Triterpenes and Aromatic Meroterpenoids with Antioxidant Activity and Neuroprotective Effects from Ganoderma lucidum

Reactive oxygen/nitrogen species generated in the human body can cause oxidative damage associated with many degenerative diseases such as atherosclerosis, dementia, coronary heart diseases, aging, and cancer. There is a great interest in developing new antioxidants from Ganoderma fungus due to its low toxicity. As part of our ongoing search for antioxidative constituents from the fruiting bodies of Ganoderma lucidum, the chemical constituents were investigated and seven secondary metabolites, including one new lanostane triterpene (1), two known aromatic meroterpenoids (6–7), and four known triterpenes (2–5), were isolated by a series of chromatographic methods. The structures of the seven compounds were elucidated by spectroscopic techniques. The isolated compounds were tested in vitro for antioxidant potencies and neuroprotective activities against H2O2 and aged Aβ-induced cell death in SH-SY5Y cells. As a result, compounds 1, 6, and 7 exhibited potent antioxidant and neuroprotective activities. Additionally, all isolated compounds were tested for radical scavenging activities. Compounds 6 and 7 showed the comparable free radical scavenging activities with the standard drug in both ABTS (2, 2’-azobis (3-ethylbenzothiazole-6-sulfonaic acid)) and ORAC (oxygen radical absorbance capacity) experiments. The results from this study suggested that G. lucidum and its metabolites (especially the meroterpenoids) may be potential functional food ingredients for the antioxidation and prevention of neurogenerative diseases.


Introduction
Reactive oxygen species (ROS) can cause extensive damage to DNA, proteins, and lipids. This could be the fundamental cause of aging and many other important diseases such as cancer, cardiovascular diseases, and neurogeneration [1]. Deposition of different physicochemical forms of amyloid β peptide (Aβ) constitutes a major neuropathological hallmark of Alzheimer's disease [2]. Several lines of evidence suggest that Aβ may exert its pathological effects in the central nervous system at least in part through ROS-mediated mechanisms [3,4]. Owing to the increased demand for and importance of antioxidants in day-to-day life, the search for effective, nontoxic, natural compounds with antioxidant activity has increasingly become a matter of interest.

ABTS .+ Radical Scavenging Assay
The ABTS .+ assay is an excellent tool for determining the antioxidant activity of hydrogen-donating antioxidants and of chain-breaking antioxidants [27]. In the present study, the ability of test samples to scavenge ABTS was assessed on the basis of their EC 50 values, defined above as an effective concentration at which the ABTS radical was scavenged by 50%. EC 50 values of the isolates and trolox (used as a reference compound) are given in Table 2. A low EC 50 value indicates strong antioxidant activity in a tested sample. Among these compounds, lingzhine E (6) and lingzhine F (7) showed comparable ABTS .+ scavenging effects with EC 50 values of 0.59 ± 0.15 and 0.27 ± 0.05 mM, respectively, which was close to the positive control (trolox) with an EC 50 value of 0.42 ± 0.03 mM. ORAC is also a widely used in vitro antioxidant capacity assay [28]. It is a chemical antioxidant assay that is based on the inhibition of the peroxyl-radical induced oxidation initiated by the thermal decomposition of 2, 2'-azobis-(2-amidinopropane) dihydrochloride (AAPH). The antioxidant capacity of the isolates was also measured by ORAC assay and the potency of the natural compound was compared with that of the positive control, quercetin, which is well known for its use as an antioxidant. The ORAC results are expressed as trolox equivalent [4] and shown in Table 2. A high ORAC value indicates strong antioxidant activity in a tested sample. Among the seven compounds, the more potent radical scavenger was lingzhine E (7) (7.24 ± 0.27 µmol TE/µmol) with a similar value to quercetin (7.78 ± 0.27 µmol TE/µmol), followed by lingzhine (6) (5.42 ± 0.20 µmol TE/µmol). This result was consistent with those of the ABTS assay.

