Plasma-Polymerized Phlorotannins and Their Enhanced Biological Activities

Article abstract of DOI:10.1021/acs.jafc.9b07077

Here, we investigated the effect of cold plasma (CP) on the biological activities of phloroglucinol. Phloroglucinol (7.92 and 15.84 mM in methanol) was treated with air dielectric barrier discharge plasma at 250 W. In vitro, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and ferrous-reducing antioxidant power (FRAP) values of phloroglucinol increased in plasma treatment in a time-dependent manner. CP treatment of phloroglucinol decreased the lipid oxidation of oil emulsion during storage and increased the antimicrobial activity against Bacillus cereus, Escherichia coli O157:H7, and Staphylococcus aureus. Angiotensin-converting enzyme (ACE) inhibitory activity of phloroglucinol increased and total phenolic content decreased based on CP treatment. The CP-induced polymerization of phloroglocinol to phlorotannin derivatives was identified using high-performance liquid chromatography with UV detector and electrospray ionization/mass spectrometry (HPLC-UV-ESI/MS) method. Consequently, the polymer structure of phloroglucinol was found in the CP-treated phloroglucinol. In addition, CP enhances the biological activity of phloroglucinol and could be applied to bioactive materials in food and related industries.

Full text of DOI:10.1021/acs.jafc.9b07077

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Bioactive Constituents, Metabolites, and Functions
Plasma-polymerized phlorotannins and their enhanced biological activities
Hyun-Joo Kim, Hae In Yong, Byong Won Lee, Sanghoo Park, Ki Ho Baek, Tae Hoon Kim, and Cheorun Jo
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b07077 • Publication Date (Web): 22 Jan 2020
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Plasma-polymerized phlorotannins and their enhanced biological activities
a
b
a
c
d
Hyun-Joo Kim , Hae In Yong , Byong Won Lee , Sanghoo Park , Ki Ho Baek ,
e
d,f*
Tae Hoon Kim , and Cheorun Jo
aCrop Post-harvest Technology Division, Department of Central Area Crop Science,
National Institute of Crop Science, Rural Development Administration, Suwon 16613, Korea
bResearch Group of Food Processing, Korea Food Research Institute, Wanju 55365, Korea
cPlasma Technology Research Center, National Fusion Research Institute, Gunsan 54004,
Korea
dDepartment of Agricultural Biotechnology and Research Institute of Agriculture and Life
Science, Seoul National University, Seoul 08826, Korea
eDepartment of Food Science and Biotechnology, Daegu University, Daegu 34141, Korea
fInstitute of Green Bio Science and Technology, Seoul National University, Pyeongchang
2
5354, Korea
*
To whom correspondence should be addressed.
Dr. Cheorun Jo, Phone: +82-2-880-4804, Fax: +82-2-873-2271, E-mail: cheorun@snu.ac.kr
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ABSTRACT
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Here, we investigated the effect of cold plasma (CP) on the biological activities of
phloroglucinol. Phloroglucinol (7.92 and 15.84 mM in methanol) was treated with air
dielectric barrier discharge plasma at 250 W. In vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical-scavenging activity and Ferrous reducing antioxidant power (FRAP) values of
phloroglucinol increased in a plasma treatment time-dependent manner. CP treatment of
phloroglucinol decreased the lipid oxidation of oil emulsion during storage and increased the
antimicrobial activity against Bacillus cereus, Escherichia coli O157:H7, and Staphylococcus
aureus. Angiotensin-converting enzyme (ACE) inhibitory activity of phloroglucinol
increased and total phenolic content decreased based on CP treatment. CP-induced
polymerization of phloroglocinol to phlorotannin derivatives was identified using high
performance liquid chromatography with UV detector and electrospray ionization/mass
spectrometry (HPLC-UV-ESI/MS) method. Consequently, the polymer structure of
phloroglucinol was found in CP-treated phloroglucinol. In addition, CP enhances the
biological activity of phloroglucinol and could be applied to bioactive materials in food and
related industries.
