An Exploration of Traditional Chinese Medicinal Plants with Anti-Inflammatory Activities

In a continuing effort to discover more anti-inflammatory medicinal plants in China, the anti-inflammatory activities of 101 extracts from different parts of 84 traditional medicinal plants were evaluated by a panel of in vitro and in vivo assays. Nuclear factor-kappa B (NF-κB) inhibitory effects were determined by luciferase assay in stably transfected Hela cells. Cytotoxic activities were assessed using the MTT assay. Inhibitory effects on LPS-induced nitric oxide production and proinflammatory mediators were assessed by Griess reaction and Real-Time PCR analysis, respectively. In vivo anti-inflammatory activities were examined by xylene-induced mice ear edema model. In total, 22 extracts showed promising NF-κB inhibitory effects whereas 9 of them did not affect the cell viability. The 9 hit extracts were active in at least one of the subsequently performed in vitro pharmacological test systems. The extract from Hemerocallis minor (root) was selected to perform the in vivo study because it demonstrated significant suppressive effects in all the in vitro assays. Results showed that the extract of Hemerocallis minor (Root) was able to alleviate ear edema effectively in xylene-induced mice ear edema mode. Collectively, our study provides evidence for the potential anti-inflammatory effects of the medicinal plants traditionally used in China. Further phytochemical and pharmacological studies remain to be clarified.


Introduction
Inflammation is an adaptive response of body tissues to external challenge or cellular injury. It is generally thought that inflammation is a beneficial host response defense system, but it can become harmful if dysregulated [1]. The complex mediators including proinflammatory and cytotoxic cytokines, growth factors, bioactive lipids, and nitric oxide in the uncontrolled inflammation reaction can sustain or induce a pathologic process involved in different diseases, such as allergy, sepsis, arthritis, the metabolic syndrome, autoimmune diseases, and cancer [2]. The inhibition of inflammatory mediators on the transcriptional level has emerged as a promising approach for the development of anti-inflammatory drugs [3]. Nuclear transcription factorkappa B (NF-B) plays a key role among the involved transcription factors to regulate various genes involved in immune and acute phase inflammatory responses. The activation of NF-B induces the transcriptions of iNOS, COX-2, TNF-, and proinflammatory cytokines [4,5]. Therefore, the NF-B signaling pathway has long been considered the "holy grail" as a target for anti-inflammatory drug [3].
Traditional Chinese medicinal plants work well for many diseases and have been used for centuries in China. Its increasing use in recent years is evidence of a public interest in having alternatives to conventional medicine [6]. A variety of medicinal plants have been reported to have antiinflammatory properties. However, a comprehensive investigation on exploring more anti-inflammatory medicinal plants and their action mode is lacking. In a continuing effort to discover more medicinal plants with anti-inflammatory effects in China, 101 extracts from different parts of 84 TCMs (Table 1) were screened for their potential inhibitory activities on NF-B activation. In order to further determine their antiinflammatory activity, the inhibition of the proinflammatory cytokines (TNF-, IL-6, and IL-1 ) or the NO production induced in LPS-stimulated murine RAW 264.7 was investigated. Mouse ear edema assay was used to verify the in vivo anti-inflammatory activity of the selected plant extracts.   The cells were cultured in DMEM medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 g/mL) in a 37 ∘ C and 5% CO 2 incubator. The human cervical carcinoma cell line Hela was transfected with pNF-B-Luc reporter plasmid, which contains five copies of NF-B binding sequence (Stratagene, La Jolla, CA). The transfected cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

NF-B Transactivation
Assay. The examination of NF-B activity was performed by a modification of methods as described previously [7]. HeLa cells expressing NF-B-Luciferase were seeded in 96-well plates at a density of 1.5 × 10 4 cells per well. After cells became adherent, they were serum-starved in DMEM without phenol red overnight before screening assay. On the next morning, cells were pretreated with the investigated samples, positive control (parthenolide, 10 M), or solvent vehicle (0.1% DMSO in culture medium) for 1 h and stimulated with 5 ng/mL TNFfor 4 h [7]. Afterwards, the cells were lysed with luciferase lysis buffer and the luminescence of the firefly luciferase was measured with the microplate reader (FlexStation 3; Molecular Devices, Silicon Valley, CA, USA).
2.6. The MTT Assay for Cell Viability. Cells were plated overnight in 96-well plate (5 × 10 3 /well). Then, the cells were treated with 0.1% DMSO (vehicle control) or the plant extract samples with various concentrations for 24 h. Following another 4 h incubation with 20 L of MTT solution (5 mg/mL), the culture supernatant was discarded and 100 L of DMSO was added to each well to dissolve the formazan crystal. OD absorbance was recorded at 490 nm with the microplate reader (FlexStation 3, Molecular Devices, Silicon Valley, CA, USA).

