Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Cellular Antioxidant and Antiproliferative Activities of Morchella conica Pers. Polyphenols in vitro

Xia Liao, Fuhua Li, Yurong Tan, Keke Lu, Surui Wu, Ran Yin, Jian Ming*

College of Food Science, Southwest University, Chongqing, P. R. China

Abstract: This study analyzed the cellular antioxidant and antiproliferative activities, as well as the phenolic composition of three Morchella conica Pers. cultures. Results showed that the free phenolic contents of the three Morchella conica Pers. ranged from 4.928 to 6.157 mg GAE/g DW and their bound phenolic contents ranged from 0.188 to 0.250 mg GAE/g DW. Polyphenols in M. conica Pers. were dominated by phenolic acids, particularly for gallic acid. The free phenolic extracts exhibited higher cellular antioxidant activity than the bound phenolic extracts. Free phenolics in M. conica Pers. cultured from Yunnan China showed the highest antiproliferative activity against HepG₂ cells, whereas bound phenolics in M. conica cultured from Tibet China showed the highest antiproliferative activity. Results confirmed that Morchella conica Pers. (growing in Yunnan China especially) could be a new source of natural antioxidant and a potential inhibitor for the growth of HepG₂ cells.

Keywords: Morchella conica Pers., polyphenols, antioxidation, antiproliferation

Citation: Xia Liao, Fuhua Li, Yurong Tan, Keke Lu, Surui Wu, Ran Yin, Jian Ming, Cellular Antioxidant and Antiproliferative Activities of Morchella conica Pers. Polyphenols in vitro. Journal of Food and Nutrition Research, 5 (10). 742-749. January 2017. doi:10.12691/jfnr-5-10-4

1. Introduction

Morchella conica Pers., belonging to the Morchella genus, is rich of nutrients such as proteins, polysaccharides, minerals, vitamins, amino acids, δ-carotene and ergosterol [1]. Morchella conica Pers. is also rich of bioactive compounds, such as polysaccharide, glutathione, etc [2,3,4]. Polysaccharide of Morchella importuna could significantly increase the viability of PC12 cells by enhancing the activity of antioxidant enzyme [5]. Studies also confirmed the antioxidant activity in vivo of Morchella esculenta polysaccharides activity [6].

Recently, a growing number of studies have indicated that wild fungus is a good source of phenolics [3,7]. The phenolic content of M. conica Pers. was 25.38 μg GAE (gallic acid equivalents)/mg extract [8]. Serbian M. conica Pers., compared with Portuguese M. conica Pers., had a higher level of phenolic compounds, which displayed a higher antioxidant capacity, while Serbian M. conica Pers. displayed a higher antibacterial capacity than Serbian M. conica Pers. [9]. Turkoglu et al. [10] found that the phenolic content of M. conica Pers. was 41.93 μg PE (pyrocatechol equivalents)/ mg extract, and the phenolic extract exhibited high DPPH· scavenging capacity, antioxidant capacity (β-carotene-linoleic acid system), and anti-inflammatory activity. The ethyl acetate extract of wild Inonotus sanghuang displayed potent antiproliferative, antioxidant, and antimicrobial activities [11]. The extracts of wild Suillus bellinii showed high reducing power, scavenging DPPH radicals capacity, and high antiproliferative activity against MCF7, NCI-H460 and HepG2 cells [12]. The aqueous-ethanolic extract of M. esculenta mycelium showed hepatoprotective activity in vivo [13]. Phenolic extracts of Ganoderma lucidum fruiting body even showed higher antioxidant capacity than its polysaccharidic extracts [14]. The researches mentioned above indicated that Morchella conica Pers. was also a potential source of antioxidative phenolic compounds.

Investigations on the genetic resources of Morchella reported that Yunnan, Tibet and Xinjiang were the major producing regions of wild Morchella in China [15,16,17]. It is necessary to screen the wild Morchella conica Pers. growing in Yunnan, Tibet and Xinjiang of China to provide the basic information serving for the breeding research.

To the best of our knowledge, studies on Morchella were mainly focused on its morphological and ultrastructural properties, genetics, and bioactive polysaccharides [3,18,19]. However, information on the composition, concentration, cellular antioxidant activity, and antiproliferative capacity against human hepatoma HepG2 cells of polyphenols extracted from wild M. conica Pers. is still poor. The objective of this study was to analyze the composition of polyphenols extracted from M. conica Pers. collected from Yunnan, Tibet, and Xinjiang, China. Human hepatoma HepG2 cells were used as a model to comprehensively evaluate the cellular antioxidant and antiproliferative activities of polyphenols in the three M. conica Pers. species.