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KEYWORDS: cold plasma, phloroglucinol, polymerization, biological activities
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INTRODUCTION
Seaweed is one of the most abundant sources of novel biologically active compounds
known to exhibit biological active characteristics and has potential industrial applications as
functional foods, nutraceuticals, and cosmeceuticals. Among the bioactive compounds, algal
phlorotannins are one of the most important natural-generated secondary metabolites with
extensive nutritional, functional, and bioactive properties.1,2
Phloroglucinol, a phenolic compound, is a natural phlorotannin components of brown
algae, comprises an aromatic ring with three hydroxyl groups. Upon polymerization to
phlorotannins, its concentration in Fucales was found to be around 10% and may reach as
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high as 20% dry weight. The antioxidant capacity of a monomeric phenolic compounds is
determined by the extent of conjugation and degree of hydroxylation. Phloroglucinol shows a
high level of hydroxylation by aromatic nucleus surrounded by three hydroxyl groups.3,4
Polymers of phloroglucinol exhibit various properties, including antioxidant, anticancer,
antimicrobial, and antiviral activities.5,6 Many studies have demonstrated the ability of
phloroglucinol to bind with various chemical compounds such as dimeric- and polycyclic-
groups.7,8 However, the present methods to produce polymeric phlorotannins are inefficient
and expensive.
Plasma, an ionized quasi-neutral gas generated by electric discharge, comprises ions,
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electrons, free radicals, atomic species, UV photons, and so on. Plasma exerts bactericidal,
virucidal, and fungicidal effects owing to the presence of various antibacterial substances,
including ultraviolet photons, reactive species, and charged particles.10,11 In particular, cold
plasma (CP) has been extensively utilized and optimized for applications in food processing
industries. CP has been evaluated to preserve the quality characteristics of various food items
such as cheese, brown rice, and raw meat.12-14 In addition, it has been applied for meat curing
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as a nitrite source and microbial inactivation without the external addition of sodium or
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potassium nitrite. In regard to the safety as foods, recent studies using CP-treated chicken
and water did not show any genotoxicity by Ames test.15,16 The sausage made with CP-
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treated water also did not show any immune toxicity. In addition, feeding CP-treated edible
film coating to rats did not induce any significant toxicological effects.17
Previous studies have shown that CP-induced structural modification of naringin and
quercetin could improve their antioxidant, antimicrobial, and tyrosinase inhibition
activities.18,19 These authors have demonstrated that the structural modifications is mainly
degradation of original compounds. However, very few studies have attempted to utilize CP
to improve the physiological activities of natural materials via polymerization, for example,
the polymerization of phloroglucinol to phlorotannin. Therefore, the purpose of the present
study was to estimate changes in physiological activities and elucidate the structural
condensation of phloroglucinol solution by CP.
MATERIALS AND METHODS
Sample preparation and plasma treatment
Phloroglucinol was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and samples
(7.92 and 15.84 mM, dissolved in methanol) were prepared before plasma treatment. The CP
apparatus was specially designed to remotely treat phloroglucinol samples. We employed an
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airtight, six-sided dielectric container (137 × 100 × 53 mm ), and polytetrafluoroethylene
(PTFE) sheets sandwiched by copper sheets were installed at inner walls of the container to
produce surface dielectric barrier discharge. A 15-kHz bipolar square wave power supply was
applied. Air plasma was created with 250 W of input power using atmospheric air at the
PTFE surface inside the container. Each sample was put in the container and the plasma was
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then treated for 5, 10, and 20 min, respectively. Detailed schematics of the CP system can be
found in Ref. 12.
Total phenolic content (TPC)
The TPC of samples was colorimetrically estimated using the Folin-Ciocalteu method.20
Sample (0.1 mL) was mixed with 0.2 mL of Folin-Ciocalteu reagent and 3 mL of a 5%
sodium carbonate solution was added and vortexed. The samples were incubated for 2 h at
room temperature and then the absorbance was measured at 765 nm using a
spectrophotometer (DU 530; Beckman Instruments Inc., Fullerton, CA, USA). Results are
expressed as micrograms gallic acid equivalents per mililiter (GAE/mL sample).
Antioxidant properties
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity
DPPH radical-scavenging activity was analyzed based on a previous study21 with slight
modifications. One milliliter of sample was added to test tube containing 1 mL of DPPH
solution (0.2 mM in methanol), and methanol was used as a blank. The mixture was shaken
and then reacted for 30 min at room temperature. The absorbance was measured using a
spectrophotometer (DU 530; Beckman Instruments Inc) at 517 nm wavelength. The DPPH
radical-scavenging activity was calculated by the following equation:
DPPH radical-scavenging activity (%) = (1 – A /A ) × 100
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where A is the absorbance of the blank and A is the absorbance of the sample.
