Direct human DNA protection by Coriolus versicolor (Yunzhi) extract

Abstract

Context and objective: Scientific evidence has shown Coriolus versicolor (L. ex Fr.) Quel (also known as Yunzhi) has the role of immunomodulator in therapeutic effect. The aim of this in vitro study was to investigate the antioxidative effect of Yunzhi and to explore the mechanisms behind its DNA protection.

Materials and methods: Commercial Yunzhi extract was dissolved in water and diluted in five concentrations (10¹–10⁵ μg/L) with appropriate buffers. Lymphocytes harvested from three healthy subjects were incubated with Yunzhi extract for 30 min. Cells were then subjected to 5 min oxidant challenge by 45 μM hydrogen peroxide. The standard alkaline comet (SAC) assay and lysed cell comet (LCC) assay were performed in parallel. DNA damage of each treatment was scored under a fluorescence microscope and compared with the cells without Yunzhi pretreatment.

Results: U-shaped dose–response was seen in both versions of the comet assay. Yunzhi at 10⁴ μg/L demonstrated a genoprotective effect against oxidative damage in the SAC assay (25% decrease in comet score). In the LCC assay, a trend of protection in lymphocytes was observed but it did not reach statistical significance.

Conclusion: A direct antioxidant effect of Yunzhi against oxidant challenge on the DNA of lymphocytes was evidenced. The active component in Yunzhi was likely to be membrane permeable.

• Antioxidant
• comet assay
• genoprotection
• hydrogen peroxide
• lymphocyte

Introduction

As the worldwide population is enjoying increasing lifespan, there is a trend of reliance on medicine and supplements for a better quality of life due to an increasing prevalence of chronic diseases. A shift from infectious disease to noncommunicable diseases such as cancer, asthma and cardiac disease has been observed with a gradual increase ton the demand of medicines (Kaphle et al., 2006).

Oxidative alterations of DNA have been linked with the development of cancer and have been reported to increase in elderly people. Elevated free radical production and decreased endogenous antioxidant defenses are suggested to be associated with aging (Essick & Sam, 2010; Gautam et al., 2010). There is evidence showing that our body, particularly the organs or cells that consume a large amount of oxygen, is vulnerable to oxidative damage from free radicals and reactive oxygen species (ROS) (William & Serge, 2003). Free radicals inevitably exist as normal products of cellular metabolism in our body. The predominant cellular free radicals are the superoxide (O²•⁻) and hydroxyl (OH•) species. In the presence of reduced metal, a hydrogen peroxide (H₂O₂) molecule forms the highly reactive OH• via the Fenton reaction (Esposito et al., 2002).

Integrated medicine that combines Chinese herbs with the practice of Western medicine as a means of enhancing medical efficacy and improving health for patients with cancer or chronic disease has enjoyed increased popularity over the last decade. Chinese herbs that are well known for their anticancer properties may be a potential source of potent natural antioxidants (Cai et al., 2004). Fungi or mushrooms are important natural source of medicines. Coriolus versicolor (L. ex Fr.) Quel (also known as Yunzhi) is one among the 650 species of higher basidiomycetes that possess medicinal properties in the aqueous extract of polysaccharides or polysaccharide–protein complexes (Rai et al., 2005). The Yunzhi extract which is soluble in water and stable in hot water has been widely used as an adjunct to chemotherapy and as an immuno-stimulator (Cui & Chisti, 2003). Furthermore, Yunzhi extract has previously been studied for its antioxidant activities and demonstrated as an effective antioxidant on animal cells (Tzianabos, 2000; Zhou et al., 1998). It is ideal for sustainable and large-scale production of antioxidants because of the fast growing property. Better understanding of the potential genoprotective effect on human cells could widen the medicinal application of Yunzhi.

The standard alkaline comet (SAC) assay is employed to assess DNA damage in the form of strand breaks in a cell. However, it provides little information on the specific form of damage. Hence, many modified versions of the comet assay have been developed and suggested to be used to study the effects of antioxidants (Wasson et al., 2008). A modified comet assay, the lysed cell comet (LCC) assay, may be useful to investigate the ability of a substance to interact with DNA directly and the possible mechanism behind the genoprotection (Szeto, 2007b; Tice et al., 2000).