2. Materials and Methods

2.1. Materials

M. conica Pers. from Yunnan (MCP-Y), Tibet (MCP-T), and Xinjiang (MCP-X) were provided by the Kunming Institute of Edible Fungi. Morchella samples were oven-dried at 50°C to a constant weight, crushed with a pulverizer, passed through an 80-mesh sieve, sealed, and then stored in the dark at room temperature (20°C) in desiccators over silica gel-self indicator prior to analysis.

2.2. Chemicals

Folin-Ciocalteu reagent and quercetin were purchased from Sigma, Inc.. 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAP), 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and William’s medium E were purchased from Wako Chemicals. Antibiotics, trypsin, fetal bovine serum, Hank’s balanced salt solutions Dulbecco's modiﬁed Eagle's medium, hydrocortisone, antibacterial-antifungal agents, and phenanthroline (biochemical reagent) were purchased from Invitrogen. Methanol and acetonitrile (HPLC grade) were purchased from the Tianjin Shield Fine Chemical Product Co., Ltd. Other reagents were all of analytical grade. Human hepatoma HepG2 cells were provided by the American Type Culture Collection.

2.3. Methods

2.3.1. Extraction Phenolic Compounds

Extraction was performed following the methods reported by Okarter et al. [20] with slight modifications. Briefly, 2.00 g of sample was accurately weighed into a 100 mL centrifuge tube, and 50 mL of chilled acetone (80%, v/v) solution was added. The mixture was homogenized for 2 min and then stirred for 10 min. The sample was centrifuged at 2500×g for 10 min, and the supernatant was collected. The residue was re-extracted, and the supernatants were combined, suction filtered, and concentrated by evaporation at 45°C. The sample was adjusted to a constant volume of 25 mL with double-distilled water to obtain the free phenolic extract, which was stored at -80 °C until use within two weeks.

The residue was collected after the extraction of free phenolic compounds, and 20 mL of NaOH (2 mol/L, w/v) solution was added. The mixture was digested by oscillation for 1.5 hours and then adjusted to a pH of 2 using concentrated hydrochloric acid. Subsequently, 25mL of n-hexane was added to remove the fat layer. Then, 20 mL of ethyl acetate was added and fully stirred for 10 min. The mixture was centrifuged at 2500×g, and the supernatant was collected. The extraction procedure was repeated five times, with the centrifugation rate increasing by 500×g every time, and the supernatants were combined. After suction filtration, the extract was rotary evaporated at 45°C and adjusted to a constant volume of 10 mL with double-distilled water to obtain the bound phenolic extract. The extract was stored at –80°C until use within two weeks.

2.3.2. Measurement of the Total Phenolic Content (TPC)

TPC was detected using the Folin-Ciocalteu method [21]. Briefly, 200 µL of extract was absorbed, followed by the successive addition of 800 μL of deionized water and 200 μL of Folin-Ciocalteu agent. The sample was thoroughly mixed by shaking the tube, and then, it was placed in the dark for 6 min. Then, 2 mL of 7% Na₂CO₃ solution and 1.6 mL of deionized water were added, and the sample was placed in the dark for another 90 min before the absorbance was measured at 760 nm. Gallic acid was used as the reference standard to plot the standard curve over a concentration range of 0-400 μg/mL. The results are expressed as mg gallic acid equivalents/g dry-weight basis (mg GAE/g DW).

2.3.3. HPLC Analysis of Phenolic Compounds

The method previously reported by Liang et al. [22] was used with slight modifications. Briefly, the polyphenol extracts and 0.1 mg/mL standards (gallic acid, protocatechuic acid, p-hydroxybenzoic acid, catechin, and chlorogenic acid) were filtered through a 0.45 μm organic membrane filter and analyzed using an LC-20A HPLC (Shimadzu Corp., Japan) equipped with a Shimadzu LC-20AD pump, a Shimadzu SIL-20A Autosampler, and a Shimadzu SPD-M20A diode array detector (DAD). A Thermo BDS C₁₈ reverse-phase column (250 × 4.6 mm i.d., 5 μm grain size) was applied. Mobile phase A was 0.2% (v/v) formic acid, and mobile phase B was pure acetonitrile. The gradient elution program was as follows: 0-5 min, 10% B; 5-25 min, 10-40% B; 25-35 min, 40-90% B; 35-40 min, 90% B; 40-45 min, 90-10% B; and 45-50 min, 10% B. The other conditions were as follows: flow velocity, 0.7 mL/min; injection volume, 8 μL; column temperature, 40°C; and detection wavelength, 280 nm. Date was analyzed by LCsolution Version 1.25.

2.3.4. Cell Culture

HepG2 cells were maintained in the growth medium CM (William’s Medium E, WEM) containing 5% FBS, 2 mmol/L glutamate, 10 mmol/L HEPES, 5 µg/mL insulin, 0.05 µg/mL hydrocortisone, 50 units/mL penicillin, 50 µg/mL streptomycin, and 100 µg/mL gentamicin. The cells were incubated at 37°C and 5% CO₂ [23,24]. The number of cell passages was 12-35.