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Ferric reducing antioxidant power (FRAP)
The FRAP was estimated using a spectrophotometer (Beckman Instruments Inc.) according
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to a previous study. FRAP assay measures the changes in absorbance 593 nm due to the
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formation of the blue colored Fe -tripyridyltriazine compound from colorless oxidized Fe
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form by the action of electron donating antioxidants. The world FRAP reagent was prepared
by mixing 10 volumes of 300 mM acetate buffer (pH 3.6), with 1 volume of 10 mM TPTZ
(2,4,6-tripyridyl-s-triazine) in 40 mM hydrochloric acid and with 1 volume of 20 mM ferric
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chloride. Aqueous solutions of known Fe concentration, in range of 100-1,000 μM (FeSO
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ㆍ7H O) were used for calibration.
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Lipid oxidation
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A 10% oil-in-water emulsion was prepared for the 2-thiobarbituric acid reactive substances
(TBARS) measurement. Soybean oil (2.0 mL) was homogenized (T10 basic, Ika Works,
Staufen, Germany) in 18 mL of distilled water at 23,000 rpm for 2 min with 100 μL of
Tween-20. Samples were prepared by mixing oil-in-water emulsion (8 mL) and plasma-
treated phloroglucinol (1 mL). The samples were incubated for 3 h at 37°C, and TBARS
value was measured every hour. In total, sample (2 mL) was added to 4 mL of thiobarbituric
acid (TBA)/trichloroacetic acid (TCA) solutions (20 mM TBA/15% TCA). The mixture was
heated in a water bath at 90°C for 15 min to allow color development. Following incubation,
the sample was cooled in cold water for 10 min and centrifuged (UNION 32R, Hanil Science
Industrial, Co., Ltd., Korea) at 2,265 ×g for 10 min at 4°C. The absorbance of the supernatant
was measured at 532 nm against a blank containing 1 mL of distilled water and 2 mL
TBA/TCA solution. The amounts of TBARS used to express the results were calculated from
the standard curve prepared with known concentrations of tetraethoxypropane (TEP). The
results were expressed as TBARS values, mg malondialdehyde (MDA)/kg sample. Methanol
was used as a negative control.
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Antimicrobial activity
Bacillus cereus (KCTC 3624), Escherichia coli O157:H7 (ATCC 43889), and
Staphylococcus aureus (KCTC 1916) were provided from the Korean Collection for Type
Culture (KCTC, Daejeon, Korea). B. cereus was cultured in fresh nutrient broth, while E. coli
O157:H7 and S. aureus were cultured in fresh tryptic soy broth. Broth was inoculated using
agar slant culture and incubated at 37°C for 24 h. After incubation, the 100 μL culture
solution was added to fresh broth and cultured for 18 h. The cultures were washed twice with
sterile saline solution by centrifugation at 2,265 ×g for 15 min at 4°C in a centrifuge (UNION
32R, Hanil Science Industrial, Co., Ltd., Korea). The cell pellets were resuspended in a saline
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solution for a final concentration of 10 to 10 colony forming units (CFU)/mL.
The antimicrobial activity was estimated by agar disc diffusion assay. A 100 μL of
bacterial suspension was spread onto the agar plates. Sterilized paper disc (8 mm diameter)
containing 50 μL of the filtered sample (0.22 μm syringe filter) was placed over the surface
of the agar and incubated for 48 h at 37°C. The diameters of the clear zones around the disc
were measured. Methanol was used as control.
