Introduction
Inflammation is a defense response of body
against injury, pathological infection, noxious stimuli or trauma (Choi et
al. 2012). Intense inflammation is often associated with several diseases
such as diabetes, arthritis, Alzheimer's and cancer (Pereira and Alvarez-Leite
2014; Torres-Rodríguez et al. 2016; Qian 2017). In response of
inflammation macrophages are activated by endotoxin like lipopolysaccharide
(LPS). These LPS activated macrophage cells excrete several pro-inflammatory
cytokines (tumor necrosis factor-α (TNF-α); interleukin-1β
(IL-1β); interleukin-6 (IL-6) and mediators (e.g., free radicals)
such as nitric oxide (NO) (Li et al. 2014). Unregulated of excessive production
of these mediators involved in arbitrating or exacerbating several diseases
such as chronic pulmonary inflammatory disease, arthritis, osteoarthritis,
ulcerative colitis and carcinogenesis (Choi et al. 2014).
Cancer (severe
metabolic disorder) is one of the major causes of human deaths globally (Iqbal et
al. 2017); involves uncontrolled normal cells proliferation caused by
genetic instabilities and alterations, which lead to malignant cells generation
and metastasis initiation or tissue invasion (Krishnamurthi 2007). At present
many therapies for cancer treatment include surgery, chemotherapy and
radiotherapy have proved effective in saving lives of numerous cancer patients.
Nevertheless, recurrence and severe side effects make
these treatments partially effective, pressing the demand for treatments with low
toxicity and minimal side effects. Plant derived chemicals compared to
chemotherapy drugs are high target specific with low toxicity and can be used
to unravel anticancer chemical agents (Cragg and Newman 2007). Rheum (Rheum emodi Wall. ex Meissn) is an
endemic, perennial, medicinal herb, commonly known as Himalayan Rhubarb;
distributed in the Himalayas (subtropical and
temperate regions), is widely used as traditional medicine against several diseases
(Rokaya et al. 2012). The R. emodi contain several bioactive
compounds such as anthraquinones, anthocyanins, flavonoids, stilbenes and
desoxyrhapontigenin (Gao et al. 2011) and is known for its strong
anti-inflammatory, anticancer, anti-oxidative and antimutagenic effects (Li et
al. 2008). Among these phytochemicals, flavonoids (cannot synthesized by human
and animal) is very important class of compounds which
is essential constituent of animal and human diet and possess a therapeutic
potential against cancer and inflammation (Raffa et al. 2017).
Few studies have reported that
aqueous and methanolic extracts of R. emodi found to possess several
anticancer (Rajkumar et al. 2011)
and anti-inflammatory metabolites (Kounsar
and Afzal 2010). However, there is very limited information
on the relative efficacy of different organic solvent extracts of R. emodi on the anticancer and anti-inflammatory
activities. Moreover, the isolation of flavonoids and their anti-inflammatory and anticancer
activities in R. emodi
is not well characterized. Therefore, the present study was carried
out to evaluate the in vitro anticancer and anti-inflammatory activities
of the rhizome extracts fractions of R. emodi
using different organic solvents and to isolate and identify the flavonoid
compounds from ethyl acetate extracts and to evaluate the anti-inflammatory and
anti-cancer potential of isolated compounds.
Materials and Methods
Plant material
R. emodi was collected from Langtang at an altitude of 3,500 m in
Nepal. The plant was identified using standard references and authenticated by
a botanist. The voucher specimen was deposited in the gene bank of Dankook
University.
Chemicals
LPS
(Lipopolysaccharide), DMSO and Griess’ reagent for nitrite were procured from Sigma
Chemical Co. (St. Louis, MO, USA). The ELISA kit (IL-6, IL-1β, TNF-α)
was purchased from BD Biosciences (USA). BHT, L-ascorbic acid, Rutin, Gallic
acid, Trizma base, NADH,
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), PMS (phenazine methosulphate),
NBT (nitroblue tetrazolium), TBA (thiobarbituric acid), TCA (trichloroacetic
acid), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),
ammonium thiocyanate and Foli-Ciocalteu's phenol reagent were procured from
Sigma Chemical Co. (St. Louis, MO, USA). The
HPLC reagents were water, methanol and acetonitrile from J. T. Baker (USA). All other chemicals and
solvents were commercial analytical grade.
