Antioxidant activity against H2O2-induced cytotoxicity of the ethanol extract and compounds from Pyrola decorate leaves

Abstract Context: The leaves of Pyrola decorate H. Andr (Pyrolaceae), known as Luxiancao, have long been used for treating kidney deficiency, gastric haemorrhage and rheumatic arthritic diseases in traditional Chinese medicine. Objective: The phytochemicals and antioxidant capacities in vitro of P. decorate leaves were investigated. Materials and methods: Ethanol, petroleum ether, acetidin, n-butyl alcohol and aqueous extracts of Pyrola decorate leaves were prepared by solvent sequential process, and then isolated and purified to obtain phytochemicals. Cell viability was measured by MTT assay. PC12 cells were pretreated for 24 h with different extractions of P. decorate leaves at concentrations of 0.1, 0.5, 1, 5 and 10 mg/mL, then H2O2 of 0.4 mM was added in all samples for an additional 2 h. The antioxidant capacities of betulin, ursolic acid and monotropein were determined in PC12 cells against H2O2 induced cytotoxicity in vitro as well. Results: Nine compounds (1–9) were isolated and structurally determined by spectroscopic methods, especially 2D NMR analyses. Ethanol extract treated groups showed inhibitory activity with IC50 value of 10.83 mg/mL. Betulin, ursolic acid and monotropein were isolated from P. decorate, and demonstrated with IC50 values of 6.88, 6.15 and 6.13 μg/mL, respectively. Discussion and conclusions: In conclusion, Pyrola decorate is a potential antioxidative natural plant and worth testing for further pharmacological investigation in the treatment of oxidative stress related neurological disease.


Introduction
Pyrola herb, with about 30 species, has the widest distribution occurring in the northern hemisphere in temperate and cold temperate regions around the world, while 27 species are distributed mainly in the west and northeast part of China . As a Yang-tonic agent, Pyrola decorate H. Andr (Pyrolaceae), also known as Luxiancao or Luticao, has been extensively used as a valuable tonifying agent for more than 2000 years in China. It has been included in Shengnong's herbal classic and came out of the top grade lists. According to traditional Chinese medicine (TCM) theory, tonic herbs have been used for various patterns of body deficiency and anti-aging (Ho et al. 2009). P. decorate has been used to nourish 'kidney' and strengthen 'bone and muscle' for long history. Therefore, it has been used as treatment for kidney deficiency, gastric haemorrhage and rheumatic arthritic diseases in Chinese medicine .
As a tonifying agent, P. decorate is an important component in many Chinese prescription formulas for aging-associated diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and other neurodegenerative diseases (Luo et al. 2004). Leaves of Pyrola are rich in a variety of active components, such as triterpenoids, flavones polysaccharides, phenolics glycosides, quinines and tannic acid (Ptitsyn et al. 2011;Zhang et al. 2012;Kirillov et al. 2015). However, detailed pharmacological evidence on Pyrola decorate need to be further elucidated. Our previous studies have shown the different polar solvent extracts from the leaves of P. decorate showed various neuroprotective effects against the Ab 25-35 induced apoptosis in PC12 cells, with the petroleum extraction (PE) and acetidin extraction (AE) showing better neuroprotective effects than other extractions. Oxidative stress is an early and sustained event in neurodegenerative disease progression (Uttara et al. 2009), and plays a significant role in many neurological diseases, such as AD, PD and cerebral ischemia (Saeidnia & Abdollahi 2013). The 'free radical theory of aging' shows promise in helping to understand the process of aging and in treating age-related diseases. Therefore, the antioxidant effect of P. decorate leaves might be one of the underlying mechanisms for neuroprotective effects. It is very important to protect neuronal cells from oxidative injury for the treatment of neurodegenerative disorders.
As our continuous investigation of P. decorate active constituents, further purification and isolation study was conducted. Nine compounds were isolated and identified from the extracts of P. decorate leaves, including five triterpenoids, an iridoid, a flavone, a sterol and an aliphatic acid ( Figure 1). In addition, we investigated in vitro antioxidant capacities of P. decorate decoction, as well as the isolated phytochemicals, in protecting PC12 cells from hydrogen peroxide induced oxidative stress. Our study provides some basis of mechanisms in prevention and treatment of neurodegenerative diseases including AD and PD by P. decorate.

