foods
Article
Potential Use of Plant Waste from the Moth Orchid
(Phalaenopsis Sogo Yukidian “V3”) as
an Antioxidant Source
Truong Ngoc Minh 1 ID , Phung Thi Tuyen 1 , Do Tan Khang 1 ID , Nguyen Van Quan 1 ,
Pham Thi Thu Ha 1 , Nguyen Thanh Quan 1 , Yusuf Andriana 1 ID , Xinyan Fan 1 , Truong Mai Van 1 ,
Tran Dang Khanh 2 and Tran Dang Xuan 1, * ID
1
2
*
Graduate School for International Development and Cooperation, Hiroshima University,
Hiroshima 739-8529, Japan; minhtn689@gmail.com (T.N.M.); phungtuyen@gmail.com (P.T.T.);
dtkhang@ctu.edu.vn (D.T.K.); nguyenquan26@gmail.com (N.V.Q.); phamthithuhabt@gmail.com (P.T.T.H.);
quanagi@gmail.com (N.T.Q.); yusufandriana@yahoo.com (Y.A.); xinyanfan5160@gmail.com (X.F.);
truongmaivan1991@gmail.com (T.M.V.)
Division of Genetic Engineering, Agricultural Genetics Institute, Pham Van Dong Street, Hanoi 10000,
Vietnam; khanhkonkuk@gmail.com
Correspondence: tdxuan@hiroshima-u.ac.jp; Tel./Fax: +81-82-424-6927
Received: 16 July 2017; Accepted: 23 September 2017; Published: 27 September 2017
Abstract: This research was conducted to exploit the waste of used plant parts from the widely
marketed moth orchid cultivar (Phalaenopsis Sogo Yukidian “V3”). Various extracts of roots, stems, and
leaves were evaluated for total phenolics, total flavonoids, and antioxidant capacity. The bound extract
from stems contained the highest total phenolics (5.092 ± 0.739 mg GAE (gallic acid equivalent)/g DW
(dry weight)). The maximum total flavonoids (2.218 ± 0.021 mg RE (rutin equivalent)/g DW)
were found in the hexane extract of leaves. Ethyl acetate extract from roots showed the greatest
antioxidant activity compared to other extracts. Of these extracts, the IC50 values of these samples
were 0.070 mg/mL, and 0.450 mg/mL in a free radical 1,-diphenyl-picryl-hydrazyl (DPPH) assay
and reducing power method, respectively. The lipid peroxidation inhibition (LPI) was found to
be 94.2% using the β-carotene bleaching method. Five phenolic compounds including caffeic acid,
syringic acid, vanillin, ellagic acid, and cinnamic acid were quantified by high performance liquid
chromatography (HPLC). It is suggested that the roots of the hybrid Phalaenopsis Sogo Yukidian “V3”
cultivar may be exploited as an effective source of antioxidants.
Keywords: Phalaenopsis Sogo Yukidian “V3”; antioxidant activity; moth orchids; phenolic acids; plant
part waste
1. Introduction
Phytoplankton is a rich source of phytochemicals and antioxidants. A considerable number
of studies have been conducted regarding new forms of antioxidants due to their known health
benefits [1]. Natural antioxidants are an important source for preventing or scavenging free radicals
and reducing chronic and degenerative diseases by slowing the aging process and treating cancer.
Secondary metabolites, of which phenolic compounds are a key component, have diverse applications
in antioxidant production. Thus, recent replacement of synthetic antioxidants with natural compounds
and the identification of new antioxidants have become very important [2].
The genus Phalaenopsis (moth orchids) comprises about 66 species belonging to the Orchidaceae
family (estimated > 25,000 species) [3,4]. Among them, the Phalaenopsis (Phal.) Sogo Yukidian “V3”
cultivar (moth orchid), which is a hybrid of Phal. Yukimai crossed with Phal. Taisuco Kochdian
Foods 2017, 6, 85; doi:10.3390/foods6100085
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varieties, is white-colored, has a large flower, and is common in the market [4]. The Phal. crinkled
flowers indicates relatively short flower longevity, with young seedlings typically propagated by tissue
culture [5]. This cultivar of orchid is also a popular potted plant in Japan, America, and Europe [6].