2.3.5. Cellular Antioxidant Activity (CAA) Assay

According to the method reported by Wolfe, & Liu [25], the HepG2 cells (6×10⁴ cells/well) were seeded into a 96-well plate containing 100 µL of growth medium per well. The cell culture was incubated at 37 °C and 5% CO₂ for 24 h. The medium was removed, and the plate was washed with 100 μL of phosphate-buffered saline (PBS) per well. Subsequently, 100 μL/well of different concentrations of quercetin reference standard (control group), polyphenol extracts (treatment group), or water (blank group) containing a 2',7'-dichlorofluorescin diacetate (DCFH-DA) working solution was added. All groups were cultured under the same conditions for another hour, and each well was then washed with 100 µL of PBS. Then, either the PBS wash protocol between antioxidant and ABAP treatment (PBS wash protocol) was performed or not (no PBS wash protocol). Then, 100 μL of ABAP working solution was added. The blank group also received 100 μL of oxidant-treated medium. The 96-well plate was immediately placed on a microplate reader (Fluoroskan Ascent FL) and read at 37°C. The measurement was performed for 60 min at 5 min intervals (emission 538 nm; excitation 485 nm).

After subtraction of the blank from the fluorescence readings of the control and treatment groups, the integral of the curve of fluorescence over time was used to calculate the CAA value of reference standards or polyphenol extracts at each concentration as follows:

Where is the integral of the curve of fluorescence for the sample and is the integral of the curve of fluorescence for the control. The half-maximal effective concentrations (EC₅₀) of the reference standards and polyphenol extracts were calculated according to the linear relationship between log (fa/fu) versus log (dose), where fa is the affected portion after treatment (CAA unit) and fu is the unaffected portion (1-CAA unit). Quercetin was used as the reference standard. The CAA values, expressed as μmol quercetin equivalents (QE)/100 g DW, were converted from the EC₅₀ values of quercetin and the samples.

2.3.6. Cytotoxicity

Following the methods reported by Yoon et al. [26] with slight modifications, the HepG2 cells were seeded into a 96-well plate (4 ×10⁴ cells/well) and incubated at 37°C and 5% CO₂ for 24 h. Then, the plate was washed with PBS (100 μL/well). Subsequently, medium containing different concentrations of free polyphenol extract (25, 50, 75, 100, 125, 150, 175, 200, 225, 250 μg/mL) and bound polyphenol extract (10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/mL) was added (100 μL/well), followed by incubation at 37°C for another 24 h. The medium was then removed, and the plate was washed with PBS (100 μL/well). Thereafter, 50 μL/well of methylene blue solution (prepared with 0.6% methylene blue, 0.67% glutaraldehyde, and 98% HBSS) was added for staining. Cells were incubated at 37°C for one hour before the staining solution was removed. The plate was washed with deionized water until the water was clear. Next, 100 µL/well of elution solution (prepared with 1% acetic acid, 49% PBS, and 50% ethanol) was added. The plate was rotary oscillated for 20 min. Then, the 96-well plate was placed on a microplate reader to measure the absorbance at 570 nm. Compared with the blank wells, when the decrease in the ratio of the absorbance in the treatment wells was greater than 10%, the solution was considered to be cytotoxic.

2.3.7. Measurement of Cell Proliferation

Following the method reported by Yang et al. [27], HepG2 cells were seeded into a 96-well plate, and 100 µL of growth medium was added to each well. The cells (2.5×10⁴ cells/per well) were incubated at 37°C under 5% CO₂ for 4 hours. The medium was then removed, and the plate was washed with 100 µL of PBS. Next, 100 μL of medium containing different concentrations of polyphenol extracts was added per well, and cells were incubated at 37°C for another 72 h. The same procedure was performed for the cytotoxicity test. The cell proliferation rate was calculated as the ratio of the absorbance in the treatment well to the absorbance in the blank well.

2.4. Statistical Analyses

The results are expressed as the mean ± SD (n=3) and statistically analyzed using SPSS 19.0 software. The figures were generated using Sigma Plot 12.0. Significant differences were evaluated at the p<0.05 level.

3. Results and Discussion

3.1. Total Phenolic Content in Morchella conica Pers

The free, bound, and total phenolic contents of M. conica Pers. from the three species were summarized in Table 1. With regards to the free phenolics, M. conica Pers. from Yunnan (MCP-Y) contained the highest concentration (6.157 mg GAE/g DW), followed by M. conica Pers. from Xinjiang (MCP-X, 6.145 mg GAE/g DW), and M. conica Pers. from Tibet (MCP-T, 4.928 mg GAE/g DW). With regards to bound phenolics, MCP-Y contained the highest concentration (0.250 mg GAE/g DW), followed by MCP-X (0.206 mg GAE/g DW) and MCP-T (0.188 mg GAE/g DW). Free phenolics comprised approximately 96% of the total phenolic content. Other dietary fungi varieties, such as Lentinula edodes [28] and Pleurotus ostreatus [29], also contained large proportions of free phenolics. The total phenolic contents of M. conica Pers. were higher than those of Pleurotus ostreatus (1.44 mg GAE/g DW), Cantharellus cibarius (0.77 mg GAE/g DW) [30], Agaricus arvensis (2.83 mg GAE/g DW) and Saarcodon imbricatum (3.76 mg GAE/g DW) [31], but lower than the ethanol extracts of Portabella (10.65 mg GAE/g DW) and Crimini (9.89 mg GAE/g DW) [32].