Lymphocytes can be used for investigating the potential protective effect of antioxidant in the in vitro environment. Previous studies demonstrated increased resistance to oxidant DNA damage in lymphocytes with the antioxidant pretreatment by using vitamin E and plant-derived compound such as quercetin, epicatechin and ginseng (Szeto, 2007b; Szeto et al., 2002, 2011a).

Under normal intracellular conditions with continuous oxidant challenge of varying intensity, there are four important intracellular mechanisms of DNA protection for the maintenance of low baseline level of DNA damage including scavenging of damaging ROS, enzymatic inactivation of ROS, binding of iron and activation of DNA repair enzymes (Szeto et al., 2002). In this study, both SAC assay and LCC assay were used to elucidate the effect of Yunzhi on human lymphocytes. The effect of Yunzhi on the whole cell was compared with that at the subcellular level to provide the insight of complementary or synergistic intracellular mechanism for antioxidant functions of Yunzhi, if any (Szeto et al., 2002).

Materials and methods

Chemicals and reagents

Type VII low gelling point agarose, standard agarose, phosphate buffered saline (PBS), sodium chloride, disodium ethylenediaminetetraacetic acid dihydrate (EDTA), Tris [hydroxymethyl] aminomethane, hydrogen peroxide solution, ethidium bromide, Triton X-100, sodium chloride, Histopaque 1077 and quercetin were from Sigma-Aldrich (St. Louis, MO); disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide and hydrochloric acid were from Merck (Darmstadt, Germany); capsule of Yunzhi extract (Chinese Medicine Council of Hong Kong Transitional Registration Number HKP-01010) which was claimed to be manufactured with 100% natural Chinese herbs was from a local pharmacy.

Blood samples

The study was approved by the Human Research Ethics Committee of Charles Sturt University (2011/148) and Macao Society for the Study of Women’s Health. Three blood donors (two males and one female, aged 35–54) were requested to sign an informed consent. Information regarding their age, gender and recent medical history were provided for reference of the study. They were asked to maintain a normal dietary pattern but stop taking supplements, if any, for at least two weeks. One milliliter of venous blood was collected into an EDTA bottle. All procedures for collection and handling of blood were performed according to the protocol, which met the requirements of the Australian National Statement on Ethical Conduct in research involving humans.

Blood (100 μL) was mixed with 1 mL PBS in a microtube and gently mixed. Then 100 μL of Histopaque 1077 was underlaid as described in previous studies (Szeto, 2007b; Szeto et al., 2002). The microtubes were centrifuged at 1500 rpm for 5 min. Immediately, lymphocytes retrieved from just above the boundary between the PBS and Histopaque layer were added to another 1 mL PBS in other microtubes. The same centrifugation step was taken and as much supernatant was removed from the pelleted lymphocytes. Cells harvested were tested in the subsequent comet assay.

Treatment

Preparation of Yunzhi solutions of different concentrations was required to be done 2 h before the experiment for the pretreatment of cells with the solution and subsequent stressing with freshly prepared 45 μM H₂O₂ solution.

Preparation of Yunzhi solutions

In both assays, five testing concentrations ranging from 10⁵ to 10 μg/L from 1% (w/v) stock solution were prepared. PBS (pH 7.4) was used to dissolve the extract in the SAC assay while phosphate buffer (0.4 M, pH 7.4) was used in the LCC assay. Powder of Yunzhi extract from capsules was weighed and dissolved in respective solvents at 37 °C. The mixture was continuously agitated for 2 h to assist dissolving of powder. After centrifugation at 3000 rpm for 10 min, only the clear upper portion of the solution was taken out as a stock solution for further serial dilutions. Quercetin solutions at 12.5 and 3 μM were used as positive controls in SAC and LCC assays, respectively.

Oxidative challenge

The lymphocytes pretreated with the Yunzhi extract were subjected to oxidant challenge with H₂O₂. One milliliter of Yunzhi extract solution was added to the microtube containing washed lymphocytes and was mixed gently. PBS without Yunzhi extract was added as control. The cell suspensions were incubated at 37 °C for 30 min. The microtubes were centrifuged at 1500 rpm for 5 min at room temperature. After discarding the supernatant, cells were washed with 1 mL cold of PBS and spun again. Yunzhi extract-treated lymphocytes were then stressed with 1 mL of cold H₂O₂ (45 μM) in PBS for 5 min. The cell suspension was washed and ready for use in the comet assay, which was performed with the following procedure (Szeto et al., 2002).

