Anti-Inflammatory Triterpene Glycosides from the Roots of Ilex dunniana Levl

A new triterpene glycoside ilexdunnoside A (1) and a new sulfated triterpene derivative ilexdunnoside B (2), together with five known analogues 3–7 were isolated from the roots of Ilex dunniana Levl. The structures were established by NMR spectroscopic analysis and acid hydrolysis. Results of an in vivo study of the biological activity showed that 75% ethanol and n-butanol extracts of the plant displayed anti-inflammatory activities against ear edema in mice, with inhibition rates of 23.5% and 37.5%, respectively, at a dose of 50 mg/kg. Furthermore, Compounds 1, 2 and 3 exhibited moderate indirect inhibitory effects on lipopolysaccharide-induced NO production in BV2 microglial cells in vitro, with IC50 values of 11.60, 12.30 and 9.70 μM, respectively.


Introduction
Inflammation is an important pathophysiological factor of many diseases, such as pneumonia, intestinal catarrh, rheumatoid arthritis, infectious diseases and so on. Plants from the genus Ilex (Aquifoliaceae) have been used in the clinic for clearing away inflammation, relieving cough, eliminating phlegm, and so on [1]. In order to search for anti-inflammatory active ingredients, we have performed systematical chemical and pharmacological investigations of Ilex dunniana Levl, an evergreen shrub or dungarunga widely distributed to the south of the Qinling mountains and Yangtze Valley in China. Herein we report the isolation and structural elucidation of two new triterpene glycosides, ilexdunnoside A (1) and ilexdunnoside B (2) (Figure 1), together with five known analogues 3-7 from the roots of Ilex dunniana Levl, as well as the anti-inflammatory activity of all the isolated compounds.
Microglial cells, which are regarded as the most important immune cells in the central nervous system (CNS), are activated by brain injuries. Following activation by bacterial toxins, microglial cells secrete a wide range of inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and prostanoids [7]. Nitric oxide (NO) plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent [8]. NO is a physiological messenger that triggers a variety of actions in several systems [9]. It can modulate the release of various inflammatory mediators from a wide range of cells participating in inflammatory responses. Because of its anti-inflammatory properties and cytoprotective effects, adjunctive NO has been considered a plausible means for improving the anti-inflammatory activity. In recent years, NO-releasing drugs have been developed, usually as derivatives of other drugs, which exhibit very powerful anti-inflammatory effects [10][11][12][13].
In order to identify the biological activity, an in vivo anti-inflammatory model were used, the results showed that the 75% ethanol and n-butanol extracts of the plant exhibited anti-inflammatory activities against ear edema in vivo, with inhibition rates of 23.5% and 37.5%, respectively, at a dose of 50 mg/kg (as shown in Table 2). Furthermore, as shown in Table 3, in vitro results showed that compounds 1-7 obtained from the n-butanol extract were tested for their inhibitory effects on lipopolysaccharide-induced NO production in mouse microglial cells. Compounds 1-3 displayed moderate indirect anti-inflammatory activity, in which, compound 3 showed cytotoxic activity against microglial cell in vitro (Table 4), so the indirect anti-inflammatory activity of 3 partly may be caused by the cytotoxic activity. The other compounds exhibited weak activities for the inhibition of NO production, and did not show cytotoxic activity.

General Information
Optical rotations were obtained on a P2000 automatic digital polarimeter (JASCO, Tokyo, Japan). NMR spectra were measured on a Mercury-400 spectrometer (Varian, Palo Alto, CA, USA). HRESIMS spectra were acquired with an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent, Santa Clara, CA, USA). The MPLC system (Biotage, Uppsala, Sweden) was equipped with an YMC-Pack ODS-A column (500 mm × 50 mm, 50 µm, YMC, Tokyo, Japan). Column chromatography was conducted with MCI GEL CHP20P resin (75-150 µm, Mitsubishi, Tokyo, Japan) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). TLC was carried out with glass precoated with silica gel GF 254 . BV2 microglial cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). A microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used for the cytotoxicity assays.

Plant Material
The roots of Ilex dunniana Levl were collected in Mount Emei, Sichun Province, People's Republic of China, in August 2015, and were identified by Associate Prof. Xiao-Zhong Chen from Heilongjiang University of Chinese Medicine. A voucher specimen (ID-g-20150828) was deposited at the herbarium of the Jiamusi College, Heilongjiang University of Chinese Medicine.