Table 1. Phenolic contents of free, bound and total phenolics extracted from M. conica Pers. growing in three different habits. (mean ± SD, n=3)

3.2. Characterization of the Phenolic Compounds

The compositions of free and bound phenolic compounds from the three varieties were identified by RP-HPLC-DAD. As shown in Table 2, gallic acid was the major free phenolic compound in the three M. conica Pers. varieties, and the contents of free gallic acid in the three varieties followed the order: MCP-T (951.20 μg/g) < MCP-Y (1,043.49 μg/g) < MCP-X (1,124.13 μg/g) (p<0.05). The bound gallic acid contents were: MCP-Y (11.41 μg/g), MCP-X (10.96 μg/g), and MCP-T (8.83 μg/g) (p<0.05).

Table 2. Composition and content (μg/g) of detected polyphenolics extracted from M. Conica Pers. growing in three different habits. (mean±SD, n=3)

The free protocatechuic acid content was the highest (p<0.05) in MCP-Y (795.06 μg/g), followed by MCP-X (707.52 μg/g) and MCP-T (505.26 μg/g). The bound protocatechuic acid contents in the three M. conica Pers. were as follows: MCP-X (7.11 μg/g) <MCP-T (8.65 μg/g) <MCP-Y (9.02 μg/g).

The free p-hydroxybenzoic acid content in MCP-Y (777.60 μg/g) was significantly higher (p<0.05) than in MCP-X (615.67 μg/g) and MCP-T (599.28 μg/g). However, no p-hydroxybenzoic acid was detected in the bound extracts of the M. conica Pers. varieties.

The free chlorogenic acid content in MCP-Y was the highest (827.22 μg/g), followed by MCP-T (517.85 μg/g) and MCP-X (510.30 μg/g) (p<0.05). The content of bound chlorogenic acid was relatively high in MCP-Y (20.21 μg/g), followed by MCP-X (13.18 μg/g) and MCP-T (13.01 μg/g).

The free catechin content was the highest in MCP-Y (555.69 μg/g), followed by MCP-X (518.30 μg/g) and (MCP-T) 396.51 μg/g (p<0.05). No catechin was detected in the bound of the three M. conica Pers. varieties.

Accordingly, the polyphenol compositions of the three varieties of M. conica Pers. were similar, but the contents varied significantly (p<0.05). It was found that the content or composition of phenolics was influenced by the breading environment and varieties [33]. We found that gallic acid, protocatechuic acid, p-hydroxybenzoic acid, catechin, and chlorogenic acid were the major free phenolics in the three M. conica Pers., whereas gallic acid, protocatechuic acid, and chlorogenic acid were the major bound phenolics. Gallic acid, protocatechuic acid, chlorogenic acid and p-hydroxybenzoic acid were associated with the antioxidant acitivity of foods [34,35,36,37]. Catechin showed cellular antioxidant activity [38]. In another side, gallic acid, catechin and chlorogenic acid could inhibit the proliferation of HepG2 cells [39,40,41]. Therefore, it could be inferred that these phenolic compounds identifed in M. conica Pers might be responsible for its bioactive activities.

3.3. Cellular Antioxidant Activities

The kinetic curve of DCFH oxidation induced by peroxyl radicals produced by ABAP in HepG2 cells was illustrated in Figure 1. Results showed that both the quercetin standard (Figure 1 A,B) and the polyphenol extracts of M. conica Pers. (Figure 1 C,D,E,F) significantly inhibited the production of DCF, as reflected by the increase in the fluorescence value.