Extraction and
solvent fraction of R. emodi
The air-dried R. emodi rhizomes
were crushed in a grinder for 2 min, the process was stopped for 15 s intervals
to prevent samples from heating. The R.
emodi samples (800 g) were extracted with 70% EtOH under reflux (3 × 2.5 L,
2 h each time), followed by filtration through filter paper (Whatman No. 2).
Later, rotary vacuum-evaporator at 50°C under reduced pressure was used to
evaporate the solvent of combined extract; while freeze drying helped in
removal of remaining water. The crude extract obtained by concentrating the
EtOH extract under vacuum was suspended in water and later sequentially
extracted with n-hexane (room
temperature, 2× 1 L) followed by dichloromethane
(2× 1 L), EtOAc (2× 1 L), n-BuOH (2 × 1 L) and water, in order (Fig. S1). The
solvents from each step were removed by evaporation and then freeze-dried. The
dried solvent fractions were stored below -18°C in deep freezer.
Isolation and
identification of bioactive compound
The method of Lin and Harnly (2007) was followed for identification of
phenolic compounds. This protocol obtains mass spectrometric data as negative
and positive ionization mode at high (250 V) and low (70–100 V) and high
excitation energy. The molecular ions are obtained from the negative ionization
with low excitation; whereas high energy liberates fragments which show
cinnamoyl quinic acids (mono-, di-, or trihydroxy) by
successive loss of hydroxy cinnnamoyls. However, UV and MS data and retention
time comparison from sample peaks with standards helps in positive compound
identification. The method has accumulated a large database of phenolic
compounds collected from routine profiling and analysis of standards and is
being used for their presence and identification in new plant sources (Lin and
Harnly 2007).
Isolation and
identification of antioxidant compound by LC-DAD-ESI/MS
The compounds
present in R. emodi fractions were isolated
using Micromass ZQ MS and an Alliance e2695
HPLC system (Waters Co., Milford, M.A., USA) equipped with a 2998 photodiode
array detector in addition to a YMC PACK ODS-AM
reversed-phase column (4.6 x 250 nm I.D., 5 μm;
YMC Co. Ltd., Japan). The analysis was conducted at 190–600 nm (the
characteristic wavelength of 254, 350 nm) detection wavelengths with 1 mL/min
flow rate and oven temperature of 30°C. Trifluoroacetic acid (0.1%) in water
(phase A) and acetonitrile (phase B) were used as mobile phases. The pretreated
samples were analyzed by using a gradient of 10 to 30% of phase B over 25-min period;
30% of phase B for five minutes; gradient of 30 to 10% of phase B for three
minutes and then final wash with 10% phase B for seven minutes. The
electrospray ionization source was used to run the MS analysis in a positive
ionization mode and MS parameters were set to 30 V (cone voltage), 120°C
(source temperature), 350°C (desolvation temperature) and 500 L/h (desolvation N2 gas flow). The molecular weight
range was 100–1200 m/z in the full scan mode.
Preparative
antioxidant compounds by LC/MS system
The analysis protocol was similar to isolation and
identification of antioxidant compound by LC-DAD-ESI/MS. In addition, the
fractionation parameters were set to maximum fractions and tubes per injection
of 78, solvent front delay of 60 s, spilt/collector delay of 10 s and maximum
fraction width of 60 s. The Ethyl acetate fraction from R. emodi with concentration of 50,000 ppm
was used for 500 μL per injection. In two step purification, the
fractionation parameters were different with the first step as fallow: maximum
tubes per injection of 114, maximum fraction width of 30 s. The Ethyl acetate
fraction from R. emodi
with concentration of 50,000 ppm was used for 200 μL per
injection.
