Background
Rhus verniciflua Stokes (RVS), the lacquer tree, has been used as a traditional medicine and food supplement for a long time in Eastern Asia. In Korea, RVS has been used as a herbal medicine for the treatment of abdominal pain, including pain caused by stomach disorders such as gastritis, and as a hemostatic agent [
1]. RVS has been reported to exhibit anticancer, antioxidant, antimicrobial, and anti-inflammatory activities [
2‐
6]. Moreover, RVS contains a wide variety of flavonoids and polyphenols, including fustin, fisetin, quercetin, butein, p-coumaric acid, kaempferol, sulfuretin, catechol, and ethyl gallate. However, despite the various biological activities of RVS, its use has been limited because of a component called urushiol, which is known to cause allergies. Therefore, urushiol should be removed before using RVS as a food supplement or medicine. Several detoxification methods have been developed to produce urushiol-free RVS, such as heat treatment, solvent extraction, and enzyme treatment by microbial or mushroom mycelium fermentation [
7‐
9]. Currently, allergen-free RVS extracts are being marketed as health functional food in Korea. However, there are no comparative studies on the components and bioactivities of RVS extracts obtained by various detoxification methods. It is important to develop efficient and cost-effective food processing methods in order to enhance the content of bioactive components.
We previously reported that RVS detoxified via a microbial method could alleviate oleic acid (OA)-induced steatosis in HepG2 cells, and that it contained phenolics and cosanols with lipid-lowering potential. In the present study, we compared the effects of three RVS extracts detoxified by different methods with regard to their antioxidant, antimicrobial, and anticancer properties, their suppressive effect on hepatic lipogenesis, and their main components.
Methods
Preparation of DRVE, FRVE, and FFRVE powders
The three types of detoxified RVS extracts used in this study are products that are commercially available in Korea, and were purchased from Okkane (Seoul, Korea). The following three extracts were used: an allergen-free RVS extract detoxified by hot air drying (dried R. verniciflua extract, DRVE), an allergen-free RVS extract fermented with Saccharomyces carlsbergensis (fermented R. verniciflua extract, FRVE), and an allergen-free RVS extract fermented with mushroom mycelium (Fomitella fraxinea-fermented R. verniciflua extract, FFRVE). The extracts (one bottle = 1.5 L) were dried to a powder by freeze-drying.
Determination of antimicrobial activity by agar diffusion method
The antimicrobial activities of the three RVS extracts were determined via modified Kirby-Bauer disk diffusion method [
10,
11]. The test microorganisms used in this experiment were
Propionibacterium acnes (ATCC 6919) and
Trichophyton rubrum (ATCC 22402), and were obtained from the Korean Culture Center of Microorganisms.
P. acnes was cultured anaerobically at 37 °C in Mueller Hinton broth (Difco, USA) and Mueller Hinton agar (0.75% agar), while
T. rubrum was cultured at 26 °C in Sabouraud Dextrose broth and Sabouraud Dextrose agar (0.75% agar). The three powdered extracts, DRVE, FRVE, and FFRVE, were dissolved in water to obtain concentrations of 100 and 1000 mg/mL for each extract. Fifty microliters of each extract was injected into a sterile disk of 6-mm diameter (Toyo Roshi Kaisha Ltd., Tokyo, Japan), and the solvent was allowed to dry off in an aseptic hood. Accordingly, disks were loaded with 5 and 50 mg of each crude extract. Standard disks containing distilled water served as negative controls for the antimicrobial test.
After 24 h, P. acnes or T. rubrum cultures were adjusted to 1 × 108 CFU/mL using 0.5 McFarland standards and inoculated into Mueller Hinton agar and Sabouraud Dextrose agar, respectively. Next, disks containing the extracts were placed on a plate and incubated at 37 and 26 °C, respectively. P. acnes and T. rubrum plates were incubated for 2 and 5 days, respectively. The antimicrobial activities of the extracts were then determined by measuring the diameter of the clear zones around the disks in millimeters. This measurement was carried out in triplicate.