Figure 1. Peroxyl radical-induced oxidation of DCFH to DCF in HepG2 cells and the inhibition of oxidation by quercetin (A, B) and free phenolic (C, D), as well as bound phenolic (E, F) extracts of M. Conica Pers. over time. A, C, and D provided the results of the PBS wash protocol; B, D, and F provide the results of the no PBS wash protocol

The EC₅₀ and CAA values for the cellular antioxidant activity of polyphenols from the three varieties of M. conica Pers. and the CC₅₀ values for the cytotoxicities of the samples were listed in Table 3. With the PBS wash, the EC₅₀ values of free phenolic extracts ranged from 112.60 to 582.03 mg/mL, and the CAA values ranged from 2.24 to 7.09 μmol QE/100 g. Without the PBS wash, the EC₅₀ values ranged from 24.51 to 71.00 mg/mL, and the CCA values ranged from 13.04 to 32.10 μmol QE/100 g. These results indicated that within the nontoxic concentration (< 250 mg/mL), the free phenolic extracts exhibited antioxidant activity on the cell membrane surface (no PBS wash), and the antioxidant activity was enhanced with increasing phenolic contents. However, in the interior of the cells (PBS wash), only the free phenolic extract of MCP-Y showed cellular antioxidant activity. The free phenolic extract of MCP-Y produced the lowest EC₅₀ (112.60 mg/mL and 24.51 mg/mL) and highest CAA (7.09 μmol QE/100 g and 32.10 μmol QE/100 g), when the protocols of no PBS wash and PBS wash were performed. The free phenolic extract of MCP-X produced the highest EC₅₀ (582.03 mg/mL and 71.00 mg/mL) and lowest CAA (0.26 μmol QE/100 g and 0.47 μmol QE/100 g). These data indicated the great potential of the free phenolic extract of MCP-Y in scavenging cellular reactive oxgen species (ROS). In both the protocols of PBS wash ad no PBS wash, the CAA values of free phenolic extracts were significantly higher than those of bound phenolic extracts, and the polyphenol extract of MCP-Y showed the best cellular antioxidant capacity.

Table 3. Cellular antioxidant activities and cytotoxicity of polyphenols extracted from M. Conica Pers. growing in three different habits. (mean±SD, n=3)

Generally, the polyphenol extracts of Morchella showed significantly higher capacity to inhibit the DCF production in the protocol of PBS wash than in the PBS wash protocol. It means that the Morchella polyphenols took more advantage in scavenging ROS on/ in cell membrane than intracellular ROS. Similar results were found in the polyphenols of Semen coicis [42] and polyphenols of M. umbrina Boud [43]. The macromolecules in the polyphenol extracts of Morchella might prevent phenolic compounds from penetrating through the cell membranes, resulting in relatively high levels of extracellular antioxidant activity. While, when the PBS wash protocol was performed, both the macromolecules and phenolic compounds were washed off, and the capacity to inhibit DCF production was lower than that without the PBS wash.

Chlorogenic acid and gallic acid contributed the most to the antioxidant capacity [44,45]. As gallic acid and chlorogenic acid showed high levels in M. conica Pers. (Table 2). Therefore, it could be inferred that gallic acid and chlorogenic acid might be the major contributors to the antioxidant activities of M. conica Pers. varieties.

3.4. Antiproliferative Effect on HepG2 Cells

The antiproliferative capacity and cytotoxicity of polyphenol extracts of M. conica Pers. against HepG2 cells were shown in Figure 2. The cell proliferation rate was approximately 100% in the control group. At the 200 μg/mL of free phenolic extracts of M. conica Pers., the proliferation inhibition rate increased from 33.80% (MCP-X) to 54.42% (MCP-Y). As 200 μg/mL was a non-cytotoxic concentration (Table 4), it indicated that the inhibitory effect of free phenolic extracts on HepG2 cells was primarily induced by the inherent anti-tumor effect rather than the cytotoxicity. Within the non-cytotoxic concentrations, free phenolic extracts of MCP-Y showed the highest antiproliferative capacity against HepG2 cells, whereas free phenolic extracts of MCP-X exhibited the lowest antiproliferative capacity. The antiproliferative capacity increased in a dose- dependent manner.

Figure 2. The inhibited proliferation and cytotoxicity of HepG2 human liver cancer cells treated by M. Conica Pers. polyphenol extracts. M. Conica Pers. from Yunnan (A, B), M. Conica Pers. from Tibet (C, D), and M. Conica Pers. from Xinjiang (E, F); free phenolics (A, C, E) and bound phenolics (B, D, F)

At the concentration of 100 μg/mL (non-cytotoxic concentration, Table 4), the bound phenolic extract of MCP-T, inhibited the proliferation of HepG2 cells by 83.53%, suggesting that bound phenolic extracts of MCP-T had higher capacity to inhibit HepG2 cell proliferation than the three of free phenolic extracts. For the bound phenolic extracts of MCP-Y and MCP-X, their highest proliferation inhibition rates were 22.23% and 29.17%, respectively, which were much weaker than MCP-T.

HepG2 cells have been extensively used in the research of cancer mechanisms, genetics and nutriology [42,46]. In this study, the proliferation of HepG2 cells was inhibited in a dose-depended manner by M. conica Pers. polyphenols. Within the non-cytotoxic concentrations, the EC₅₀ values of free phenolic extracts ranged from 186.43 to 271.38 μg/mL. Specifically, MCP-Y showed the highest antiproliferative capacity with a 54.42% of the inhibiting rate against HepG2 cells. For bound phenolic extracts, MCP-T showed the highest antiproliferative capacity against HepG2 cells, with an inhibition rate of 83.53% and an EC₅₀ value of 49.84 μg/mL. However, the other two varieties showed almost no inhibition of HepG2 cell proliferation.