Angiotensin-converting enzyme (ACE) inhibitory activity
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ACE inhibitory activity of samples was measured using the method of Jang et al. with some
modifications. An aliquot of sample (10 μL) was mixed with 30 μL of 0.1 M sodium borate
buffer (pH 8.3) containing 10 mM of N-Hippuryl-l-Histidil-l-Leucine (HHL) and incubated at
37°C for 10 min. After incubation, 10 μL of ACE (0.5 unit/mL, Sigma, buffer information)
was added and the mixture was incubated for 30 min at 37°C. The enzymatic reaction was
stopped with the addition of 50 μL of 1 M hydrochloric acid (HCl). Hippuric acid formed by
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the action of the angiotensin-converting enzyme on HHL was extracted with 300 μL of ethyl
acetate and centrifuged at 1,500 ×g for 10 min at 4°C. The supernatant (250 μL) was
transferred to a culture tube and incubated at 37°C for 1 h. The sample was dissolved in 300
μL of distilled water, vortex mixed, and analyzed for its absorbance at 228 nm against a blank
prepared with 10 μL of distilled water. ACE inhibitory activity was measured using the
following equation:
ACE inhibitory activity = [(blank abs − sample abs)/blank abs]  100
High-performance liquid chromatography (HPLC)-UV and liquid chromatography and
electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-
MS)
The identification of the phlorotannins derived from phloroglucinol was carried out using a
Thermo Scientific HPLC UltiMate 3000 system with UV detector coupled on-line with a
Bruker Daltonics micrOTOF-QII mass spectrometer. Phlorotannins were separated on the
Agilent Eclipse XOB-C18 column (5 μm, 4.6  150 mm) at 30C. Samples (10 μL) were
injected, and elution was completed in 40 min with a sequence of linear gradients and an
isocratic flow rate of 0.5 mL/min. The mobile phase comprised 0.1% formic acid in water (A)
and 0.1% formic acid in acetonitrile (B). The gradient elution program was as follows: 0 min,
10% B; 10 min, 20% B; 20 min, 35% B; 30 min, 55% B; 35 min, 70; 38 min, 90% B; 38-40
min, 90% B. The UV detector was set at 280 nm. The separated molecules were directly
introduced into the mass spectrometer by electrospray. The mass spectra were scanned
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between 50-1000 m/z in anion mode (ESI ) using a nebulizing gas pressure set at 1.5 bar.
Drying gas flow rate was 8 L/min and drying gas temperature was set at 200C. Other mass
spectrometric conditions were programmed as follows: collision energy 10.0 eV; collision RF
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300.0 Vpp; transfer time 60 μs; pre pulse storage 6.0 μs. The control of the instrument was
carried out using microTOF control 2.3.
Statistical analyses
Each experiment was performed in three replicates. Data were analyzed using SAS software
(Release 8.01, SAS Institute, Inc., Cary, NC, USA). Statistical analysis was performed with
one-way analysis of variance (ANOVA). The differences among the mean values were
determined using the Student Newman Keul’s multiple-comparison test at a confidence level
of p < 0.05. Mean values and standard deviations are reported.
RESULTS AND DISCUSSION
Total phenolic content
Several studies have reported the correlation between antioxidant activity and phenolic
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contents. Polyphenols, the plant’s secondary metabolite, exert its antioxidant property by
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scavenging reactive oxygen/nitrogen species such as OH, NO , and OONO . Irradiation
tends to increase the TPC and antioxidant activities of quercetin in tomato and lettuce.25,26
Previous studies have shown that the increase in phenolic content after CP treatment may be
associated with the breakage of multimeric polyphenols.18,19 In the present study, however,
the TPC of phloroglucinol solution decreased after CP treatment (Table 1), but biological
activities such as antioxidant activities showed reverse results. Thus, the extracts would exert
protective effect against reactive radical formation and oxidative damage to biomolecules.24
This result provides the evidence of CP-induced polymerized compounds, phlorotannins.
Thereafter, we identified the structure of compounds after CP treatment.
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Antioxidant properties
The DPPH radical-scavenging activities of phloroglucinol solutions increased after plasma
treatment in a time-dependent manner (Table 2). The DPPH radical-scavenging activity of
phloroglucinol solution after CP treatment for 20 min was almost twice the activity of the
untreated solution. At both concentrations, DPPH radical scavenging activity increased with
an increase in plasma treatment time, and the highest activity was recorded for 20 min
plasma-treated samples.
FRAP values are generally utilized to evaluate antioxidant activity. The FRAP value of
phloroglucinol solutions increased with increased plasma treatment time (Table 3).
Regardless of the concentration, FRAP values of phloroglucinol solutions significantly
increased after 20 min of plasma treatment. Here, antioxidants could have act as reductant.