The plant becomes very weak after flowering one or two times, subsequently turning into waste [2].
Moreover, although this orchid has great economic value, its disposal has proven to bring about
detrimental effects on the environment [2,7,8]. Therefore, the objective of this study was to evaluate
the antioxidant properties of plant part waste of the moth orchid.
Phenolic compounds exist in both free and bound forms in plant cells. Free phenolic compounds
are extractable with solvents, while bound phenolic compounds are covalently bound to the plant
matrix [9]. Therefore, different extraction techniques using increasingly polar solvents, including
hexane, chloroform, ethyl acetate, and water, were applied to examine their efficacy for extracting
antioxidants and evaluate the antioxidant capacity, total phenolics, and total flavonoids in the orchid.
The identification and quantification of individual phenolic acids were also conducted.
2. Materials and Methods
2.1. Materials
The hybrid Phal. Sogo Yukidian “V3” cultivar used in this study was grown in a greenhouse at
the Kurousu Orchid Company, Saitama Prefecture, Japan from April 2014 to July 2015. After flowering,
the whole plant was collected and used for the experiment. Leaves, roots, and stems of this hybrid
orchid were cut separately into small pieces and dried in an oven at 30 ◦ C. After drying, the samples
were ground into powder using a kitchen grinder.
2.2. Preparation of Free Phenolics and Bound Phenolics
Different extracting solvents with increasing polarity were used to examine the efficacy of different
extracts on orchid plant parts. The solvents used included the following compounds: hexane,
chloroform, ethyl acetate, and water (Table 1). Dried plant part powder was first separated into
groups of stems, roots, and leaves. Next, these groups were placed in ethanol and were concentrated
under reduced pressure at 40 ◦ C. The resulting dried ethanol extracts were then dissolved in water
and successively extracted with each of the solvent compounds, hexane, chloroform, and ethyl
acetate. The resulting mixtures were added to the aqueous solution that remained following extraction.
These mixtures were filtered and concentrated under decreased pressure to obtain extracts in hexane,
chloroform, ethyl acetate, and aqueous extracts [10]. The ethanol residues were hydrolyzed with 4 M
NaOH at 50 ◦ C and extracted with ethyl acetate to obtain bound phenolics [11].
Table 1. Names and abbreviations of plant extracts with different solvents.
Name of Plant and Extraction Solvent
Abbreviation
Sogo Yukidian “V3” stem free hexane
Sogo Yukidian “V3” stem free chloroform
Sogo Yukidian “V3” stem free ethyl acetate
Sogo Yukidian “V3” stem free aqueous
Sogo Yukidian “V3” root free hexane
Sogo Yukidian “V3” root free chloroform
Sogo Yukidian “V3” root free ethyl acetate
Sogo Yukidian “V3” root free aqueous
Sogo Yukidian “V3” leaf free hexane
Sogo Yukidian “V3” leaf free chloroform
Sogo Yukidian “V3” leaf free ethyl acetate
Sogo Yukidian “V3” leaf free aqueous
Sogo Yukidian “V3” stem bound
Sogo Yukidian “V3” root bound
Sogo Yukidian “V3” leaf bound
SFH
SFC
SFEA
SFA
RFH
RFC
RFEA
RFA
LFH
LFC
LFEA
LFA
SB
RB
LB
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2.3. Total Phenolic Content
The Folin—Ciocalteu method was used to determine the phenolic content of both free phenolics
or bound phenolics of plant extracts, of which, the total phenolic content was calculated using a
calibration curve and presented as mg of gallic acid equivalent (GAE)/g dry weight (DW) [12].
2.4. Estimation of Flavonoid Content
The aluminum chloride colorimetric assay was used to determine the flavonoid content of both
free phenolics and bound phenolics of plant extracts, of which, the total flavonoid content was
established using a calibration curve and expressed as mg rutin equivalent/g DW [13].
2.5. Antioxidant Activity Measurement by DPPH Scavenging Assay
Experiments were carried out according to the method described in Elzaawely et al. [14].