Materials and methods
General experimental procedures 1 H-, 13 C NMR, COSY, HMQC spectra were recorded on a Bruker AV500 NMR spectrometer (TMS as internal standard, 1 H 400 MHz, 13 C 100 MHz). HR-ESI-MS was conducted on an Agilent (Santa Clara, CA) 1100 LC/TOF/MSD system (ESI model), Shimadzu (Kyoto, Japan) LC-10 ATVP HPLC with ODS-AGG12 (YMC, Kyoto, Japan) column, column chromatography and TLC silica gel (Qingdao Marine Chemical Group Co., Qingdao, China). All the solvents used in the isolation and purification studies were analytical grade.

Plant material
The P. decorate leaves were collected in September 2013 in Wuhan, China, and were identified by Professor Yongzhong Zhang (Department of Pharmacognosy, Medical College, Wuhan University of Science and Technology, Wuhan, China). A voucher specimen (No. 130901-01) was deposited in Department of Phytochemistry, Medical College, Wuhan University of Science and Technology, Wuhan, China.

Extraction and isolation
The P. decorate leaves were washed and crushed prior to extraction. The air-dried leaves (5 kg) were extracted thrice with 30 L of 95% ethanol after maceration in 24 h. The supernatant was collected, combined and filtered through paper filter. Then the filtrate was concentrated in rotary evaporator to yield 238.6 g of a gummy residue, which was stored afterwards in vacuo in the dark.
Samples (1.0 g) of ethanol extract (EE) were reserved for pharmacological tests; the remaining crude EE was dissolved in 1500 mL water and extracted with petroleum, acetidin and nbutyl alcohol, successively, to afford 60.1 g (PE), 103.6 g (AE) and 45.5 g (n-butyl alcohol extraction, BE), respectively. The residue of extract was also been retained as the aqueous extraction (WE) for isolation. PE, AE and BE had been reserved 1.0 g for the following pharmacological screening. PE (59.1 g) was subjected to silica gel column chromatography (12 Â 70 cm), eluting with petroleum containing increasing amounts of acetidin with a flow rate of 16 mL/min to yield fraction A (petroleum:acetidin ¼ 100:1, v/v), fraction B (100:3, v/v), fraction C (100:6, v/v). Fractions B and C were subject to silica gel, eluting with petroleum/acetidin gradient to obtain compound 4 (33 mg), 6 (21 mg) and 9 (18 mg) based on monitoring by thin layer chromatography (TLC). AE (102.6 g) was subjected to silica gel column chromatography fraction using petroleum/acetidin/methanol as eluent to yield fractions D-F. Fraction D was further chromatographed eluting with petroleum/acetidin (100:20, v/v) to give sub-fraction D1, followed by separation on Agilent 1100 HPLC using a gradient of methanol/H 2 O/acetic acid (19:1:0.01, v/v) to give compound 1 (38 mg, t R 12.9 min) and 2 (20 mg, t R 20.3 min). Fraction E was further purified by preparative TLC and recrystallized to afford compound 3 (182 mg) and 5 (28 mg).
Bioactive assay PC12 cells were maintained at 37 C in 5% CO 2 in DMEM medium supplemented with 10% FBS, 150 U/mL penicillin and 50 lg/mL streptomycin before experiments. PC12 cells were seeded in 96-well culture plates (0.8 Â 10 4 cells per well) for 24 h. The resultant products were weighed, then resuspended with dimethyl sulphoxide (DMSO) at a predetermined concentration, so that DMSO content in culture medium, at concentration selected for the plant extracts, never exceeded 1% in the growth medium. At this concentration, DMSO had negligible effects on PC12. Cells were pretreated for 24 h with petroleum, acetidin, nbutyl alcohol and aqueous extracts of P. decorate at concentration of 0.1, 0.5, 1, 5 and 10 mg/mL. Then, 0.4 mM H 2 O 2 was added in all samples for an additional 2 h except DMSO vehicle control. Cell viability was measured by the 3-(4,5-dimehthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Wong et al. 2012). Following treatment, the medium was removed and MTT solution (0.5 mg/mL in medium, Sigma Chemical Co., St. Louis, MO) was added. The incubation was kept for 4 h at 37 C and then the culture medium was removed. Vitamin E (0.5 mg/mL) was used as positive control in MTT assay. Experiments were carried out in triplicate (n ¼ 5). Absorbance was analysed at 490 nm with a Multiskan Ex microplate absorbance reader.

