QR inducing activity of tested extracts was represented by the maximum folds of QR inducing activity (MQI) compared with the untreated control group. SF as a positive control displayed an approximately 1.7-fold induction at 2.0 μM.  (Table 1) Table S1 and Figure S1.
During the chronic inflammation process, excessive NO have been produced and involved in the tissue injury through damages to proteins, lipids, DNA, and the modulation of leukocyte activity [15]. Accordingly, inhibiting NO production is regarded to be an effective strategy for the therapy of inflammation-related diseases. Herein, we detected NO level in LPS-stimulated RAW264.7 macrophages to evaluate anti-inflammatory function of TCM extracts. Cytotoxicities of tested TCM extracts were simultaneously evaluated by a MTT assay to confirm that the decrease of NO production was not attributed to inhibition of cell proliferation. The maximum inhibition rate (MIR) of NO production under the nontoxic tested concentration, which was calculated by comparing the decreased NO concentration in TCM-treated group with that in LPS-stimulated group, was adopted to evaluate the anti-inflammatory property. Didox with an approximately 60 % inhibition of NO production at 100 μM was used as a positive control. The inhibitory potency of TCM extracts on NO production was ranked according to the criteria as follows: strong (MIR ≥ 80 %); moderate (80 % > MIR ≥ 50 %); weak (50 % > MIR ≥ 30 %); undetected (MIR <30 %).
Since oxidative stress and inflammatory response have the synergistic reactions in the pathophysiology of COPD, TCMs having dual inhibitions on the two targets are apt to be the resource for discovering lead molecules [5,7]. Our results indicated that the extracts of Artemisia scoparia (aerial part, 27), Aucklandia lappa  (Table 1 and Additional file 1: Table S1). Most of all, both QR inducing effects and NO inhibitory activities of the extracts of Aucklandia lappa (31), Cimicifuga heracleifolia (40), and Glycyrrhiza uralensis (85) are labelled as the level of strong. In addition, the extracts of Inula helenium (92) and Physalis alkekengi L. var. franchetii (132) also demonstrated the potencies that are closed to the strong level.
TCMs have been adopted for the therapy of inflammation-related diseases with a long history in China. Compared with QR inducing assay, NO inhibitory effect assay and other in vitro and in vivo anti-inflammatory models are classical and commonly adopted biological research methods, and accordingly more literatures concerning inflammation of TCMs have been published. Based on our findings, we carried out a systemic search of reported inflammation-related literatures of our observed 55 active TCM extracts, and concluded that: some TCMs have been comprehensively investigated for their antiinflammatory property and resulted in the discovery of diverse types of natural products, covering agrimonolide from Agrimonia pilosa (8) [24], (-)-nyasol from Anemarrhena asphodeloides (17) [25], alantolactone from Aucklandia lappa (31) [26], berberine from Coptis chinensis (53) [27], forsythiaside from Forsythia suspensa (79) [28], resveratrol from Polygonum cuspidatum (141) [29], etc. Beside these comprehensively investigated molecules, a great deal of constituents have been isolated from these active TCMs, and required further confirmation of their anti-inflammatory function. Meanwhile, a number of TCMs [e.g. Alisma orientalis (11), Equisetum hiemale (72), Cirsium setosum (43)] have not been investigated in the field of inflammation. Significantly, little research on the therapeutic effect of COPD has been performed, and thus these active TCMs are still being researched.
In the present screening assay, we only adopted two typical markers, QR and NO, to evaluate the potential of TCMs as oxidative stress and inflammation inhibitory agents. Based on our preliminary results, active TCM extracts could be subjected to further research in the field of phytochemistry and pharmacology, however, solid evidences on their biological functions are required before a systemic investigation [14]. With regard to the inhibition on oxidative stress, the levels of endogenous glutathione (GSH) and reactive ROS, as well as the protein level of key intracellular redox-balancing protein GCLM, are suggested to be detected to estimate the intracellular redox state and antioxidant capacity when exposed to TCM extracts [30][31][32]. Concerning the inhibition of inflammation by the active TCMs, the levels of crucial inflammatory mediators in the COPD pathology, including TNFα, LTB4, and IL-8, should be determined to confirm their anti-inflammatory potential [33].
Additionally, the pivotal regulators for oxidative stress and inflammation should be sufficiently investigated to verify action of mechanism of the active TCMs. The transcription factor Nrf2 plays a dominant role for regulating oxidative stress. It is ubiquitously expressed in human organs, particularly rich in lung, and counteracts oxidative injury through activating intracellular redox-balancing proteins (e.g. GCLM, GST, HO-1) and up-regulating endogenous antioxidants (e.g. GSH) [11,34]. NF-kB regulates the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules, and its inhibition therefore definitely relieves the inflammatory response of COPD [7,35]. It has also been verified that phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) are involved in the regulation of inflammatory response [36,37]. Hence, the further research on active TCM extracts and purified ingredients could focus on their action of mechanism on Nrf2, NF-kB, PI3K, and MAPK signaling pathways, as well as the cross talk between these pathways.

Conclusion
Although the present research indicates that some TCMs possessed inhibitory effects on inflammation and oxidative stress, further pharmacological investigations in vitro and in vivo models are warranted. Furthermore, bioassay-guided fractionations and identifications of active ingredients should be launched to help us illustrate the mechanism of these active species, and discover new lead molecules with unknown mechanisms and potent functions on oxidative stress-and inflammationrelated diseases, especially COPD. Accordingly, these results may give new insight in research and development of COPD therapeutic agents.

Additional file
Additional file 1: Figure S1. NADP(H): quinone oxidoreductase (QR) inducing effects of 38 bioactive TCM extracts in hepa 1c1c7 cells. The QR inducing effect was determined after 24h treatment of the hepa 1c1c7 cells in the presence or absence of tested TCMs. The data of the untreated control group was normalized as 1, and then the QR inducing activity of tested extracts was represented by the maximum folds of QR inducing activity (MQI) compared with the untreated control group. Sulforaphane (SF, 2.0 μM) was used as a positive control. The data are reported the means ± SD from three independent experiments. Figure  S2. Inhibitory effects on NO production of 55 bioactive TCM extracts in RAW 264.7 cells. The NO concentration in the RAW 264.7 cell culture media was determined through the Griess reaction 24 h after treated in the presence or absence of tested TCMs and lipopolysaccharides (LPS, 1.0 μg/mL). Didox (100 μM) was adopted as a positive control. The data are reported the means ± SD from three independent experiments. The maximum inhibition rates (MIRs) of NO production under the untoxic tested concentration were calculated by comparing the decreased NO concentration in TCM-treated group with that in LPS-stimulated group. Table S1.