Cell culture
Human hepatocellular carcinoma (HepG2, ATCC HB-8065) and murine normal hepatocyte (AML12, ATCC CRL-2254) cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Welgene, Daegu, Korea) with 1% antibiotics (Welgene). Human prostate cancer (PC3, ATCC CRL-1435), breast cancer (MDA-MB-231, ATCC HTB-26), and colon cancer (HCT116, ATCC CCL-247) cell lines were maintained in RPMI 1640 containing 10% FBS (Welgene) with 1% antibiotics (Welgene).
Cell viability assay
HepG2, PC3, MDA-MB-231, HCT116, and AML12 cells were incubated in 96-well plates (5 × 104 cells/well) and treated with various doses of DRVE, FRVE, and FFRVE (0, 50, 100, 200, and 400 μg/mL) for 24 h. Next, 50 μL of 3-(4–5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 1 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added. After 2 h, formazan crystals in viable cells were dissolved in dimethyl sulfoxide (DMSO), and cell viability was calculated by measuring the optical density (OD) at 570 nm using a microplate reader (Molecular Devices Co, Sunnyvale, CA, USA).
Urushiol in the three extracts was analyzed by high-performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, CA, USA) using Hichrome HPLC columns (5 μm, 250 mm × 4.6 mm, Hichrome, Ltd., Theale, UK). A flow rate of 0.3 mL/min and an injection volume of 10 μL were used. The solvent used was 100% methanol, and the detection wavelength was set to 254 nm.
Moreover, the polyphenols gallic acid, fustin, fisetin, quercetin, butein, and sulfuretin were analyzed in the three RVS extracts via HPLC. The mobile phases were composed of 0.1% formic acid in water (solvent A) and 100% methanol (solvent B), delivered at a flow rate of 0.7 mL/min. The following gradient conditions were used: 0–17 min, 100% B; 17–20 min, 100% B; 20–23 min, 0% B; and 23–30 min, 0% B. The detection wavelength was set to 254 nm, and an injection volume of 10 μL was used.
Oil red O staining
HepG2 cells were incubated in a 6-well plate (5 × 105 cells/well) for 24 h. The cells were then treated with DRVE, FRVE, and FFRVE (400 μg/mL), and stimulated with OA (200 μM) for a further 24 h. After incubation, the supernatants were discarded and cells were washed with phosphate-buffered saline (PBS). Cell fixation was done by treating with 4% formalin for 10 min, followed by washing with PBS and staining with Oil Red O (ORO). ORO stain was extracted using isopropanol and the OD was measured at 510 nm using a microplate reader (Molecular Devices Co).
Western blot analysis
HepG2 cells were incubated in a 6-well plate (5 × 105 cells/well) for 24 h. The cells were then treated with DRVE, FRVE, and FFRVE (400 μg/mL), and stimulated with OA (200 μM) for a further 24 h at 37 °C. Next, cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 M EDTA, 1 mM Na3VO4, 1 mM NaF, and protease-inhibitor cocktail). Protein samples were quantitated using a Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA, USA), resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% polyacrylamide gel, and electrotransferred onto a BioTrace NT transfer membrane (Pall, Gelman Laboratory, Port Washington, NY, USA). After electrotransfer, membranes were blocked with 5% skimmed milk (BD, NJ, USA) and probed with primary antibodies for sterol regulatory element-binding protein 1 (SREBP-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), peroxisome proliferator-activated receptor alpha (PPAR-α, Santa Cruz Biotechnology), AMP-activated protein kinase (AMPK, Cell Signaling, Denvers, MA, USA), phosphorylated AMPK (p-AMPK, Cell Signaling), and β-actin (Sigma-Aldrich) overnight, and exposed to horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies. Protein expression levels were detected using an EZ-Western Lumi Pico kit (DOGEN, Seoul, Korea).
Measurement of cellular triglyceride levels
HepG2 cells were incubated in a 6-well plate (5 × 10
5 cells/well) for 24 h. The cells were then treated with DRVE, FRVE, and FFRVE (400 μg/mL), and stimulated with OA (200 μM) for a further 24 h. To measure cellular triglyceride (TG) levels, a chloroform-methanol extraction method was applied with some modifications, as described in a previous protocol [
12,
13]. Cells were collected and mixed with 1 mL of a 2:1 chloroform:methanol mixture at room temperature for 20 min. After centrifugation at 500×
g for 10 min, the lower layers were collected and dried overnight at 4 °C, and TG levels were measured using a TG assay kit (Asan Pharm, Hwaseong-si, Korea). The OD was measured at 550 nm using a microplate reader (Molecular Devices Co).