The inactivation of the oxidant by the reducing agent can be explained by a redox reaction,
wherein one reactive species is reduced at the expense of oxidation of another species,
antioxidants.27
TBARS value of oil emulsion containing phloroglucinol solution was lower than that of
the control (Fig. 1). CP treatment of phloroglucinol resulted in a decrease in lipid oxidation
during storage. This phenomenon can be described as free radical formation by the molecules,
which can be decreased after CP treatment.28
Several studies have reported that phlorotannin derived from brown algae has antioxidant
activities against oxidative damage caused by free radicals. The antioxidant activities may be
the result of the scavenging of oxygen-containing compounds, scavenging of radicals
produced by peroxidation, or metal-chelating ability.29,30 Eckol, dieckol, phlorofucofuroeckol
A, and 8,8-bieckol exhibit a strong inhibitory effects on phospholipid peroxidation at
concentrations of 1 µM in liposome system, and these phlorotannins have significant radical-
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scavenging activities against DPPH radicals and superoxides as compared to α-tocopherol
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and ascorbic acid. Hermund et al. studied antioxidative phlorotannins of Icelandic brown
algae, Fucus vesiculosus, and reported that, generally, low molecular weight phlorotannins
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had higher antioxidant capacity. Another study reported that the majority of low molecular
weight phlorotanin with antioxidative activity was found to have a molecular weight range
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equivalent to the 4-12 monomer of phloroglucinol, which are the similar range of the CP-
polymerized phlorotannins in the present study.
The treatment of natural materials with CP was carried out by our previous studies. For
instance, DPPH radical-scavenging activity of naringin was originally 1.45% but increased to
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38.20% after CP treatment. In addition, the TBARS value of oil emulsion containing CP-
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treated quercetin solution was higher than that of the non-treated quercetin solution. Kim et
al.35 found that a new flavanone glycoside 2, containing a methoxyalkyl group, showed
enhanced antioxidant properties compared to naringin after CP treatment. Their results
suggest that CP could modify the chemical interactions and induce phenol production.
Antimicrobial activity
The agar disc diffusion assay was performed to investigate the antimicrobial activity of
phloroglucinol solutions against three pathogens, B. cereus, E. coli O157:H7, and S. aureus
(Table 4). Untreated samples and samples treated with plasma for 5 min showed no
antimicrobial activity. However, the samples treated with plasma for more than 10 min
showed an increase in antimicrobial activities against these pathogens tested. A time-
dependent increase in the antimicrobial activity was reported, consistent with the results of
antioxidative activity assay. The diameters of clear zone for 20 min plasma-treated samples
were the highest for B. cereus, which were ranged from 24 to 35 mm. However, the
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antimicrobial activity after CP treatment was low against E. coli O157:H7. The increase in
the antimicrobial activity of natural materials after CP treatment is consistent with the result
reported by previous studies.18,19
Previous studies have reported that certain physicochemical treatments can affect the
structure or chemical group of natural substances and increase its antibacterial activity. An et
al.36 suggested that gamma-irradiated polyphenols exhibit changes in the activities of
functional groups, such as –COOH or –OH, resulting in enhanced antimicrobial activity.
When chitosan was irradiated at 100 kGy, the chitosan gives degradation and lower
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molecular weight, and which results in increased antibacterial activity. Our previous study
also proposed that CP treatment can increase antimicrobial activity of quercetin and it could
be explaining by structural modifications.19
In this study, the mechanism of increase in antimicrobial activity can also elucidated
through the determination of structural modifications after CP treatment. In Fig 4, when CP
was treated to phloroglucinol, it was polymerized to phlorotannins which is well known
bactericidal material. Nagayama et al.38 and Eom et al.39 reported the strong bactericidal
activity of phlorotannins from Ecklonia kurome and marine brown algae. Due to
phlorotannins produced after CP treatment, antimicrobial activity could be increased as Table
4.
Polyphenols are the most important active compounds to control bacteria.40,41 Nagayama et
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al. reported the strong bactericidal activity of phlorotannins from Ecklonia kurome. An et
al.36 suggested that gamma-irradiated polyphenols may not exhibit any change in their
chemical structures but undergo changes in the activities of functional groups, such as –
COOH or –OH, resulting in enhanced antimicrobial activity. The mechanism underlying the
increase in antimicrobial activity could be elucidated through the determination of structural
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modifications after CP treatment.
ACE inhibitory activity
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Hypertension is a major risk factor for various cardiovascular diseases. In particular, ACE
is involved in the developing hypertension because it can inactivates the vasodilator
bradykinin and produces a potent angiotensin Ⅱ. Therefore, ACE inhibition is an in vitro
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indicator of the antihypertensive properties of food or natural materials.