The fractions collected in tubes were directly analyzed
without any additional liquid handling. Proper wash of the injection was found
to be most important procedure for the proper purity check of collected
fractions after preparative purification. Before analytical runs of each
series, needle was washed and blank injection with MeOH 70% was used.
Peak purity of collected fractions with their main
component was evaluated using relative peak area in mass chromatograms, thus
assuming comparable response factor for the impurities and the major component.
Chemical structures of bio active compounds from R. emodi
ChemDraw Ultra 8.0 software
(CambridgeSoft, USA) was used for drawing the
chemical structures of compounds.
Anti-inflammatory
activity assays
Cell culture: The RAW 264.7 (mouse macrophage cell line) obtained from Korean cell line
bank, Seoul, Korea was grown in Dulbecco’s modified Eagle’s medium having fetal
bovine serum (10%). The macrophages were kept at 37°C in humidified atmosphere
having 5% CO2. For the experiment, RAW 264.7 cells were subculture
for every 2~3 days.
Cell viability
To evaluate the effect of extracts on the cell viability, cytotoxicity
assay was performed. The RAW 264.7 macrophage cell was seeded onto 96-well
plate at 104 ~105 cell/well then cultured in 5% CO2
incubator at 37°C for 24 h.
The cells were treated with final concentration of 10, 25 and 50 µg/mL of the extracts. The cells were incubated
for further 24 h. The medium was exchanged with 180 µL fresh medium
contained 20 µL of 0.5 mg/mL MTT solution in each well. The absorbance
for isolated compounds and solvent extract fractions was measured at 450 and
570 nm respectively using microplate reader after 1~4 h.
NO inhibition
activity
The RAW 264.7 macrophage cell was seeded onto 96-well plate at 104
~105cell/well then cultured in 5% CO2 incubator at 37°C for 24 h. The cells were incubated in 1 µg/mL of medium
containing LPS (Sigma Co.) and final concentration of 10, 25 and 50 µg/mL each sample. The cells were incubated
for further 24 h. The NO inhibition activity was analyzed by Griess assay. The
Griess’ regent (0.1% naphthyethylenediamine and 1% sulfanilamide in 5% H3PO4
solution) was added to each of the supernatant from the cells treated with
samples. Sodium nitrite was used as positive control. No contents were read at
540 nm against a standard sodium nitrite curve.
IL-6, IL-1β, and TNF-α inhibition activity
The RAW 264.7 macrophage cell was seeded onto 96-well plate at 104~105
cell/well then, cultured in 5% CO2 incubator at 37°C for 24 h. The cell were
incubated in a medium containing LPS (Sigma Co.) 1 µg/mL and final concentration 10, 25 and 50 µg/mL each samples. The cells were incubated
for an additional 24 h. The IL-6, IL-1β,
TNF-α inhibition activity was analyzed by
commercially available ELISA kit (BD OptEIATM set Mouse IL-6, IL-1β, TNF-α). The assay
was performed as described by the manufacturer’s recommendations.
Anticancer activity
assays
Cell culture: Cytotoxicity of the extracts were determined in MCF7 (human breast
carcinoma) and A549 (lung carcinoma) from Korean cell line bank, Seoul, Korea,
HGF (Human gingival fibroblast) form Department of Dentistry from Dankook
University. The cells were kept at 37°C in humidified atmosphere containing 5% CO2; grown in minimum
essential medium and RPMI 1640 medium with fetal bovine serum (10%). The cells
were subcultured every 2~3 days.
Cytotoxicity assay
To evaluate effects of extracts on the cell viability, a cytotoxicity assay was
performed. The cancer cells (MCF7, A549) were seeded onto 96-well plate at 104
~105 cell/well then, cultured in 5% CO2 incubator at 37°C for 24 h and cells were treated with final
concentration 20, 50, 100, 200 mg/mL of
extracts. The cells were incubated for further 24 h and medium was changed with
200 µL fresh medium contained 10 µL of CCK-8 (Dojindo, Japan)
solution in each well. The absorbance was measured at 570 nm using microplate
reader after further 1~4 h.