Comet assays

The SAC assay was used to detect the DNA strand breaks in lymphocytes. Whole cells were embedded on standard agarose, then lysed and electrophoresed. In the LCC assay, lymphocytes were lysed prior to pretreatment with Yunzhi extract and stressed with H₂O₂ for determining chemical and biological characteristics of Yunzhi extract. Experiments were mostly performed at room temperature unless stated, and under dim light to minimize the occurrence of additional DNA damage.

SAC assay

The current SAC protocol was adopted from the previous study (Szeto et al., 2002). Cells for slide preparation were mixed well with low melting agarose and added on the top of a layer of standard agarose. Cell membrane and other proteins were removed in the lysis solution containing high salt level and detergent. DNA was then unwound at high pH followed by electrophoresis at 25 V for 20 min. Electrophoresis solution was removed by immersing the slides in tap water. The coded slides were stained with ethidium bromide, and scored under a fluorescent microscope by other team member.

LCC assay

The LCC assay was performed following the procedure of Szeto et al. (2002). Isolated and washed lymphocytes were embedded in agarose gel on microscopic slides. These slides were transferred to a Coplin jar with 0.4 M phosphate buffer for 10 min at room temperature before and after the cell lysing step, pretreatment with Yunzhi extract and stressing with 45 μM H₂O₂. Slides with stressed and unstressed (control) cells were transferred to a Coplin jar for the rest of comet assay procedure as described previously (Szeto et al., 2002).

Scoring of DNA damage/Comet scoring

DNA damage was determined with visual scoring. Cells were classified into five categories ranging from 0 to 4 on the basis of tail length and shape where score 4 signified the greatest damage (Szeto et al., 2011b). One hundred cells were scored in every single gel. Each assay using 300 cells from three different subjects was scored at each concentration for each treatment. Reading of comets was performed using a fluorescence microscope (Nikon, Eclipse-Ni, with filter combinations encompassed excitation wavelength: 510–560 nm). Results of comet scores were presented as mean ± SD in treated and untreated cells.

Statistical analysis

One-way ANOVA with post hoc Dunnett’s t-test was used to investigate the differences in DNA damage score of cells that were pretreated with various concentrations of Yunzhi extract. A p < 0.05 was considered as significantly different.

Results

There was a significant genoprotection by aqueous Yunzhi extract pretreatment at 10⁴ μg/L with 25% decrease of DNA damage in SAC assay (p < 0.05, Figure 1). A similar trend was seen in the LCC assay but it did not reach the significant level. About a 23% reduction of DNA damage was observed at 10⁴ μg/L (Figure 2). In both versions of the comet assay, a U-shaped dose–response was observed. Qucrcetin (postive control) was able to decrease DNA damage by 30% and 56% in SAC and LCC assays, respectively.

Figure 1. Effects of the aqueous extract of Yunzhi and data are expressed as the comet score of DNA damage relative to that in control cells in the SAC assay. Data were obtained in three separate experiments. Lymphocytes in microtubes were separately pre-incubated with five different concentrations (w/v) of Yunzhi extract followed by H₂O₂ challenge. Signifcant decrease in DNA damage at 10⁴ µg/L concentration was seen (p < 0.05).

Figure 2. Effects of aqueous extract of Yunzhi and data are expressed as the comet score of DNA damage relative to that in control cells in the LCC assay. Lysed lymphocytes embedded in gel were separately pre-incubated with five different concentrations (w/v) of Yunzhi extract followed by H₂O₂ challenge. The trend of decreased DNA damage was seen but did not reach a signifcant level.

Discussion

Antioxidants can be divided into two groups, enzymatic/nonenzymatic and endogenous/exogenous. Exogenous and nonenzymatic antioxidants are the focus of most of the researchers in the field of nutritional studies. The standard comet assay responds to a wide range of antioxidants but gives little information on the mechanism of genoprotective action. Certain antioxidants had shown their protection against oxidant challenge, while others showed no effect or even induced DNA damage in subjects with exposure to metals (Flora, 2009). With the aid of the LCC assay, more information on genoprotection or genotoxic property of a compound can be revealed. Two versions of the comet assay are therefore useful in investigating the efficacy of putative antioxidants. Previous studies attempted to find the potential role of various dietary antioxidants in DNA protection and the mechanism of their protection. Concepts regarding the action of antioxidant treatment and possible mechanisms of the effects on DNA by respective treatment have been mapped to possible experimental outcome (Szeto et al, 2002; Ulrich-Merzenich et al., 2010). These are helpful in the planning of further antioxidant studies and facilitating interpretation of results.