The ACE inhibitory activities of untreated phloroglucinol solutions of 7.92 and 15.84 mM
were 7.78% and 18.33%, respectively (Fig. 2). After 20 min of CP treatment, the ACE
inhibitory activities of these samples increased to 24.63% and 49.07%, respectively. These
samples showed higher ACE inhibitory activities as plasma treatment time increased at both
concentrations. The ACE inhibitory activity of natural material from algae has been widely
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reported. Wijesing et al. showed that ACE inhibition can be closely related to the protein
binding capacity of phlorotannins; tannin has the ability to produce a strong complexes with
proteins by covalent condensation, or by hydrogen bonding via peptide or amine linkage.
However, no previous study has shown the increase in ACE inhibition activity after CP
treatment.
HPLC-UV and LC-ESI-QTOF-MS analysis
A sample of pure phloroglucinol in methanol was treated with CP for 20 min, and the ion
generation pattern was directly monitored using QTOF-MS and HPLC-UV systems.
Considering that the peak area of untreated phloroglucinol was 100%, only 55.45% of
phloroglucinol was presented after 20 min of CP treatment, while 9 new peaks were detected
except for peak area of <1% (Fig. 3). These chromatograms indicate that CP treatment
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induced structural changes in phloroglucinol that were closely associated with enhancement
in biological activities. Choi et al. 44 also showed that degree of oligomerization increased
with increasing CP treatment time on phloroglucinol. In addition, oligomerization of
phloroglucinol induced by CP increased the antidiabetic activities measured by α-glucosidase
inhibitory activity. 44 However, there may be an appropriate range of polymerization for
optimum bioactivities.32, 33
−
MS analyses (Fig. 4) of peaks 1, 2, 5, 6, 7, and 8 revealed [M-H] ion at m/z 263.1, 401.1,
539.1, 677.2, 815.2, and 953.2, respectively. Molecular weights of peaks sequentially
increased by m/z 138 (methyl phloroglucinol), and their retention times were 12.3, 15.9, 19.8,
21.4, 22.5, and 23.5 min, respectively (Table 5). In MS/MS analyses, peaks 2 and 5 had an
MS/MS fragment anion at m/z 263.1 derived from a dimer, while peak 6 had MS/MS
fragment anions at m/z 263.1 (dimer) and 401.1 (trimer). Peaks 7 and 8 had MS/MS fragment
anions at m/z 263.1 (dimer), 401.1 (trimer), and 539.1 (tetramer). On the basis of the above
results, these molecules were tentatively assigned as phloroglucinol polymers, dimer (1),
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trimer (2), tetramer (5), pentamer (6), hexamer (7), and heptamer (8) (Fig. 5). Peak 3 (Rt =
−
17.5 min) with an [M-H] ion at m/z 825.2 and MS/MS anions at m/z 263.1 (dimer) and 401.1
(trimer) was tentatively identified as compound 3 (hexamer derivative). Peak 4 showing an
−
[M-H] ion at m/z 473.1 and an MS/MS anion at m/z 197.0 was tentatively identified as
−
compound 4 (trimer derivative). Peak 9 produced an [M-H] ion at m/z 689.2 and MS/MS
ions at m/z 275.1 (dimer derivative) and 401.1 (trimer) was identified as compound 9
(pentamer derivative).
These results show that physiological properties such as antioxidant, antimicrobial, and
ACE inhibition activities of phloroglucinol could be substantially improved by treatment with
CP. This increase in bioactivity may be related to the polymerization of phloroglucinol to
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phlorotannins by CP-induced polymerization and structural modification. Thus, CP treatment
has the potential to increase the biological activity of natural compounds. Future work with
seaweed will be worth studying.
Abbreviations
ACE, angiotensin-converting enzyme; CP, cold plasma; DPPH, 1,1-diphenyl-2-
picrylhydrazyl; FRAP, ferric reducing antioxidant power; HPLC, high-performance liquid
chromatography; LC-ESI-QTOF-MS, liquid chromatography and electrospray ionization
quadrupole time-of-flight mass spectrometry; TBARS, 2-thiobarbituric acid reactive
substances
Acknowledgment
This work was supported by the R&D Program of Plasma Advanced Technology for
Agriculture and Food (Plasma Farming) through the National Fusion Research Institute of
Korea (NFRI) funded by government funds. Also, this work was supported by the BK21 Plus
Program of the Department of Agricultural Biotechnology, Seoul National University, Seoul,
Korea.