Antioxidant Effects on H 2 O 2 -Induced ROS Production in SH-SY5Y Cells
The in vitro antioxidant assays, based on chemical reactions, are easy to operate and widely used to evaluate antioxidant capacities. However, their main disadvantage is that they do not reflect cellular physiological conditions. Therefore, a cell-based antioxidant activity assay to evaluate the antioxidant potential is necessary. ROS, such as the superoxide anion radical, hydrogen peroxide, and hydroxyl radical, are generated during many physiological and pathological processes and reported to function in an array of intracellular signaling cascades [29]. H 2 O 2 is used extensively as an inducer of oxidative stress in vitro because its cellular actions and pathophysiological roles have been well studied [30]. SH-SY5Y human neuroblastoma cells are highly sensitive to oxidative stressors such as H 2 O 2 . The protective activity of compounds 1-7 against H 2 O 2 -induced oxidative stress was evaluated on SH-SY5Y cells at the concentration of 40 µM. After incubation with 200 µM H 2 O 2 for 12 h, only 41.54 ± 2.04% of cultured cells survived. Compounds 1, 6, and 7 (10-40 µM) could protect H 2 O 2 -induced cell damage in a dose-dependent relationship, and the survival rates at 40 µM were 62.68 ± 2.81%, 72.57 ± 2.12%, and 78.96 ± 1.86%, respectively. Luteolin is used as positive control with a survival rate of 73.59 ± 2.19% at 40 µM ( Figure 2). 2',-7'-dichlorofluorescin diacetate (DCFH-DA) is one of the most widely used techniques for directly measuring the redox state of a cell. When SH-SY5Y cells were challenged with 200 µM H 2 O 2 , ROS were generated over twofold compared to the unchallenged control, while the treatment with compounds 1, 6, and 7 dose-dependently decreased the H 2 O 2 -mediated ROS formation ( Figure 3). These results imply that methyl ganoderate G1 (1), lingzhine E (6), and lingzhine F (7) may have an ability to directly scavenger ROS and/or free radicals.  The columns represent % of change in fluorescence intensity with respect to H 2 O 2 -treated cells. All data were expressed as mean ± S.D., n = 4. **p < 0.01, *p < 0.05 compared with H 2 O 2 -treated control.

Protection of SH-SY5Y Cells Against Aβ-Induced Damage
There is abundant evidence suggesting that an excess of Aβ, which aggregates into toxic fibrillar deposits, plays a central role in the etiology of Alzheimer's disease (AD) [31]. In support of this hypothesis, numerous in vitro and in vivo studies have reported on the neurotoxic effects of Aβ-related fragments in neurons derived from regions severely affected in AD [32]. Although the precise mechanisms mediating the toxic properties of Aβ have yet to be extensively understood, it has been proposed that they are associated with oxidative stress-dependent apoptosis [33]. Hence, the capacity of methyl ganoderate G1 (1), lingzhine E (6), and lingzhine F (7) in protecting neuroblastoma SH-SY5Y cells against Aβ-induced damage was examined. Aβ [25][26][27][28][29][30][31][32][33][34][35] , a synthetic peptide that possesses most of the physical and biological properties of full-length Aβ and is often used to study the neuroprotective effects of various compounds, was predicted to modulate Aβ toxicity in vitro [34]. In this study, the neuroprotective activity of compounds 1, 6, and 7 against Aβ-induced oxidative stress was evaluated on SH-SY5Y cells. After incubation with 25 µM aged Aβ 25

Inhibition of ROS Generation Induced by Aβ 25-35 in SH-SY5Y Cells
The neurotoxicity of Aβ has been reported to be mediated with oxygen free radicals and attenuated by antioxidants and free radical scavengers. Many reports have demonstrated the involvement of ROS formation in Aβ-induced neurotoxicity [35]. We investigated whether compounds 1, 6, and 7 affect ROS formation by Aβ using DCFH-DA probe staining. As shown in Figure 5, exposure to Aβ induced an elevation of the intracellular ROS levels. Treatment with compounds 1, 6, and 7 ameliorated the intracellular ROS elevation. These results indicated that methyl ganoderate G1 (1), lingzhine E (6), and lingzhine F (7) have the ability to scavenge Aβ-induced ROS increase.

Discussion
Since 2000, many structurally diverse aromatic meroterpenoids were found from different species of Ganoderma and have attracted the interest of chemists and pharmacologists [11,15,26]. In the present study, two aromatic meroterpenoids (6-7) were isolated and identified from the fruiting bodies of G. lucidum. Our previous and present research exhibited that aromatic meroterpenoids had excellent in vitro antioxidant effects [11]. Hence, the antioxidant potencies of the two compounds were first investigated employing various in vitro systems. The results showed that lingzhine E (6) and lingzhine F (7) had strong antioxidant activities. Moreover, the lanostanoid triterpenes obtained from the Ganoderma family have been reported to show strong antioxidant activities and neuroprotective activities [17,18]. Hence, the isolated compounds were evaluated in vitro for their antioxidant potencies and neuroprotective activities against H 2 O 2 -and Aβ-induced cell death in SH-SY5Y cells. Methyl ganoderate (1), lingzhine E (6), and lingzhine F (7) possessed significant neuroprotective activities. These results will lay the foundation for further in vivo bioactive research and provide a theoretical basis to the application of G. lucidum on antineurological disease.