The LCC assay provided a fast way to measure the endpoint of Yunzhi pretreatment and oxidative challenge with H₂O₂. In the LCC assay, the DNA of lymphocytes could neither be repaired nor preceded to apoptosis once stress was applied. A slightly lower comet scores across all concentrations of Yunzhi tested suggested less mechanical damage caused by centrifugation steps in the course procedure. However, a similar percentage of DNA damage reduction in both SAC and LCC assays implied that the action was direct and was unlikely owing to enzymatic origin.

The occurrence of such U-shaped dose–response relationships, also termed as hormesis, has been documented in many biological, toxicological and pharmacological investigations (Calabrese & Baldwin, 2001). Whereas hormesis has been defined as a dose–response relationship in which there is beneficial effect or stimulatory response at low doses, but little effect or inhibitory response at high doses, resulting in a U-shaped dose–response (Calabrese & Baldwin, 2001). This phenomenon sometimes may be explained in one generic mechanism arisen as a consequence of overcompensation by adaptive, homeostatic mechanisms functioning in biological systems. Another example was found in resveratrol, a polyphenolic antioxidant present in red wine which provides cardioprotection at lower doses, but can kill cancer cells at relatively higher doses by exerting a death signal. Important issues raised by U-shaped dose–response curves including the identification of no-observed-effect levels and the evaluation of different beneficial outcomes and the tradeoffs of a given agent. The possibility of deleterious effects of Yunzhi is small as the potential toxicity of water extract of Yunzhi was evaluated in the rat. It did not cause remarkable adverse effect in rats after acute and subchronic administration (Hor et al., 2011).

The antitumor activity of the Yunzhi extract in animal models has been extensively demonstrated with in vivo studies. An immunomodulating mode of action was suggested to be related to this activity (Ooi & Liu, 2000). With in vitro studies, Yunzhi extract has shown an antiproliferating effect on various cancer cell lines (Ho et al., 2006; Zhou et al., 2007). Moreover, direct cytotoxic effects on the cancer cells by some of the polysaccharides found in Yunzhi extract have also been shown (Cheng & Leung, 2008). In addition, the hot water extract of Yunzhi has been shown to have hepatoprotective activity (Kim et al., 2000). Recently, a study suggested that the consumption of Yunzhi extract may be able to prevent or reduce hypercholesterolemia which is an important risk factor for cardiovascular diseases (Hor et al., 2011). In Asian countries, Yunzhi extract is in clinical use for adjuvant cancer therapy with conventional radiotherapy and chemotherapy (Kidd, 2000).

Besides a desirable DNA protection effect, side effects of Yunzhi may include nausea, vomiting, diarrhea and loss of appetite, but they are rarely reported (Mitomi et al., 1992). When the extracts were used in conjunction with chemotherapy agents, low-grade hematologic and gastrointestinal toxicities (Ohwada et al., 2004), passage of dark-colored stools (Shiu et al., 1992) and darkening of fingernails (Kidd, 2000) have been reported. However, the root cause of these effects were possibly caused by the chemotherapy (Szeto, 2007a). More clinical studies for comprehensive data are needed for the better use of this potential antioxidant. Further studies on the bioavailability and in vivo efficacy are deemed to be necessary. The ultimate goal should be contribution to efforts for the prevention and control of acute or chronic disease in the worldwide aging and urbanizing global population.

Conclusions

In conclusion, direct antioxidant effect of Yunzhi against oxidant challenge on DNA was observed. Similar efficacy of the antioxidant effect in both SAC and LCC assays indicated that the active ingredient from Yunzhi was not restricted by the cellular membrane. Better understanding of the mechanism of antioxidant action is essential to ensure a consistent amount of bioactive components of Yunzhi and reproducible pharmacological actions in future preparations.

Declaration of interest

The authors declare no conflicts of interest.

Acknowledgements

The authors wish to thank Macao Society for the Study of Women’s Health and Pathology Department of Ruttonjee Hospital for supporting this study.