Calculation of half
maximal inhibitory concentration (IC50)
IC50 value, the sample concentration required to scavenge 50%
of the free radicals, were calculated using the percent scavenging activities
of the different sample concentrations.
Statistical analysis
General analysis: The experimental data were analyzed by SPSS (version
12.0 for windows XP, SPSS Inc.) using one and two-way analysis of variance,
while Duncan’s multiple range test was performed for mean separation (P < 0.05).
Multivariate analysis (partial least squares of discriminant
analysis (PLS-DA)
The data matrix was
developed by arranging and normalizing all the qualitative and quantitative
information, which is used for multivariate statistical analysis as log10-transformed
data. SIMCA-P 11.0 software (Umetrics, Umea, Sweden) was used for performing
PLS-DA models.
Results
Identification of antioxidant
compounds
Five peaks were obtained from ethyl acetate fraction of R. emodi based
on their retention time and detection
wavelength of 190–600 nm by LC-DAD-ESI/MS.
These peaks were separated into compound 1 (Rt = 5.30 min), compound 2 (Rt =
17.30 min), compound 3 (Rt = 23.30 min), compound 4 (Rt = 24.30 min) and
compound 5 (Rt = 28.30 min) using an YMC PACK ODS-AM reversed-phase column (4.6
x 250 nm I.D., 5 μm) and
narrower range of solvent gradient (Fig. 1).
%201161-1172_files/image002.jpg)
Fig. 1: LC chromatogram (254 nm and 350 nm) of ethyl
acetate fraction from R. emodi
%201161-1172_files/image004.jpg)
Fig. 2: Structure
and MS spectra about compound 1 isolated from R. emodi by LC-DAD-ESI/MS
Compound 1 was identified based on the comparison of
retention times, numerous literature sources and mass spectra data of the
samples. Retention time of the peak was observed at 4.533 min, revealed a
solvent peak. It was not possible to detect the mass even when the
LC-DAD-ESI/MS experiment was performed. Likewise, compound 2 was identified
using literature sources and mass spectra data of the samples as Myricetin
3-O-rhamnoside (Myricitrin) at the retention time of 17.80 min through
comparison of the retention times, ion was revealed at 464 m/z with a molecular formular C21H20O12
(Fig. 2). Compound 3 was identified as Myricetin 3-galloylrhamnoside at the
retention time at 23.82 min, ion was revealed at
617 m/z with a molecular formula of C28H24O16 (Fig.
3). Remarkably, this compound was found in this plant for the first time.
Compound 4 was identified as Myricetin (Fig. 4), at the retention time of 24.37
min, ion was revealed at 319 m/z with
a molecular formular of C15H10O8. Accordingly,
compound 5 was identified as unknown (Fig. 5) at retention time of 28.465 min,
ion was revealed at around 520 m/z.
It was not possible to elucidate a structure even when the LC-DAD-ESI/MS
experiment was performed. There was not enough evidence to propose the
structure of the compound.
Positive ion electrospray
analysis, total ionic current profile and reconstructed ion chromatograms of
compound 2–5 (Fig. 2–5) identified the Myricetin derivatives through comparison
of retention times and m/z values in
the total ion current chromatogram from the data gotten from the literature.
The ion chromatograms were reconstructed for each value of m/z observed for the standard compounds 2 (319 and 464 m/z), 3 (280, 299, 319, 465 and 617 m/z), and 4 (319 m/z) to advance the separation and the recognition of single
compounds. LC-DAD-ESI/MS analysis of compound 2 discovered the presence of
myricetin related compounds. Specifically, peaks at 464 m/z were observed and the reconstructed ion chromatograms revealed %201161-1172_files/image006.jpg)
Fig. 3: Structure and MS spectra about compound 2
isolated from R. emodi
by LC-DAD-ESI/MS
%201161-1172_files/image008.jpg)
Fig. 4: Structure
and MS spectra about compound 3 isolated from R. emodi by LC-DAD-ESI/MS
one peak; the value of the mass of the pseudo molecular
ion, the retention and the presence in this fraction suggested that these
compounds were Rhamnoside (145 m/z).