The reaction mixture of 0.5 mL of sample extract, BHT (butylated hydroxytoluene) (positive control),
or MeOH (methanol) (control), 0.25 mL of 0.5 mM DPPH, and 0.5 mL of 0.1 M acetate buffer (pH 5.5),
were kept in the dark at 26 ◦ C for 30 min. A HACH DR/4000U spectrophotometer (Hach, Loveland,
CO, USA) was used to evaluate the absorbance at 517 nm. The rate of inhibition percentage was
determined according to the formula: % radical scavenging activity = [(Acontrol − Atest )/Acontrol ] × 100.
The inhibitory concentration (IC50 ) of the sample extracts is the amount needed to inhibit 50% of the
DPPH radicals. As such, lower IC50 values show higher antioxidant activity.
2.6. Antioxidant Activity Measurement by β-Carotene Bleaching Method
Experiments were carried out according to the method described by Siddhuraju and Becker [15].
A one mL solution of β-carotene (2.0 mg) in 10 mL chloroform, 20 µL linoleic acid, and 200 mg Tween-40
was mixed. An aliquot of 50 mL of oxygenated water was added after concentration under reduced
pressure at 45 ◦ C. After shaking, 1 mL the β-carotene linoleic acid emulsion was mixed with 120 mL
(1000 ppm) of sample extracts, BHT (positive control), or MeOH (control). The samples were then
incubated at 50 ◦ C. The absorbance of each mixture was measured by spectrophotometer at 492 nm, at
an interval of 15–180 min to quantify the absorbance for all samples. Lipid peroxidation inhibition
(LPI) percentage was calculated using the following formula [16]: % LPI = A1 /A0 × 100.
A0 corresponds to the absorbance value estimated at time zero for the test sample, and A1
corresponds to the absorbance value at 180 min.
2.7. Reducing Power
The reducing power was determined following previously described methods [17]. A reaction
mixture of 1 mL of either extract or BHT (with concentrations 25, 50, 100, and 250 ppm in MeOH)
and 2.5 mL phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide [K3 Fe(CN)6 ] (10 g/L)
was prepared. Next, the solution was incubated at 50 ◦ C for 30 min. Following incubation, a total
of 2.5 mL trichloroacetic acid (100 g/L) was added to the mixture, which was then centrifuged at
4000 rpm for 10 min. Finally, the supernatant solution (0.5 mL), distilled water (0.5 mL), and 0.5 mL
FeCl3 (1 g/L) was measured for absorbance at 700 nm. The absorbance of the reaction mixture is
directly proportional to reducing power.
2.8. HPLC Analysis
Phenolic compound analyses were carried out using HPLC by a method described in
Xuan et al. [18]. Five mL of each sample extract was first filtered using a 0.2 µm filter (KANTO
Chemical, Tokyo, Japan) and then injected into the HPLC (JASCO PU-2089 Plus, JASCO Corporation,
Tokyo, Japan, column J–Pak Symphonia C18 column; 225 mm × 4.6 mm., i.d., 5 µm, 100 Å (Å is an
unit of length equal to 1 × 10-10 meters (m) or 0.1 nanometer (nm)). A gradient elution of two solvents
including absolute methanol (A) and 0.1% acetic acid (B) was used. The initial step began with the
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mobile phase A with gradient concentration from 5% to 10% in the first 5 min, then increased from
10% to 90% for next 45 min, and 100% for the last 10 min; wavelength: 254 nm; flow rate: 1.0 mL/min.
Each sample was measured in triplicate, and each phenolic compound was identified and quantified
in comparison with retention times and peak areas of their respective standards.
2.9. Statistical Analysis
All data from this research were analyzed using Minitab Software (version 16.0, copyright 2015,
Minitab Inc., State College, PA, USA) which performed a two-way analysis of variance (ANOVA)
with p < 0.05. All trials were conducted using a completely randomized block design with at least
three replicates.
3. Results
3.1. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)
Different chemicals have various protective and therapeutic effects and may help in the prevention
of illness and in maintaining good health [19]. Among them, flavonoids and phenolics are highly
effective antioxidants. Some diseases have been treated through the application of polyphenols and
flavonoids associated with free radical based terminators, and they have been reported to be effective
in disease control as well as involvement in many physiological functions [20,21]. Phenolic compounds
have been highlighted as antioxidants, which act as free radical oxidant binders, having been shown
to elicit pharmacological activity and express biological properties [22,23]. Das et al. [24] mentioned
that some flavonoids containing hydroxyls contribute to the narrowing effect of the roots of many
plants. The flavonoid mechanism of action is accomplished through either scavenging or chelating.