Table 4. Antiproliferation activity (EC₅₀) and cytotoxicity (CC₅₀) of polyphenols extracted from three M. Conica Pers. on HepG2 cells (mean±SD, n=3)

4. Conclusions

Above all, a total of five phenolic compounds were identified in the wild M. conica Pers. Gallic acid and chlorogenic acid represented the major free and bound phenolic compound, respectively. The free phenolic extract exhibited high CAA, whereas the bound phenolic extract showed nearly no CAA when incubated within non-cytotoxic concentrations. The wild M. conica Pers. growing in Yunnan, China showed the highest CAA values owing to its highest concentrations of free and bound polyphenols. This study confirmed the antioxidant and antitumor activities of wild Morchella polyphenols. Particularly, the wild Morchella growing in Yunnan, China might be a good choice for breeding. Our results might also contribute to the comprehensive utilization of Morchella.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no. 31471576); the National Key R&D Program of China (grant no. 2016YFD0400203); High-level Science and Technology Talent Training Project (grant no. 2008OC008), and the Fundamental Research Funds for the Central Universities (grant no. XDJK2016E113).

References

[1]   | Liu, B., Wu, S. R., Zhu, P., Zhang, L.Y., Tai, L. M., & Gui, M. Y. (2012). Nutrient analysis of morel in northwest Yunnan Province. Science and Technology of Food Industry, 33(1), 363-365.
[2]   | Kalaras, M. D., Richie, J. P., Calcagnotto, A., & Beelman, R. B. (2017). Mushrooms: A rich source of the antioxidants ergothioneine and glutathione. Food Chemistry, 233, 429-433.
[3]   | Ramírez-Anguiano, A. C., Santoyo, S., Reglero, G., & Soler-Rivas, C. (2010). Radical scavenging activities, endogenous oxidative enzymes and total phenols in edible mushrooms commonly consumed in Europe. Journal of the Science of Food and Agriculture, 87(12), 2272-2278.
[4]   | Yildiz, O., Can, Z., Laghari, A. Q., & Şahin H., Malkoç M. (2015). Wild edible mushrooms as a natural source of phenolics and antioxidants. Journal of Food Biochemistry, 39(2), 148-154.
[5]   | Xiong, C., Li, Q., Chen, C., Chen, Z. Q., & Huang, W. L. (2016). Neuroprotective effect of crude polysaccharide isolated from the fruiting bodies of Morchella importuna against H2O2-induced PC12 cell cytotoxicity by reducing oxidative stress. Biomedicine and Pharmacotherapy, 83, 569-576.
[6]   | Meng, F., Zhou, B., Lin, R., Jia, L,. Liu, X. N., Deng, P., Fan, K. M., Wang, G. Y., Wang, L,. & Zhang, J. J. (2010). Extraction optimization and in vivo antioxidant activities of exopolysaccharide by Morchella esculenta SO-01. Bioresource Technology, 101(12), 4564-4569.
[7]   | Badshah, H., Ullah, F., Khan, M. U., Mumtaz, A. S. & Malik , R. N. (2015). Pharmacological activities of selected wild mushrooms in South Waziristan (FATA), Pakistan. South African Journal of Botany, 97, 107-110.
[8]   | Gursoy, N., Sarikurkcu, C., Cengiz, M., & Solak, M. H. (2009). Antioxidant activities, metal contents, total phenolics and flavonoids of seven Morchella species. Food and Chemistry Toxicology, 47(9), 2381-2388.
[9]   | Vieira V., Fernandes Â., Barros L., Glamočlija J., Ćirić A., Stojković D., Martins A., Soković M., & Ferreira I. C. (2016). Wild Morchella conica Pers. from different origins: a comparative study of nutritional and bioactive properties. Journal of Science of Food and Agriculture, 96 (1), 90-98.
[10]   | Turkoglu, A., Kivrak, I., Mercan, N., Duru, M., Gezer, K., & Turkoglu, H. (2006). Antioxidant and antimicrobial activities of Morchella conica Pers. African Journal of Biotechnology, 5(11), 1146-1150.
[11]   | Liu, K., Xiao, X., Wang, J., Chen, C-Y. O., & Hu, H. (2017). Polyphenolic composition and antioxidant, antiproliferative, and antimicrobial activities of mushroom Inonotus sanghuang. LWT - Food Science and Technology, 82, 154-161.
[12]   | Souilem, F., Fernandes, Â., Calhelha, R. C., Barreira, J. C. M., Barros, L., Skhiri, F., Martins, A., & Ferreira, I. C. F. R. (2017). Wild mushrooms and their mycelia as sources of bioactive compounds: Antioxidant, anti-inflammatory and cytotoxic properties. Food Chemistry, 230, 40-48.
[13]   | Nitha, B., Fijesh, P. V., & Janardhanan, K. K. (2013). Hepatoprotective activity of cultured mycelium of Morel mushroom, Morchella esculenta. Experimental and Toxicologic Pathology, 65(1-2), 105-112.