The presences of the fragment ion at 463.0853 m/z indicate the loss of a galloyl moiety and the presence of
myricetin rhamnoside (Amani et
al. 2014). The ion at 464, which corresponds to the myricetin
(319 m/z) and rhamnoside (145 m/z), confirmed that this compound had
the natural of Myricetin 3-O-rhamnoside (Myricitrin).
The LC-DAD-ESI/MS spectrum of compound 3 gave base peak
of the [M+H]+ ion at 617m/z and molecular weight 616. The ion at
617m /z, which corresponds to the myricetin (m/z 464) and
galloic acid (m/z 152), which eluted at 14.90 min and showed 615.0988 m/z, it was confirmed that this compound was characterized as
Myricetin 3-O-galloylrhamnoside according to the MS and MS/MS data and the
literature. LC-DAD-ESI/MS analysis of compound 3 revealed the presence of other
compounds related to myricetin. Specifically, peaks at 319 m/z were identified as myricetin. The LC-DAD-ESI/MS spectrum of
compound 3 gave base peak of the [M+H]+ ion
at 319 m/z and molecular weight of
317. In addition, another fragment was presented at 280 m/z, which corresponds to the consequent loss of water molecule.
Finally, the ion at 299 m/z, which
corresponds to the subsequent loss of a rhamnoside (145 m/z) and galloic acid (152 m/z),
it was confirmed that this compound was the basic structure.
%201161-1172_files/image010.jpg)
Fig. 5: Structure and MS spectra about compound 4 isolated from R. emodi by
LC-DAD-ESI/MS
Table 1: Comparison of anticancer activity
of antioxidant compounds from R. emodi in A549
and MCF-7 cells
|
Cell Lines |
Fraction |
IC50 (μg/mL) |
|
Myricetin 3-O-rhamnoside |
64.04 ± 9.82c |
|
|
A549 |
Myricetin 3-O-galloyrhamnoside |
62.21 ± 6.07c |
|
Myricetin |
67.49 ± 8.63c |
|
|
Unknown |
56.15 ± 9.37b |
|
|
Doxorubicin* |
41.68 ± 5.79a |
|
|
|
Myricetin 3-O-rhamnoside |
104.76 ± 11.8d |
|
MCF-7 |
Myricetin 3-O-galloyrhamnoside |
93.35 ± 9.09c |
|
Myricetin |
84.35 ±8 .08b |
|
|
Unknown |
80.49 ± 8.39b |
|
|
|
Doxorubicin* |
70.17 ± 4.70a |
Each value represents means ± SD (n = 3)
a-d Values within a column followed by different
letters are significant different (P <
0.05)
* Compound used as a positive control
%201161-1172_files/image012.png)
Fig. 6: Cytotoxic activity of isolated compounds from R. emodi fractions on (a) A549 and (b) MCF-7
cancer cell lines
Anticancer
activities of R. emodi
Cytotoxic activity
of isolated compounds on A549 and MCF-7 cell: The cytotoxic activity on A548 and MCF-7 were performed to estimate the
anticancer activity potential of isolated compounds. Doxorubicin was used as a
positive control. The IC50 (concentration needed for inhibition of
50% radical-scavenging effect) was verified from the results of a series of
concentration tested. Lower IC50 value corresponds to a greater
scavenging activity. The cytotoxic activity on A549 cell lines varied from IC50 56.15 to 67.49 µg/mL (Table 1). The IC50 values of the cytotoxicity activities on A549 cells
can be ranked as unknown (56.15 µg/mL) > myricetin 3-galloylrhamnoside (62.21 µg/mL) >
Myricetin 3-rhamnoside (64.04 µg/mL) > Myricetin (67.49 µg/mL) (Table 1). All the compounds exhibited an excellent cytotoxicity
activity. However, their IC50 values were greater than that of Doxorubicin (41.68 µg/mL). Unknown compound showed very strong
anticancer activity against A549 cells, with an IC50 of
56.15 µg/mL. The cytotoxicity
activity on A549 cell lines by concentration from compounds were expressed in a dose-dependent
manner manner (Fig. 6a). The decrease in absorbance
at high dose could be justified by cytotoxicity exhibited by the fractions.
