New Iridoid Derivatives from the Fruits of Cornus officinalis and Their Neuroprotective Activities

Three previously undescribed iridoids, cornusfurals A–C, were isolated from the ethanolic extracts of fruits of Cornus officinalis. Their structures were elucidated by spectroscopic methods, including one-dimensional and two-dimensional nuclear magnetic resonance, ultraviolet spectroscopy, infrared spectroscopy, and mass spectrometry. The neuroprotective activity was evaluated by measuring corticosterone-induced damage in PC12 cells. The results showed that cornusfural B decreased corticosterone-induced PC12 cell damage compared with that in model cells.


Introduction
Cornus officinalis (Cornus) is a shrub distributed in Eastern Asia. The fruits of C. officinalis, named "shanzhuyu", have traditionally been used for nourishing the liver and kidneys for thousands of years [1]. Extracts or constituents of C. officinalis have been reported to possess neuroprotective, antidiabetic, anti-inflammatory, antioxidant, and cardiovascular effects [2][3][4][5][6]. Phytochemical investigation of the fruits of C. officinalis has demonstrated the presence of iridoid glycosides, flavonoids, terpenoids, and polyphenols [7][8][9][10][11]. Among these, iridoids were found to be the most important constituents and are considered to be active components of the extracts.
Depression is a common mental disorder characterized by persistent depression, which imposes a great burden on patients. Despite the high incidence of depression, the pathogenesis of this disease has not been fully elucidated [12]. The identification of antidepressant drugs from natural products is an important step in the development of novel therapeutics. PC12 cells, derived from the pheochromocytoma of the adrenal medulla in rats, are widely used in studies because of typical neuron characteristics [13]. Corticosterone-induced PC12 neuronal damage is useful as an in vitro experimental model for depression studies [14]. Loganin, the main iridoid glycoside from C. officinalis, has been reported to have antidepressive activity in recent studies [15,16]. Moreover, investigations of iridoid analogs from C. officinalis have revealed a number of biologically and structurally interesting compounds. In our previous pharmacology studies, we found that the macroporous resin 40% ethanol elution fraction of the ethanol extract of C. officinalis exhibited potent neuroprotective activity, and four new iridoid glycosides were isolated [17]. According to the HPLC spectroscopic characteristics, there are still many similar constituents which were suspected to have potential activities in this fraction. Thus, the 40% ethanol elution fraction was further evaluated in this study. Herein, the new iridoids were isolated, and their biological activities were discussed.
The relative stereochemistry of 3 was determined by the NOESY spectrum and coupling constants. The large coupling constant J 4,5 = 11.4 Hz between H-4 and H-5 indicated that H-4 was located in the α-oriented configuration, and J 3,4 = 8.6 Hz indicated that H-3 was β-oriented. In the NOESY spectrum, correlations between H-3 and H-5/H-9 indicated that H-5 and H-9 were both β-oriented. Moreover, NOESY correlations of H-4 with H-1 and of H-10 with H-1 and H-7 confirmed that H-1, H-10, and H-7 were all α-oriented. According to the biogenetic grounds in which the absolute configurations of H-5 and H-9 were 5S and 9R, respectively, and because the negative Cotton effects were at 234 and 281 nm, it can be said the compound 3 had an absolute configuration of (1R, 3R, 4R, 5S, 7S, 8S, and 9R). Analysis of the HSQC, 1 H-1 H COSY, and HMBC spectra led to the complete assignments of the proton and carbon signals in compound 3. Therefore, compound 3 was characterized as cornusfural C.

Neuroprotective Effects of Compounds 1-3
Corticosterone-induced PC12 neuronal damage is useful as an in vitro experimental model for depression studies [14]. The neuroprotective effects of compounds 1-3 were assessed (Table 2). Compound 2 exhibited neuroprotective activity compared with the model (complete medium with 500 µM corticosterone). Table 2. Neuroprotective effects of compounds 1-3 at a concentration of 10 −5 M (means ± SD, n = 6).