Funding
This work was supported by R&D Program of Plasma Advanced Technology for
Agriculture and Food (Plasma Farming) through the National Fusion Research Institute of
Korea (NFRI) funded by government funds.
Notes
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The authors declare no competing financial interest.
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Biosci. Biotechnol. Biochem. 2018, 82, 2019-2063.
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Table 1. Total phenolic contents of phloroglucinol treated with dielectric barrier
discharge plasma
Treatment time (min)
Total phenolic content (μg GAE/mL sample)
7
.92 mM
15.84 mM
0.32±0.01a
0.30±0.00ab
0.29±0.00ab
0.24±0.00b
0
5
0.15±0.01a
0.14±0.00ab
0.13±0.00b
0.12±0.00b
1
2
0
0
4
4
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69
a-dValues with different letters within the same column differ significantly (p < 0.05).
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Table 2. DPPH radical-scavenging activity of phloroglucinol treated with dielectric
barrier discharge plasma
Treatment time (min)
DPPH radical-scavenging activity (%)
7
.92 mM
15.84 mM
66.31±0.57d
85.03±0.34c
89.29±0.72b
97.23±0.65a
0
5
54.36±0.78d
78.13±0.68c
82.55±0.83b
93.26±0.88a
1
2
0
0
4
73
a-dValues with different letters within the same column differ significantly (p < 0.05).
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Table 3. FRAP values of phloroglucinol treated with dielectric barrier discharge plasma
Treatment time (min)
FRAP value (μmol/g)
.92 mM
7
15.84 mM
0
5
20.55±0.96d
245.55±9.18c
372.22±10.05b
650.56±45.71a
292.22±31.90d
434.44±13.33c
736.11±20.48b
881.67±15.84a
1
2
0
0
4
76
a-dValues with different letters within the same column differ significantly (p < 0.05).
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Table 4. Antimicrobial activity of phloroglucinol treated with dielectric barrier
discharge plasma
Concentration
mM)
Treatment time
(min)
Pathogens
(
Bacillus cereus
Escherichia coli Staphylococcus
O157:H7
-
aureus
Methanol
-
-
7
.92
0
5
-c
- a
-c
-c
- a
-c
1
0
0
25.00±0.25b
34.00±0.56a
-c
- a
14.00±0.20b
19.00±0.30a
-c
2
- a
1
5.84
0
-b
5
-c
-b
-b
-c
1
0
0
24.03±0.25b
35.00±±0.30a
14.00±0.10b
20.00±0.20a
2
17.97±0.25a
4
4
4
80
81
82
aNo inhibition.
bClear zone diameter (disc diameter = 9 mm).
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Table 5. Peak assignments, retention times, MS, and MS/MS data of phlorotannins
derived from cold plasma-treated phloroglucinol using HPLC-QTOP-MS/MS
Rt
[M-H]-
(m/z)
2
Peak
MS ions (m/z)
Identify
(
min)
1
2
3
4
5
6
12.3
15.6
17.5
19.1
19.8
21.4
263.1
401.1
825.2
473.1
539.1
677.2
196, 230
263.1 (dimer), 356.0
263.1(dimer), 401.1 (trimer)
197.0
Dimer (1)
Trimer (2)
Hexamer derivative (3)
Trimer derivative (4)
Tetramer (5)
263.1 (dimer)
263.1 (dimer), 401.1 (trimer)
Pentamer (6)
2
63.1 (dimer), 401.1 (trimer),
39.1 (tetramer)
63.1 (dimer), 401.1 (trimer),
39.1 (tetramer)
75.1 (dimer derivatives), 401.1
trimer)
7
8
9
22.5
23.5
30.3
815.2
953.2
689.2
Hexamer (7)
Heptamer (8)
5
2
5
2
Pentamer derivative (9)
(
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Fig. 1. TBARS value (mg malondialdehyde/kg sample) of oil emulsion containing a
plasma-treated phloroglucinol solution dissolved in methanol during storage at
37°C.
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Fig. 2. ACE inhibition activity (%) of phloroglucinol treated with dielectric barrier
discharge
plasma.
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