The cytotoxic activity on MCF-7 cell lines varied from IC50 of 80.49
to 104.76
µg/mL. The IC50 values of the
cytotoxicity activities on MCF-7 cells can be ranked as unknown (80.49 µg/mL) >
myricetin (84.35 µg/mL) > Myricetin 3-galloylrhamnoside (93.35 µg/mL) > Myricetin 3-rhamnoside (104.76 µg/mL) (Table 1).
Their IC50 values were smaller than that of Doxorubicin (70.17 µg/mL). Unknown showed very strong anticancer
activity against A549 and MCF-7 cells. The cytotoxic activities on MCF-7 cell lines by different
concentration of compounds were expressed in the dose-dependent
(Fig. 6b).
Cytotoxic activity
of solvent extract fractions of R. emodi on
A549 and MCF-7 cells
The R. emodi
fractions significantly differed for cytotoxic activity on A549 cells. The
IC50 values of R. emodi fractions on A549 cell lines and Doxorubicin
(positive controls) varied from 74.9
to 128.5
µg/mL (Table 2). The
aqueous fraction exhibited the highest cytotoxic
activity on A549 cell lines (74.9 µg/mL) followed by
dichloromethane (93.6 µg/mL) > n-butanol (104.0 µg/mL)> n-hexane (106.14 µg/mL) > ethyl acetate (128.5 µg/mL) fractions.
The cytotoxicity activity of R. emodi fractions on A549 cell lines increased in the dose-dependent manner. In this regard, the water fraction of R. emodi inhibited
A549 cell lines growth at 20, 50 and 100 µg/mL while n-Hexane showed highest
cytotoxicity at 200 µg/mL on A549 cell lines (Fig. S3a).
The cytotoxic activity of R. emodi
fractions significantly differed against MCF-7 cells. All fractions exhibited
lower cytotoxic activity than Doxorubicin (control) (IC50 50.82 µg/mL) (Table 2). The cytotoxic activity on MCF-7
cell lines varied from IC50 114.72 to 139.36 µg/mL. However,
among the tested fractions highest cytotoxic activity was observed for dichloromethane (114.72 µg/mL) > n-hexane
(116.34 µg/mL)
> water (122.6 µg/mL)
> n-butanol (124.24 µg/mL) > ethyl acetate (139.36 µg/mL) (Table 2). The concentration of
fractions on cytotoxicity activity against MCF-7 showed a linear rise with
increase in fractions concentration (Fig. S3b). However, the dichloromethane
fraction showed higher cytotoxic activity at all concentration than other
fractions (Fig. S3a, b).
In vitro cytotoxicity of R. emodi fractions on A549 and MCF-7 cells
The In vitro study
revealed that treatment with 50 mg/mL,
100 mg/mL and 200 mg/mL fractions dramatically induced black spot in a
dose-dependent manner in A549 cells. The viability of A549 cells showed a
significant decrease even at high concentration of n-Hexane fraction. The
destruction of A549 cells was concentrated at 50 mg/mL in n-Hexane fraction. The water fraction was found to have the highest cytotoxic activity on A549 cell lines as visible from the destructed
nucleus and nuclear compartment at 100 mg/mL of water fraction (Table 2; Fig.
7).
Treatment of MCF-7 cells with higher concentrations (50, 100 and 200
mg/mL) of R. emodi fractions induced the
apoptosis. In this regard, n-Hexane fraction showed a significant inhibition of
morphologic changes in MCF-7 cell. The data suggest that apoptosis of MCF-7
cancer cells was induced in dose-dependent manner, and these morphologic
changes appeared with 50 mg/mL treatment (Fig. 8).