Materials and Reagent
The

Apparatus and Chemicals
1D and 2D NMR spectra were recorded in CDCl 3 using a Bruker AVANCE III-600 spectrometer (Bruker Corp., Switzerland), and tetramethyl silane (TMS) was used as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to TMS. Optical rotations were obtained with a Jasco P-1020 polarimeter (Japan). HRESIMS spectral data were recorded on an ultra performance liquid chromatography-time of flight-mass spectrometry (UPLC-TOF-MS) Waters, MA, USA), carried out on Waters Acquity UPLC columns at 35 • C (Zorbax eclipse plus C18: 2.1 mm × 50 mm, 1.8 µm). Column chromatography was conducted using silica gel (Qingdao Marine Chemistry Company, China) and Sephadex LH-20 (Pharmacia, Sweden). Activity data were recorded on a microplate reader (Infinite M200 PRO, Tecan, Sweden).

ABTS Radical Cation Scavenging Activity
ABTS radical cation scavenging activity was assayed according to instructions by the Beyotime Institute of Biotechnology. The stock solutions included ABTS solution and oxidant solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 16 h at room temperature in the dark. The solution was then diluted by mixing 1 mL working solution with 90 ml 80% ethanol in order to obtain an absorbance of 0.7 ± 0.05 at 734 nm. A fresh ABTS solution was prepared for each assay. Samples (10 µL) with a concentration range of 0.05-3.00 mM were mixed with 200 µL of fresh ABTS solution and the mixture was left at room temperature for 6 min. The absorbance was then measured at 734 nm. Trolox was used as a reference compound. The radical scavenging activity of each sample was expressed in terms of the EC 50 (the effective concentration at which ABTS .+ radicals were scavenged by 50%), which was calculated from the log-dose inhibition curve.

Oxygen Radical Absorbance Capacity Assay (ORAC Assay)
The ORAC assay was carried out based on the previously described procedure with slight modification [36]. In brief, the sample/blank (175 µL) was dissolved in phosphate buffer saline (PBS) at the concentration of 160 µg/mL at pH 7.4. The trolox standard was prepared in serial dilutions starting from 75 mM. Standard 96-well black microplates were used for the assay, and 25 µL each of the samples, standard (trolox), blank (solvent/PBS), or positive control (quercetin) were added to the wells. Fluorescent sodium salt solution was added at 150 µL per well, followed by incubation at 37 • C for 45 min. The total volume of each well was made up to 200 µL by adding 2, 20-azobis-(2-amidinopropane) dihydrochloride (AAPH) solution. Fluorescence value was recorded at 37 • C (excitation at 485 nm, emission at 535 nm) using a fluorescence spectrophotometer (Infinite M200 PRO) equipped with an automatic thermostatic autocell holder. Data were collected every 2 min for 2 h and the data analysis was subsequently done by calculating the differences of AUC between the blank and the sample. Results were expressed as µmol of Trolox Equivalent/µmol of pure compound.

Cell Culture
The human neuroblastoma cell line (SH-SY5Y) was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. SH-SY5Y cells were cultured in MEM/F12 (1:1) medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 • C and 5% CO2.

Measurement of Intracellular ROS Level
The generation of intracellular reactive oxygen species (ROS) was measured using the DCFH-DA method [37]. Briefly, SH-SY5Y cells were cultured in 96-well plates (1 × 10 4 cells per well). Drug treatment, H 2 O 2 stimulation, and Aβ 25-35 stimulation were carried out as described in Sections 4.4.4 and 4.4.5. The cells were washed with warm PBS and were incubated with DCFH-DA for 30 min at 37 • C in darkness. After the cells were washed twice with PBS, the fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The level of intracellular ROS was expressed as a percentage of value against the nontreated control group.

Statistical Analysis
Data analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, USA). All results are presented as the mean ± S.D. and a two-tailed test or a one-way analysis of variance (ANOVA) was used to determine the statistical significance. Differences were considered to be significant for p-value <0.05.

Conflicts of Interest:
The authors declare no conflict of interest.