It is well known that many plants are highly effective in the collection of free radicals and antioxidants.
Thus, plant phenolics are generally highly effective in removing free radicals [25]. The contents of total
phenolics and flavonoids in hexane, chloroform, ethyl acetate, and aqueous extracts prepared from
the whole plant of the Phal. Sogo Yukidian “V3” cultivar vary between 0.66 and 9.10 mg GAE/g DW
and 0.21 to 3.00 mg rutin equivalent (RE)/g DW, as shown in Figure 1. These results suggest that the
highest amount of phenolics was detected in bound extract in comparison with flavonoids and were
observed principally in the hexane extracts.
10
Total phenolics content (GAE mg/g DW)
9.10 ± 1.28
Total flavonoids (RE mg/g DW)
8
6
4
3.00 ± 0.03
2.80 ± 0.08
1.70 ± 0.05
2
0.65 ± 0.01
1.16 ± 0.04
0.66 ± 0.05
0.30 ± 0.01
0.21 ± 0.01
1.19 ± 0.07
0
Hexane
CHCl3
EtOAC
Aqueous
Bound form
Figure 1. Total phenolic and flavonoid content in extracts of plant parts of Phal. Sogo Yukidian “V3”
cultivar. Values represent each mean ± standard deviation (SD) (p < 0.05) (n = 3); GAE: gallic acid
equivalent; RE: rutin equivalent; DW: dry weight.
Foods 2017, 6, 85
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3.2. Antioxidant Activity by the DPPH Radical Scavenging Assay
At room temperature, a stable, free radical DPPH creates a violet solution in ethanol, while an
antioxidant shows a colorless solution in aqueous ethanol [2]. DPPH is an easy and accurate way to
determine antioxidant presence in plant specimens [14]. DPPH reagents provide a convenient and
accurate method for titrating a collective oxidation of natural or synthetic antioxidants [26]. The DPPH
free radical scavenging activity of the orchid extracts is shown in Table 2, and is exhibited through
the IC50 value, of which a smaller value indicates greater activity [27]. Consequently, the IC50 values
of free phenolic extracts prepared from an aqueous extract of stems and leaves were higher than
3 mg/mL, indicating a lower antioxidant activity. Contrarily, the corresponding IC50 values of the
ethyl acetate extracts were in lower antioxidant levels, ranging from 0.070 mg/mL in the root samples
to 0.699 mg/mL in the leaf samples. The ethyl acetate root extract only required 0.07 mg/mL to reduce
the DPPH radicals by 50%. On the other hand, the IC50 of the chloroform and hexane extracts in each
plant part varied from 0.175 to 1.737 mg/mL.
Table 2. DPPH radical scavenging activity of Phal. Sogo Yukidian “V3” cultivar extracts in terms of
IC50 values.
Samples
Hexane
Chloroform
Ethyl Acetate
Aqueous
Bound Extract
BHT
DPPH IC50 (mg/mL)
Stems
Roots
b
1.634 ± 0.020
0.175 ± 0.002 d
0.195 ± 0.001 d
3.197 ± 0.046 a
0.377 ± 0.007 c
Leaves
a
1.663 ± 0.083
0.210 ± 0.005 d
0.070 ± 0.011 e
1.537 ± 0.007 b
0.959 ± 0.054 c
0.021 ± 0.012
ns
1.737 ± 0.040 b
0.699 ± 0.009 d
6.697 ± 0.025 a
1.348 ± 0.134 c
Values represent means ± SD (standard deviation). Values with no letter in common in each column are not
significantly different (p < 0.05) (n = 3); Different lowercase letters denote significant differences in the same column;
ns: not significant; BHT: Butylated hydroxytoluene.