2.7.
Determination of NO Production. RAW 264.7 macrophages were plated in 48-well plate (1.5 × 10 5 /well). After cells became adherent, they were pretreated with the test samples, positive control (parthenolide, 10 M), or solvent vehicle (0.1% DMSO in culture medium) for 1 h and then stimulated with 100 ng/mL LPS for additional 12 h. The nitrite accumulated in culture medium was measured as an indicator of NO production based on Griess reaction. Briefly, 50 L of cell culture medium was mixed with 50 L of Griess reagent (1% sulfanilamide in 5% phosphoric acid, 1%naphthylamide in H 2 O) in a 96-well plate, incubated at room temperature for 10 min, and then measured at 540 nm using a microplate reader (FlexStation 3, Molecular Devices, Silicon Valley, CA, USA) [8].

Xylene-Induced Ear Edema in Mice.
Xylene-induced ear edema in mice was initiated according to a previously described method [10]. Briefly, the male mice were randomly divided into four groups ( = 6/group). Mice were intraperitoneally (i.p.) given twice vehicle, dexamethasone (3 mg/kg), and two doses (250 and 500 mg/kg) of the test sample 12 and 1 h before xylene treatment. After the final administration, ear edema was induced by applying 20 L of xylene to the outer and inner surface of the right ear. The left ear served as a control. Mice were sacrificed after 1 h by carbon dioxide suffocation, and both ears were removed. 7 mm diameter ear punch biopsies were collected by using a metal punch and  Table 1 for full names). The produced luciferase activity was assayed as described in Materials and Methods. Data points represent mean ± SEM; = 3, * < 0.05 (ANOVA) compared with negative control.
weighed. Edema (Δ ) was defined as the difference in weight (mg) between the right and the left ear sample Δ = R − L . The extent of the edema was expressed by percentage of increase in the ear tissue weight (%), using the following formula [10]: percentage of edema weight (%) = ( R − L ) × 100/ L .

Statistical Analysis.
The results are expressed as mean ± SEM. Statistical differences were compared with one-way ANOVA. values < 0.05 were considered to be significant. All statistical tests were carried out using the GraphPad Software (California, USA).

Inhibitory Effects of the Chinese Medicinal Plant Extracts on TNF--Induced NF-B Activation.
In a primary screening, 101 different extracts from 84 plants were assessed for their potential to inhibit TNF-induced NF-B activation in Hela cells at the concentrations of 50, 25, and 12.5 g/mL. As shown in Figure 1 (Figure 1(b)). These extracts were including 6-Alangium chinense ssp. triangulare (Stem), 26-Capparis masaikai (Fruit),

Effects of the Extracts on Cell Viability.
In order to exclude the potential influence of cytotoxicity, the effects of these 22 effective plant extracts at the concentrations of 50, 25, and 12.5 g/mL along with parthenolide (2.5∽15 M) on proliferation of Hela cells and RAW 264.7 cells were evaluated using MTT assay. As shown in Table 2 (Herb). Therefore, the remaining nine plant extracts were selected for further investigation.    Table 1 for full names). NO levels were determined with Griess reagent. Data points represent mean ± SEM; = 3, * < 0.05 (ANOVA) compared with negative control.

Inhibitory Effects of the Extracts on LPS-Induced NO
showed significant inhibitory effects at both low (12.5 g/mL) and high concentrations (50 g/mL).  Figures 3(b) and 3(c)). Among these effective extracts, the extract 62-Hemerocallis minor (Root) was observed to suppress all the proinflammatory cytokines TNF-, IL-6, and IL-1 .

Inhibitory Effects of 62-Hemerocallis Minor (Root) on
Xylene-Induced Mice Ear Edema. Hemerocallis minor, which was effective in all the above in vitro assays, was selected to further evaluate the in vivo anti-inflammatory effect. Xyleneinduced mice ear edema model was used in this study. Xylene can stimulate local inflammation and then induce edema of tissues. Edema was determined by the increase in ear weight due to an inflammatory challenge. Figure 4 exhibited that Hemerocallis minor extract was effectively inhibiting ear edema in a dose-dependent manner. Compared with the control group, Hemerocallis minor extract at the doses of 250 mg/kg and 500 mg/kg significantly alleviated xyleneinduced edema, and the percentage of edema weight was 69% and 42%, respectively. Similarly, dexamethasone (3 mg/kg) led to a percentage of edema weight value of 15%. The results indicated that the Hemerocallis minor extract has a significant anti-acute-inflammation activity.