Acid Hydrolysis of Saponins
Each saponin (1.0 mg) was refluxed with 2 N aqueous CF 3 COOH (10 mL) at 100 • C for 2 h. The reaction mixture was partitioned between H 2 O (10 mL) and CH 2 Cl 2 (3 × 4 mL). The CH 2 Cl 2 layer were washed with H 2 O and evaporated to obtain the aglycone. After evaporation in vacuo and removing the acid by adding MeOH, the aqueous extracts were analyzed by GC. Thus, the absolute configuration of the crude sugar was demonstrated as described in the previous papers [14,15].

Detection of the Sulfate Group
Each compound (2-3 mg) was refluxed with 10% HCl (4 mL) for 4 h. After that the reaction mixture was extracted with Et 2 O. Then, an aliquot of the aqueous layer of each sample was treated with 70% BaCl 2 to yield a white precipitate (BaSO 4 ) [16].

In Vitro Anti-Inflammatory Activity Assays
BV2 microglial cells were maintained in RPMI1640 medium at 37 • C in 5% CO 2 . The cells were placed in 48-well plates and preincubated for 24 h, treated with tested triterpene glycosides dissolved in DMSO at various final concentrations (5.0, 10.0, 20.0, 40.0, 80.0 µM) in triplicate for 1 h, and continuously incubated with LPS (1 µg/mL) for 24 h. Dexamethasone (10 −6 M) was used as the positive control. From each well, the supernatants (100 µL) were mixed with an equal amount of Griess reagent at room temperature for 20 min. The concentration of NO 2 − was measured for the amount of NO by a microplate reader at 570 nm, using sodium nitrite as the standard to calculate the concentration of the nitrite [17,18].

Cytotoxicity Assays
Cell viabilities were measured using the MTT assay. Briefly, BV2 microglial cells were seeded in 96-well plates at concentrations of 1 × 10 5 cells per well. After incubation for 2 h, the cells were incubated with compounds for 24 h and then washed with PBS three times. Following the washing step, 200 µL of RPMI 1640 medium containing 0.5 mg/mL MTT were added to each well, the cells were then incubated at 37 • C for another 4 h. Finally, the culture medium was removed, and the formazan crystal was dissolved by adding 150 µL of DMSO. Absorbances at 570 nm were measured using a microplate reader.

In Vivo Anti-Inflammatory Assays
On the basis of the inhibition of croton oil-induced ear edema in mice, the topical anti-inflammatory activity of the test compounds was evaluated. Animal experiments were performed in accordance to the Institutional Guidelines for Animal Care and Use of the Chinese Academy of Medical Sciences and Peking Union Medical College. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Chinese Academy of Medical Sciences and Peking Union Medical College (permit pumber: 002973).The croton oil (Croton tiglium L., seed oil) was purchased from Sigma-Aldrich (St. Louis, MO, USA). In ICR male mice (18-20 g, n = 10 per group), croton oil (0.4 mg) was mixed with acetone (1 mL) to be applied to the left ear (10 µL each side) topically to induce ear edema. The animals were administered with candidate extracts (50 mg/kg) for 1 h, followed by treatment with croton oil for 4 h. After the application of croton oil, the mice were euthanized using sodium pentobarbital, and ear tissues (8 mm diameter punches) were collected for the measurement of the weight of ear patches (left and right). Based on the weight difference between two plugs (8 mm diameter) of the treated (left) and untreated (right) ears, the edematous response was measured. The percentage reduction in edema in treated mice compared with control mice is calculated for the expression of anti-inflammatory activity. Dexamethasone was used as the positive control [18,19].

Conclusions
The results of croton-oil ear inflammation in mice showed that 75% ethanol and n-butanol extracts of powdered root of Ilex dunniana Levl exerted anti-inflammatory activity in vivo against ear edema. Bioactivity-guided isolation of the n-butanol extract yielded two new triterpene glycoside 1 and 2 together with five known ones 3-7. Compounds 1-7 belong to the triterpene glycosides, of which compounds 4 and 5 belong to sulfated triterpene glycosides. Futhermore, in vitro anti-inflammatory activity showed that compounds 1, 2 and 3 obtained from the n-butanol extract exhibited moderate indirect anti-inflammatory activity on LPS-induced proinflammatory factors production in BV2 microglial cells.