3.3. Antioxidant Activity with the ß-Carotene Bleaching Method
Unsaturated β-carotene can be damaged by linoleic acid to produce linoleate and other free
radicals in a chemical process, namely, β-carotene-bleaching; antioxidants are capable of neutralizing
these free radicals [16]. Antioxidant tests that used the discoloration of β-carotene are widely used,
as β-carotene is extremely sensitive to intermediate oxidative free radicals [28]. All extracts of
Phal. Sogo Yukidian “V3” cultivar sampled inhibited β-carotene oxidation (Figure 2a). The polar
extracts, ethyl acetate, and aqueous solution exhibited superior inhibition to the non-polar extracts,
hexane and chloroform, and bound phenolic extracts. The LPI values of sample extracts ranged from
33.4% to 94.2% (Figure 2b). The free linoleic acid is formed when the abstraction of a hydrogen atom
from one of the dialytic methyl groups attacks the molecules of unsaturated β-carotene. When the
molecules of β-carotene lose their oxidative double bond, the resulting compound will lose its orange
color. The presence of antioxidant activity in various extracts can interfere with the bleaching of
β-carotene levels by neutralizing the free radicals formed in the system [29].
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Control
SFA
LFH
RB
(a)
BHT
RFH
LFC
LB
SFH
RFC
LFEA
SFC
RFEA
LFA
SFEA
RFA
SB
0.20
Absorbance at 492 nm
0.16
0.12
0.08
0.04
0.00
0
15
30
45
60
75
90
Time (min)
(b)
100
89.0 91.7
87.0
88.3 91.6
91.0
105
95.9
120
135
150
LPI %
63.9
180
90.8 89.3 91.8
77.4
80
165
72.8
63.3
60
44.8
40
33.4
20
0
Figure 2. Antioxidant activity of Phal. Sogo Yukidian “V3” orchid sample extracts measured by
β
β-carotene bleaching method (a) and their lipid peroxidation inhibition (%LPI) (b) Control (MeOH);
BHT: Butylated hydroxytoluene. SFH: Stem free hexane, SFC: Stem free chloroform, SFEA: Stem free
ethyl acetate, SFA: Stem free aqueous, RFH: Root free hexane, RFC: Root free chloroform, RFEA: Root
free ethyl acetate, RFA: Root free aqueous, LFH: Leaf free hexane, LFC: Leaf free chloroform, LFEA: Leaf
free ethyl acetate, LFA: Leaf free aqueous, SB: Stem bound, RB: Root bound, LB: Leaf bound.
3.4. Reducing Power
The potential antioxidant activity of a compound is indicated by its ability to transfer electrons
(reducing power). Sample extracts with high antioxidant activity also have a high reducing power [10].
Figure 3 shows the reducing power of hexane, chloroform, ethyl acetate, aqueous, and bound extracts
prepared from the stems, roots, and leaves of Phal. Sogo Yukidian “V3”. In this assay, the yellow
color of the test solution varied between green and blue, depending on the reduction capacity of each
compound. The presence of substances arising from the degradation of antioxidants contributed to the
reduction of Fe3+ to Fe2+ [30].
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SFH
RFH
LFH
SB
0.8
SFC
RFC
LFC
RB
SFEA
RFEA
LFEA
LB
SFA
RFA
LFA
BHT
Absorbance at 700 nm
0.6
0.4
0.2
0.0
0.0
0.2
0.4
0.6
Concentration (mg/mL)
0.8
1.0
Figure 3. Reducing power of Phal. Sogo Yukidian “V3” orchid sample extracts. BHT: Butylated
hydroxytoluene. SFH: Stem free hexane, SFC: Stem free chloroform, SFEA: Stem free ethyl acetate,
SFA: Stem free aqueous, RFH: Root free hexane, RFC: Root free chloroform, RFEA: Root free ethyl
acetate, RFA: Root free aqueous, LFH: Leaf free hexane, LFC: Leaf free chloroform, LFEA: Leaf free
ethyl acetate, LFA: Leaf free aqueous, SB: Stem bound, RB: Root bound, LB: Leaf bound.
The reducing power of all sample extracts was proportionate to the concentrations applied.
The ethyl acetate extracts exhibited a higher ability to reduce Fe3+ to Fe2+ than those of the chloroform,
hexane, aqueous, and bound extracts. For the stem extracts, the reducing power IC50 values ranged
from 0.871 to 4.495 mg/mL, the IC50 values ranged from 1.544 to 4.553 mg/mL for the leaf extracts,
and 0.450 to 4.354 mg/mL for the root extracts (Table 3).
Table 3. Reducing power of Phal. Sogo Yukidian “V3” orchid in terms of IC50 values.