[14]  
Heleno, S. A., Barros, L., Martins, A., Queiroz, M. J. R. P., Santos-Buelga, C., & Ferreira, I. C. F. R. (2012). Fruiting body, spores and in vitro produced mycelium of Ganoderma lucidum from Northeast Portugal: A comparative study of the antioxidant potential of phenolic and polysaccharidic extracts. Food Research International, 46(1), 135-140.

[15]   | Wu, D. M., Xu, W. T., Xie, Z. M., Luo, Y. B., & Li, Q. S. (2013). Present status and prospect of wild Morchella in Xinjiang. Science and Technology of Food Industry, 34(1), 381-384.
[16]   | Yang, M. (2014). Study on sustainable development of Tibetan edible fungi. (Master dissertation, Huazhong Agricultural University).
[17]   | Yang, M., & Jin, Q. (2014). Study on sustainable development of Tibetan edible fungi - Based on fuzzy comprehensive evaluation method. Guangdong Agricultural Sciences, 22, 18-23,34.
[18]   | He, P., Cai, Y., Liu, S., Han, Li., Huang, L., & Liu, W. (2015). Morphological and ultrastructural examination of senescence in Morchella elata. Micron, 78, 79-84.
[19]   | He, P., Wang, K., Cai, Y., & Liu, W. (2017). Live cell confocal laser imaging studies on the nuclear behavior during meiosis and ascosporogenesis in Morchella importuna under artificial cultivation. Micron, 101, 108-113.
[20]   | Okarter, N., Liu, C. S., Sorrells, M. E., & Liu, R. H. (2010). Phytochemical content and antioxidant activity of six diverse varieties of whole wheat. Food Chemistry, 119(1), 249-257.
[21]   | Chu, Y. F., Sun, J., Wu, X. Z., & Liu, R. H. (2002). Antioxidant and antiproliferative activities of common vegetables. Journal of Agriculture and Food Chemistry, 50(23), 6910-6916.
[22]   | Liang, Q., Cui, J., Li, H., Liu, J., & Zhao, G. H. (2013). Florets of Sunflower (Helianthus annuus L.): Potential new sources of dietary fiber and phenolic acids. Journal of Agriculture and Food Chemistry, 61(14), 3435-3442.
[23]   | Liu, R. H., Jacob, J. R., Tennant, B. C., & Hotchkiss, J. H. (1992). Nitrite and nitrosamine synthesis by hepatocytes isolated from normal woodchucks (Marmota monax) and woodchucks chronically infected with woodchuck hepatitis virus. Cancer Research, 52(15), 4139-4143.
[24]   | Liu, R. H., Jacob, J. R., Hotchkiss, J. H., Cote, P. J., Gerin, J. L., & Tennant, B. C. (1994). Woodchuck hepatitis virus surface antigen induces nitric oxide synthesis in hepatocytes: possible role in hepatocarcinogenesis. Carcinogenesis, 15(12), 2875-2877.
[25]   | Wolfe, K. L., & Liu, R. H. (2007). Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. Journal of Agriculture and Food Chemistry, 55(22), 8896-8907.
[26]   | Yoon, H., & Liu, R. H. (2008). Effect of 2α-hydroxyursolic acid on NF-κB activation induced by TNF-α in human breast cancer MCF-7 cells. Journal of Agriculture and Food Chemistry, 56(18), 8412-8417.
[27]   | Yang, J., Liu, R. H., & Halim, L. (2009). Antioxidant and antiproliferative activities of common edible nut seeds. LWT-Food Science and Technology, 42(1), 1-8.
[28]   | Choi, Y., Lee, S. M., Chun, J., Lee, H. B., & Lee, J. (2006). Influence of heat treatment on the antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes) mushroom. Food Chemistry, 99(2), 381-387.
[29]   | Yuan, Y., Xu, J. N., Zhang, J. F., Yang, X. L., Xia, C. Y., & Ming, J. (2014). Effects of different culture media on nutritional composition, polyphenol contents and antioxidant activity of pleurotus ostreatus. Food Science, 35(13), 137-142.
[30]   | Robaszkiewicz, A., Bartosz, G., Ławrynowicz, M., & Soszyński, M. (2010). The role of polyphenols, β-carotene, and lycopene in the antioxidative action of the extracts of dried, edible mushrooms. Journal of nutrition and metabolism, 2010, 173274.
[31]   | Barros, L., Ferreira, M. J., Queiros, B., Ferreira, I. C. F. R., & Baptista, P. (2007). Total phenols, ascorbic acid, β-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chemistry, 103(2), 413-419.
[32]   | Dubost, N. J., Ou, B., Beelman, & R. B. (2007). Quantification of polyphenols and ergothioneine in cultivated mushrooms and correlation to total antioxidant capacity. Food Chemistry, 105(2), 727-735.
[33]   | Palacios, I., Lozano, M., Moro, C., D’Arrigo, M., Rostagno, M. A., Martínez, J. A., García-Lafuente, A., Guillamón, E., & Villares, A. (2011). Antioxidant properties of phenolic compounds occurring in edible mushrooms. Food Chemistry, 128(3), 674-678.
[34]   | Xi, Y., Fan, X. G., Zhao, H. D., Li, X. H., Cao, J. K., & Jiang W. B. (2017). Postharvest fruit quality and antioxidants of nectarine fruit as influenced by chlorogenic acid. LWT - Food Science and Technology, 75, 537-544.
[35]   | Farhoosh, R., Johnny, S., Asnaashari, M., Molaahmadibahraseman, N., & Sharif, A. (2016). Structure-antioxidant activity relationships of o-hydroxyl, o-methoxy, and alkyl ester derivatives of p-hydroxybenzoic acid. Food Chemistry, 194, 128-134.
[36]   | Zhang, J., Li, D. M., Sun, W. J., Wang, X. J., & Bai, J. G. (2012). Exogenous p-hydroxybenzoic acid regulates antioxidant enzyme activity and mitigates heat stress of cucumber leaves. Scientia Horticulturae, 148(1), 235-245.
[37]   | Chen, C., Wang, L., Wang, R., Luo, X. H., Li, Y. F., Li, J., Li, Y. N., & Chen, Z. G. (2018). Phenolic contents, cellular antioxidant activity and antiproliferative capacity of different varieties of oats. Food Chemistry, 239, 260-267.
[38]   | Wolfe, K. L., & Liu, R. H. (2008). Structure-activity relationships of flavonoids in the cellular antioxidant activity assay. Journal of Agricultural and Food Chemistry, 56(18), 8404-8411.