Discussion
Traditional Chinese medicinal plants play a significant role in drug discovery and development. Many have provided the foundation for modern pharmaceuticals and drug leads [11,12]. In this study, we collected 101 extracts from 84 species of traditional medicinal plants to assess their potential anti-inflammatory effects in a panel of in vitro and in vivo assays. The purpose of this study is to provide more evidence for the plants which have been recorded with uses linked to anti-inflammatory effects and explore more species of undiscovered medicinal plants with anti-inflammatory properties. NF-B controls the expression of genes involved in a number of physiological responses, including immune and inflammatory responses [13]. Given NF-B's importance in human diseases related to inflammatory disorders and the fact that many anti-inflammatory drugs interfere with NF-B signaling, we established a screening assay specific for NF-B activity to discover medicinal plants targeting NF-B signal pathway [14]. Twenty-two out of 101 extracts exhibit significant inhibitory effects on NF-B transactivation at concentrations of 25 and 50 g/mL while ten among them are effective at even lower concentration (12.5 g/mL). Among these 22 extracts, Paris polyphylla, Liriope spicata, and Cleome gynandra have been historically used in China and have been exploited from the chemical components to biological activities including anti-inflammation and anticancer [15][16][17]. Limited information regarding other species of these medicinal plants has been reported. Our study provides the first evidence for their potential anti-inflammatory effects targeting NF-B signaling pathway. In the present study, we show that the extracts from different species even from the same genus lead to different pharmacological responses. Different clinical application of extracts from the different species of the same genus or different parts of the same plant may be explained by differences in pharmacological responses induced by them.
Macrophage activation by LPS leads to the phosphorylation, ubiquitination, and subsequent degradation of the inhibitory B (I B). This induced degradation of I Bs allows NF-B to freely enter the nucleus and to induce the production of NO and transcriptions of proinflammatory cytokines including IL-1 , IL-6, and TNF-, which are important for the inflammatory and immune response [18,19]. The pharmacological reduction of LPS-inducible inflammatory mediators is regarded as one of the essential conditions to alleviate acute and chronic inflammations caused by activation of macrophages [20]. Therefore, LPS-stimulated macrophage cells provide us with an excellent model for investigating inflammatory response and subsequently for screening anti-inflammatory drugs and their underlying   Table 1 for full names). The mRNA levels of TNF-(a), IL-6 (b), and IL-1 (c) were determined by quantitative Real-Time PCR. Data points represent mean ± SEM; = 3, * < 0.05 (ANOVA) compared with negative control. mechanisms [21,22]. In the present study, the hit extracts found in the preliminary screening with NF-B assay and without cellular toxicities were further subjected to detect the effects on the level of NO production and TNF-, IL-1 , and IL-6 mRNA expressions in LPS-stimulated RAW 264.7 macrophages. The nine hit extracts were active in at least one of the subsequently performed in vitro pharmacological test systems, which were including NO production and cytokines mRNA expressions. Hemerocallis minor extract was found to be effective in all in vitro assays and not to affect the cell viability. The extract from Hemerocallis minor also inhibited xylene-induced mice ear edema. Hemerocallis minor, frequently referred to as yellow lily, has been applied in China as food and folk remedy, particularly for heat-clearing and breast mastitis [21,22]. To our knowledge, it is the first report that Hemerocallis minor may possess possible therapeutic anti-inflammatory properties. Our results might provide a rationale for the traditional use of this plant in China and make it a highly interesting medicinal plant, having the potential to be further explored as anti-inflammatory agents. However further phytochemical and pharmacological studies on Hemerocallis minor remain to be performed.

Conclusions
In summary, the present study demonstrates that extracts from nine medicinal plants exhibit properties of antiinflammatory activity by either suppressing NO production or the major proinflammatory cytokines TNF-, IL-1 , and IL-6. Hemerocallis minor is the particularly interesting one which is effective in all the in vitro and in vivo assays and has the potential to be developed as anti-inflammatory therapeutics or preventatives.