Samples
Hexane
Chloroform
Ethyl acetate
Aqueous
Bound extract
BHT
IC50 (mg/mL)
Stems
Roots
b
2.191 ± 0.025
0.871 ± 0.012 d
0.929 ± 0.007 d
4.495 ± 0.051 a
1.523 ± 0.068 c
Leaves
b
2.018 ± 0.013
1.143 ± 0.019 c
0.450 ± 0.005 d
4.354 ± 0.087 a
4.167 ± 0.105 a
0.262 ± 0.005
2.029 ± 0.037 b
2.020 ± 0.022 b
1.544 ± 0.067 c
4.312 ± 0.112 a
4.553 ± 0.099 a
The values presented represent means ± SD (standard deviation) (p < 0.05). Values with no letter in common in
each column are not significantly different (n = 3); Different lowercase letters denote significant differences in the
same column; BHT: Butylated hydroxytoluene.
3.5. HPLC Quantification
The hexane, chloroform, ethyl acetate, aqueous, and bound extracts prepared from the stems, roots,
and leaves of Phal. Sogo Yukidian “V3” cultivar contained high amounts of phenolic compounds and
showed strong antioxidant activity. These extracts were used to identify and quantify the individual
phenolic acids.
μ
μ
Using HPLC, five compounds
μ including caffeic acid, syringic acid, vanillin, ellagic acid, and
cinnamic acid were detected in varying quantities (Table 4). Among these phenolics, caffeic and ellagic
acids were most prevalent in leaves. Caffeic acid (574.75 µg/g DW), syringic acid (16.98 µg/g DW), and
cinnamic acid (15.43 µg/g DW) were found only in the hexane extracts of stems, ethyl acetate extracts
of roots, and chloroform extracts of stems. Furthermore, vanillin was detected in the ethyl acetate
Foods 2017, 6, 85
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extracts of roots (11.06 µg/g DW) and in aqueous extracts of leaves (51.27 µg/g DW). The highest
concentration of ellagic acid (143.90 µg/g DW) was found in ethyl acetate extracts of stems, followed
by chloroform extracts of stems (134.02 µg/g DW). Several sources indicate that phenolic acids such as
ferulic, sinapic, p-coumaric, and ellagic acids are strong antioxidants [31–33]. Ellagic acid was reported
to obtain anti-inflammatory, anti-proliferating, anti-angiogenesis, anti-cancer, anti-fungal, anti-cancer,
and anti-depressant effects [34].
Table 4. Phenolic acids in Phal. Sogo Yukidian “V3” cultivar.
Samples
SFH
SFC
SFEA
SFA
RFH
RFC
RFEA
RFA
LFH
LFC
LFEA
LFA
SB
RB
LB
Phenolic Contents (µg/g DW)
Caffeic Acid
Syringic Acid
Vanillin
Ellagic Acid
Cinnamic Acid
574.75 ± 3.87
-
16.98 ± 10.35
-
11.06 ± 1.22
51.27 ± 0.09
ns
50.15 ± 5.30 b
134.02 ± 0.14 a
143.90 ± 0.14 a
7.05 ± 1.41 d
21.09 ± 21.09 c
11.00 ± 0.02 cd
71.08 ± 0.09 b
-
15.43 ± 0.10
-
Values presented represent the mean ± SD. Values with no letter in common in each column are not significantly
different (p < 0.05) (n = 3). Different lowercase letters denote significant differences in the same column; ns: not
significant. SFH: Stem free hexane, SFC: Stem free chloroform, SFEA: Stem free ethyl acetate, SFA: Stem free aqueous,
RFH: Root free hexane, RFC: Root free chloroform, RFEA: Root free ethyl acetate, RFA: Root free aqueous, LFH: Leaf
free hexane, LFC: Leaf free chloroform, LFEA: Leaf free ethyl acetate, LFA: Leaf free aqueous, SB: Stem bound,
RB: Root bound, LB: Leaf bound.
4. Discussion
Plants are known to provide substances and chemicals effective in the treatment and remedy of
various diseases [35–38]. Orchids constitute an order of royalty in the world of ornamental plants,
are important for large horticulture, and play an important role in balancing forest ecosystems.