[39]  
Lima, K. G., Krause, G. C., Schuster, A. D., Catarina, A. V., Basso, B. S. De Mesquita, F. C., Pedrazza, L., Marczak, E. S., Martha, B. A., Nunes, F. B., Chiela, E. C., Jaeger, N., Thomé, M. P., Haute, G. V., Dias, H. B., Donadio, M. V., & De Oliveira, J. R. (2016). Gallic acid reduces cell growth by induction of apoptosis and reduction of IL-8 in HepG2 cells. Biomedicine and pharmacotherapy, 84, 1282-1289.

[40]   | Yan, Y., Liu, N., Hou, N., Dong, L,. & Li, J. (2017). Chlorogenic acid inhibits hepatocellular carcinoma in vitro and in vivo. Journal of Nutritional Biochemistry, 46, 68-73.

[41]  
Jain, P., Kumar, N., Josyula, V. R., Jagani, H. V,. Udupa, N., Mallikarjuna R. C., & Vasanth, R. P. (2013). A study on the role of (+)-catechin in suppression of HepG2 proliferation via caspase dependent pathway and enhancement of its in vitro and in vivo cytotoxic potential through liposomal formulation. European Journal of Pharmaceutical Sciences, 50(3-4), 353-365.

[42]   | Wang, L. F, Chen, J. Y., Xie, H. H., Ju, X. R., & Liu, R. H. (2013). Phytochemical profiles and antioxidant activity of adlay varieties. Journal of Agriculture and Food Chemistry, 61(21), 5103-5113.
[43]   | Lu, K. K., Tan, Y. R., Zheng ,S. J., Liu, D., Wu, S. R., & Ming, J. (2015). Antioxidant and antiproliferative activities of polyphenols in Morchella umbrina Boud on the HepG2 cell model. Modern Food Science and Technology, 12(31), 6-13.
[44]   | Olszewska, M. A., & Michel, P. (2009). Antioxidant activity of inflorescences, leaves and fruits of three Sorbus species in relation to their polyphenolic composition. Natural Product Research, 23(16), 1507-1521.
[45]   | Alañón, M. E., Castro-Vázquez, L., Díaz-Maroto, M. C., Gordon, M. H., & Pérez-Coello, M. S. (2011). A study of the antioxidant capacity of oak wood used in wine ageing and the correlation with polyphenol composition. Food Chemistry, 128(4), 997-1002.
[46]   | Ehrlich, V., Darroudi, F., Uhl, M., Steinkellnera, H., Gannc, M., Majer, B. J., & Knasmuller, S. (2002). Genotoxic effects of ochratoxin A in human-derived hepatoma (HepG2) cells. Food and Chemical Toxicology, 40(8), 1085-1090.