Determination of the antioxidant capacity of the three extracts
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was measured by the Blois method [
14]. The extracts were mixed with DPPH and stabilized at room temperature for 30 min. The OD was then measured at 515 nm using a microplate reader (Molecular Devices Co).
Statistical analysis
Results are presented as means ± standard deviation of triplicates. Data were analyzed via Student’s t-test. Differences among groups were considered statistically significant if p < 0.05.
Discussion
Despite the various benefits of RVS, it is known to cause allergy, which may be due to the presence of urushiol. Nevertheless, due to its numerous biological activities, RVS is of high importance in the development of functional food and medicine. Therefore, various detoxification methods have been developed for the removal of allergens from RVS. In Korea, the Ministry of Food and Drug Safety permitted the use of detoxified RVS extracts in 2012. Since then, the types of functional foods containing detoxified RVS extracts have been steadily increasing on a yearly basis. Detoxification methods include the removal of allergens from RVS using solvents [
15], electron beam radiation [
16], high temperature [
17], and microorganisms [
9,
13]. However, RVS extracts detoxified by solvents and irradiation are not suitable for use in food.
We have been studying the biological activities of RVS along with many other researchers, and to date, several bioactivities have been reported. However, studies on detoxified RVS extracts are still inadequate.
To the best of our knowledge, there are no studies comparing RVS extracts prepared by different detoxification methods. Thus, the present study was conducted to compare the bioactive constituents and biological activities of three RVS extracts prepared by different detoxification methods. The investigated extracts (DRVE, FRVE, and FFRVE) are commercially available as allergen-free functional food in Korea.
The antioxidant activities of the three extracts were assessed via DPPH scavenging assay. Among the three extracts, DRVE was found to possess a superior antioxidant activity. Regarding antimicrobial activity, FRVE was the most effective, and inhibited the growth of both P. acnes and T. rubrum. The three extracts successfully suppressed hepatic lipogenesis in an in vitro model of non-alcoholic fatty liver. However, DRVE and FRVE were more effective than FFRVE at the same concentration. These results suggest that the different detoxification methods may induce alterations in the major components of RVS, leading to differences in their activities. Therefore, we analyzed the polyphenolic constituents of RVS in the three extracts.
Polyphenols are bioactive compounds present at high concentrations in various plants [
18]. Many studies have reported that phenolics such as a fustin, fisetin, gallic acid, and quercetin are highly abundant compounds in
R. verniciflua [
3,
19,
20].
According to a previous report, RVS extracts detoxified via heating methods show high gallic acid contents [
17]. This is consistent with our finding that DRVE, an allergen-free RVS extract detoxified by heating to a high temperature, possesses the highest gallic acid content. Moreover, another study reported that an allergen-free RVS extract detoxified by heating (by roasting in an iron pot at 240 °C for 50 min and extracting with water) contains higher amounts of fustin (130 mg/g) than fisetin (20 mg/g) [
21]. Similar to these data, our results demonstrated that DRVE contained three times more fustin than fisetin. However, FFRVE, the RVS extract detoxified by fermentation with
F. fraxinea mushroom, was found to contain more fisetin than fustin
. This result is in agreement with that reported in a previous study [
7]. Finally, FRVE, the RVS extract detoxified by fermentation with the yeast
S. carlsbergensis, contained higher levels of fustin than fisetin or gallic acid, unlike DRVE or FFRVE. As shown in Tables
2 and
3, DRVE was found to contain the highest amount of gallic acid among the three extracts, and gallic acid was found to be the second most effective antioxidant after fisetin. Therefore, gallic acid was considered to be a marker compound reflecting the antioxidant effect of DRVE, while fustin was considered to be a marker compound reflecting the bioactivity of FRVE. However, the contents of all marker compounds, including fisetin, were low. Therefore, FFRVE seems to have a low biological activity when compared to DRVE or FRVE.