Statistical analysis
Statistical analyses were done using one-way analysis of variance (ANOVA) with t-test. Values reported are mean ± SD of five repeats. Results were considered as statistical significance when p < 0.05.

Antioxidant activity against H 2 O 2 induced cytotoxicity
The antioxidant capacities of P. decorate decoction, as well as the isolated phytochemicals, in protecting PC12 cells from hydrogen peroxide induced oxidative stress were investigated. Figure 2 demonstrates 24 h treatment with 0.2 mM H 2 O 2 alone produced significantly increased apoptosis (the H 2 O 2 group 48%) compared with blank control group. Ethanol extract treated groups showed inhibitory activity with IC 50 value of 10.83 mg/mL, inhibiting 72% of oxidative damage at 0.5 mg/mL concentration compared with 47% at 10 mg/mL. On the basis of these data, the 0.5 mg/mL of effective concentration of different polar extracts of Pyrola decorate were determined in the following experiments. Figure 3 demonstrates the results that % cell viabilities were improved by different polar extracts of Pyrola decorate compared with the H 2 O 2 treated cultures. PE and BE treated groups extracts showed better results when comparing with other extracts at the same concentration. PE (0.5 mg/mL) showed the strongest protective activities among all of the extractions (Figure 3), and yielded the maximum cell viability of 94% and 82% at the dose of 0.5 and 1.0 mg/mL, respectively. While BE extracts showed the higher cell viability of 77% at the dose of 0.5 mg/mL.
Three compounds promoted % cell viability from 7.8 to 1000 lg/mL concentration ( Figure 4). All tested samples showed scavenging activity against H 2 O 2 induced cytotoxicity of PC12 cells in a concentration-dependent manner. A significant potential for antioxidation of the tested phytochemicals was observed and three compounds had the stronger inhibitory effect compared with Vitamin E as a positive control agent. Betulin (1, BE), ursolic acid (3, UA) and monotropein (8, MO) were the main active constituents isolated from P. decorate leaves, and demonstrated with IC 50 value of 6.88, 6.15 and 6.13 lg/mL, respectively.
The highest scavenging activity against H 2 O 2 was observed in monotropein (95%, 500 lg/mL), depending on the concentration, the % cell viability of ursolic acid treated groups ranged from 53 to 95% within the concentration range of 7.8-500 lg/mL. As a result, ursolic acid, betulin and monotropein have also shown to enhance the protective effects against toxicity in PC12 cells. Simultaneously, three compounds isolated from P. decorate got their maximum efficacies at the concentration of 500 lg/mL.