Discussion
As the results showed, only compound 2 exhibited neuroprotective activity in in vitro experiments. The structural analysis of these new compounds showed that compounds 1 and 3 had the same configuration of 3α, while compound 2 had a 3β configuration. The current data showed that the neuroprotective activity of these iridoid derivatives might be associated with the stereochemistry of C-3, and the 3β-substituents might be the active groups. However, the mechanism of the effect of C-3 configuration needs further confirmation by studying the structure-activity relationships of more similar compounds. Although there are many reports on the neuroprotective activities of iridoids from C. officinalis, the structure-activity relationship has not been reported [20][21][22]. Morroniside, the main active component of C. officinalis, was even evaluated in the form of a mixture in the neuroprotective study. Therefore, more efforts are suggested to explore the structure-activity relationships of iridoids from C. officinalis.

Plant Material
The fruits of C. officinalis were purchased from Tong-Ren- Tang

General Experimental Procedures
The HRESIMS data were generated on a Thermo QE Spectrometer (Thermo Scientific Inc., Waltham, MA, USA). The specific rotation data were obtained with a JASCO P-2000 polarimeter (JASCO Inc., Tokyo, Japan). IR spectra were recorded as KBr disks on a Nicolet Impact 400 FT-IR Spectrophotometer (Nicolet Instrument Inc., Madison, WI, USA). The circular dichroism spectra and UV data were recorded on a JASCO J-1005 circular dichroism spectrometer (JASCO Corporation, Tokyo, Japan). The one-and two-dimensional NMR spectra were recorded in CD 3 OD with TMS as the internal standard on Varian 500 MHz and Bruker AV500-III spectrometers (Bruker Corporation, Billerica, MA, USA). A Waters 2996 series was coupled with an RP-C18 column (Sunfire, 250 mm × 19 mm i.d.; Alltech Associates, Inc., Bannockburn, IL, USA) and a Waters 2998 dual-wavelength absorbance detector (Waters Corporation, Milford, MA, USA). Column chromatography was performed with silica gel (160-200 mesh; Qingdao Marin Chemical Inc., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Thin-layer chromatography (TLC) was used with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 8% H 2 SO 4 in 95% EtOH followed by heating.

Cell lines, Chemicals, and Biochemicals
PC12 cells (adrenal gland; pheochromocytoma) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). DMSO, corticosterone, and MTT were obtained from Sigma (St. Louis, MO, USA). The methanol used for HPLC isolation, which was of HPLC grade, was purchased from Fisher (Waltham, MA, USA). The solvents used to open the column isolation (Silica gel and Sephadex LH-20 gel column) in the study, such as chloroform, acetone, and methanol, were of ACS grade (Beijing, China).

Neuroprotection Bioassays
PC12 cells were cultured in RPMI 1640 medium supplemented with 1% streptomycin, 5% horse serum, and 5% fetal bovine serum. The cell suspensions were seeded in 96-well culture plates (2 × 10 4 cells/mL) and cultured for 24 h. Then, the medium was replaced with different fresh media, including the control (complete medium), the model (complete medium with 500 µM corticosterone), and the sample (the test compounds at a concentration of 10 µM), and the cells were cultured for 24 h. Next, 10 µL MTT (5 mg/mL) was added to each well. After incubation for 2 h, the medium was removed, and 100 µL DMSO was added to dissolve the formazan crystals generated by the reaction.
The optical density was then measured on a microplate reader (Molecular Devices, SFO, USA) at 570 nm. Cell viability was indicated as a percentage of the control.

Conclusions
Three new iridoids, that is, cornusfural A (1), cornusfural B (2), and cornusfural C (3), containing a furan ring, were isolated from the fruits of C. officinalis, and the neuroprotective effects of these compounds were evaluated. Compound 2 showed neuroprotective activities. The neuroprotective activities of iridoid glycosides have been evaluated through a variety of in vitro and in vivo studies [20][21][22]; however, iridoid aglycones isolated from C. officinalis have rarely been studied. Importantly, we identified one new iridoid aglycone exhibiting neuroprotective effects, thereby providing a potential new neuroprotective agent for further antidepressants research. According to the literature, iridoid glycosides are the main component in this plant, but the quality of iridoid aglycones are less. If the iridoid aglycones have significant biological activities, a large number of aglycones can be obtained through the hydrolysis of iridoid glycosides, which could be used for further animal experiments. This indicated that Cornus officinalis is a good resource of bioactive compounds and functional food.