Orchids are economically important as they are used not only for decorative purposes, but also for the
treatment of certain diseases in traditional medicines [35]. Tubers and pseudobulbs of several orchids
like Orchis latifolia, Orchis mascula, Cymbidium aloifolium, Zeuxine strateumatica, and some species of
Dendrobium, Eulophia, and Habenaria, can treat a host of diseases [39]. The roots of Vanda tessellate can
be used for manufacturing poison antidotes as well as for treating rheumatic and abdominal pain [40].
Dendrobium fimbriatum has been used as a treatment for liver upset and nervous debility, while
Dendrobium teretifolium has been used for headache and pain relief. Hawkes [41] and Withner et al. [42]
reported that Oberonia, Eria, Bulbophyllum, Eulophia, Geodorum, Grammatophylum, and Hetaeria could be
used as a traditional medicine against various diseases.
There are an impressive number of modern medicines that have been isolated from natural
sources [43]. In this study, the method of sequential extraction with different solvents (hexane,
chloroform, ethyl acetate, and water) revealed a rich presence of flavonoids and phenols. Although,
among 15 individual phenolic acids used as standards, there were only five constituents detected by
HPLC from the waste materials of the orchid (Table 4). The existence of other individual flavonoids
and phenols in low concentrations, and antioxidants other than flavonoids and phenolics, should
be investigated by using analytical instruments with higher sensitivity of detection such as gas
Foods 2017, 6, 85
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chromatography-mass spectrometry (GC-MS). Extracts with phenolic substance-medicated antioxidant
activity were shown in conjunction with the development of reducing power [44]. As such, the ethyl
acetate extract may contain much higher amounts of electron donors for reducing, and they can react
with free radicals to convert them into more stable products, terminating the radical chain reaction.
The findings of this study suggest that waste material from orchids resulting from the production
process for commercial flowers may be exploited as a natural source of antioxidants. In 2009, Thailand
exported $80 million US worth of orchids, of which the Phal. cultivar comprised 23% of that total [45].
In 2012, Taiwan was the foremost exporter of the Phal. plant (69%) which, of a total of $164.70 million
US, the major cultivar was the Phal. Sogo Yukidian “V3” [4]. Rich antioxidants were found in the
roots, with well-known phenolic compounds—caffeic acid at 574.75 µg/g DW, and ellagic acid at
328.07 µg/g DW—in the stems. In this study, extract solvents of increasing polarity were used to
examine their capacity for extracting antioxidants. It was found that ethyl acetate extracts of the roots
provided maximum antioxidant yield. In general, polar solvent extracts showed greater antioxidant
yield than others that were studied. One explanation is that highly polar solvents often accumulate
free substances with a hydroxyl moiety, such as phenols and flavonoids [46–48]. This suggests that the
root of the orchid was rich in compounds with the hydroxyl moiety, although only five free individual
phenolic acids were detected by HPLC. Further analysis should be concentrated on the roots of the
Phal. plant to exploit its potent antioxidant capacity.
Results of this study may be worthwhile to the principal locations of orchid production such as
Taiwan, Thailand, and the Netherlands, where orchids play an active role in flower production, but
waste disposal causes environmental problems and are costly to treat [49,50].
5. Conclusions
The results of this study verified that ethyl acetate extracts from Phalaenopsis Sogo Yukidian “V3”
plant parts have potent antioxidant activity and may be utilized as an efficient and safe antioxidant
source. Further trials examining other medicinal and pharmaceutical properties, as well as application
of further analytical instruments with higher sensitivity to determine potent constituents in the waste
of the Phalaenopsis should be conducted.
Acknowledgments: The authors thank Kurousu Orchid Company, Saitama Prefecture, Japan for supply plant
materials (Phalaenopsis Sogo Yukidian “V3).
Author Contributions: Truong Ngoc Minh conceived the idea, designed the experiments and wrote the
manuscript. Do Tan Khang, Pham Thi Thu Ha, Phung Thi Tuyen, and Xinyan Fan contributed to review
the article after comments. Nguyen Van Quan, Truong Mai Van, Nguyen Thanh Quan, and Yusuf Andriana
performed the experiences in antioxidant assay. Tran Dang Xuan and Tran Dang Khanh revised and approved the
final submission of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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