Discussion and conclusions
Oxidative stress causes endothelial dysfunction and cellular injury, which contribute to aging. Many studies indicate that oxidative stress from ROS has been widely implicated in aging related disorders (Saeidnia & Abdollahi 2013). H 2 O 2 is a Figure 2. Protective effect of the alcohol extracts of Pyrola decorate on H 2 O 2induced cytotoxicity in cultured PC12 cells (means ± SD, n ¼ 5). The data (cell viability, measured by MTT assay) were normalized and expressed as a percentage of the control group, which was set to 100%. Results were calculated from three independent experiments and are shown as mean ± SD. Compared with blank control group, #p < 0.05, ##p < 0.01; compared with H 2 O 2 model group, Ã p < 0.05, ÃÃ p < 0.01.
relatively stable reactive oxygen species (ROS) that is capable of diffusing through cellular membranes and induces production of O 2 Á by activating NADPH oxidase ). Treatment of PC12 cells with nontoxic concentrations of P. decorate extraction and the isolated phytochemicals could protect cells from H 2 O 2induced cytotoxicity with a decrease in the generation of ROS. In this study, EEs of P. decorate in different concentration had revealed the changes after treatment except in the maximum Figure 3. Protective effect of different polar extracts of Pyrola decorate on H 2 O 2 -induced cytotoxicity in cultured PC12 cells (means ± SD, n ¼ 5). The data (cell viability, measured by MTT assay) were normalized and expressed as a percentage of the control group, which was set to 100%. Results were calculated from three independent experiments and are shown as mean ± SD. Compared with blank control group, #p < 0.05, ##p < 0.01; compared with H 2 O 2 model group, Ã p < 0.05, ÃÃ p < 0.01. Figure 4. Protective effect of betulin (BE), ursolic acid (UA) and monotropein (MO), isolated from Pyrola decorate, on H 2 O 2 -induced cytotoxicity in cultured PC12 cells (means ± SD, n ¼ 5). The data (cell viability, measured by MTT assay) were normalized and expressed as a percentage of the control group, which was set to 100%. Results were calculated from three independent experiments and are shown as mean ± SD. Compared with blank control group, #p < 0.05, ##p < 0.01; compared with H 2 O 2 model group, Ã p < 0.05, ÃÃ p < 0.01. concentration of 10 lg/mL. The PE and BE extracts treated groups showed significant antioxidant capacities in vitro comparable with vitamin E as natural antioxidant, while the AE, WE and 50% EE extract groups exhibited relatively better antioxidant activity. The biological activities of the P. decorate extracts can be attributed to the secondary metabolites. 5,7-Dimethoxy-4 0 -hydroxyflavone was isolated from the PE extracts. Luteolin, kaempferol and 2 0 -O-galloyl-3-b-galactosyloxy quercetin were reported in the Pyrolaceae family by Zhen et al. (2016). Flavonoids, known as sensitive to oxidative stress, are higher in PE and lower both in BE and WE, were also found as the one of most commonly phytochemicals in P. decorate and exhibited obvious abilities of scavenging superoxide anion and hydroxyl free radical (Wang et al. 2014), and the effects of which came from their polyphenol structures (Sheng 2012). On the other hand, four of five triterpenoids in this study were isolated from the AE extract, it is noteworthy that individual compounds possessed remarkable antioxidant activities while the protection activity of AE extract against oxidative injury was not most significant, probably due to the interactions between heterogeneous ingredients even including that we have not achieved yet. BE extract also showed a better activity only second to petroleum ether compared with model group. Earlier studies have demonstrated iridoids were also one of the main biochemicals in P. decorate leaves (Liu et al. 2007), which are widely distributed among many medicinal plants possessing a range of biological activities (West et al. 2016). For example, evidence implied that the antiaging effects of catalpol, an iridoid glycoside from Rehmannia, were achieved by promoting endogenous antioxidant enzyme activities and normalizing energy disturbance (Huang et al. 2016). However, the effect of iridoids in P. decorate targeting in the treatment of oxidative stress related neurological diseases has not been reported before.
As a continuous study, betulin, ursolic acid and monotropein isolated from P. decorate were investigated with capabilities of resistance to oxygen damage, which expanded our understanding of the antioxidant role of components candidates. Betulin was found from leaves of P. decorate for the first time. Ursolic acid was the most abundant and primary constituent of pentacyclic triterpenoid carboxylic acid in leaves of P. decorate, which has wide pharmacological effects of hepatoprotection, antiinflammation, antitumor, antimicrobe and lipidemic regulation (Li et al. 2003). What is more, the neuroprotective therapeutic efficacy of ursolic acid has been confirmed by our previous studies . At cellular level, aging is associated with accumulating oxidative stress, mitochondrial dysfunction, telomere erosion and impaired DNA repair (Hung et al. 2010). One of the main mechanisms is the oxidation and antioxidation theory. The finding that ursolic acid played an important role to protect cells from oxidative injury suggested the consistency with our previous studies on its neuroprotection. Simultaneously, monotropein was one of the main components of the extracts from BE extract of P. decorate (Zhao & Tu 2010), and the bioactive tests indicated monotropein had possessed remarkable abilities against cytotoxicity induced by H 2 O 2 . In China, monotropein must be checked out in P. decorate as a crucial standard component in pharmacopeia. Furthermore, it is found Pyrola and Morinda, both are regarded as tonifying traditional medicines in China.
Oxidative stress and ROS are proposed to be major contributors to the aging process and many neurodegenerative diseases (Wang et al. 2016;Wojtunik-kulesza et al. 2016). Our study revealed that P. decorate, used as a tonic in traditional medicine, showed higher potency of anti-oxidative, which could partly explain the molecular mechanisms whereby P. decorate has neuroprotective effect of our previous study in neuronal cell models. In conclusion, Pyrola decorate is a potential antioxidative natural plant and worth testing for further pharmacological investigations in the treatment of oxidative stress related neurological disease.