Anti-inflammatory
activities of compounds isolated from R. emodi
%201161-1172_files/image014.jpg)
Fig. 7: Cytotoxic activity
on A549 cell lines by different concentration of R. emodi compound
%201161-1172_files/image016.jpg)
Fig. 8: The Cytotoxic
activity of different R. emodi fractions on MCF-7 cell lines
with different concentration
Table 2: IC50 values of
anticancer activities of Rheum emodi Wall fractions on each cancer
cell lines
|
Cell |
Fraction |
IC50 (µg/mL) |
|
n-Hexan |
106.14 ± 4.42c |
|
|
n-Butanol |
103.96 ± 2.56c |
|
|
A549 |
Ethylacetate |
128.52 ± 4.92d |
|
Dichloromethane |
93.58 ± 2.71b |
|
|
Water |
74.88 ± 2.25a |
|
|
|
Doxorubicin* |
75.66 ± 2.88a |
|
|
n-Hexan |
116.34 ± 2.47bc |
|
n-Butanol |
124.24 ± 9.90c |
|
|
MCF-7 |
Ethylacetate |
139.36 ± 7.84d |
|
Dichloromethane |
114.72 ± 2.99b |
|
|
Water |
122.68 ± 6.25bc |
|
|
|
Doxorubicin* |
50.82 ± 1.56a |
Each value represents means ± SD
Values within a column for each
parameter followed by different letters are significantly different (P < 0.05)
NO inhibition activity: The NO
production in the medium of RAW 264.7 cells
cultured with LPS was measured in the presence or absence of individual
compounds was determined by Greiss reagent assay. The results revealed that individual compounds with
concentration of 50, 100 and 200 µg/mL substantially inhibited the
LPS-induced NO production in a dose dependent manner (Table 3). The NO
inhibition activity from individual compounds varied from 40.6 to 51.3 µg/mL
at 50 µg/mL and the myricetin (3) was found to have the highest NO
inhibition activity, while at 100 µg/mL myricetin and its derivatives
significantly inhibited the NO activity. Likewise, the highest NO inhibition activity was recorded by Myricetin
3-galloylrhamnoside (2) at 200 µg/mL concentration (Table 3).
IL-6 inhibition activity of individual
compounds: The isolated
compounds concentration inhibited LPS-induced IL-6 production linearly. In this regard, at 50 µg/mL of
isolated compound treatments, mycetrin exhibited the
strongest IL-6 inhibition
activity; while myricetin 3-galloylrhamnoside (2)
showed the most potent IL-6 inhibitory effect with 0.90 µg/mL at 100 µg/mL concentration compared
with the 1.41 µg/mL of cells were viable after LPS induced stimulation
for 24 h (Table 3).
Moreover, higher concentration (200 µg/mL) of isolated compound strongly inhibited the IL-6 activity in macrophages. Among isolated compounds, myricetin (3), myricetin 3-galloylrhamnoside and unknown compound strongly inhibited the LPS
induced IL-6 production (Table 3).
IL-1β inhibition activity of isolated
compounds: The results
showed that isolated compounds significantly inhibited LPS-induced IL-1β production in a concentration-dependent manner. Low concentration
(50 µg/mL) of isolated compounds possess strong IL-1β inhibition
activity potential and at this concentration Myricetin was most effective;
while at 100 µg/mL concentration, 576.78 ρg/mL of cells unknown viable after LPS induced
stimulation for 24 h and among isolated compounds. Unknown compound strongly
inhibited the LPS induced IL-1β production in macrophages. However, Myricetin 3-rhamnoside substantially
reduced the cell viability of RAW 264.7 at 200 µg/mL (Table 3).
TNF-α
inhibition activity of isolated compounds: The increase in isolated compounds concentration significantly
inhibited LPS-induced TNF-α production in a linear pattern. %201161-1172_files/image017.png)
After
treatment with 100 µg/mL of the compounds. myricetin 3-galloylrhamnoside
(2) exhibited the greatest potent TNF-α inhibitory
effect with 458.61ρg/mL,
compared with the 742.79 ρg/mL of cells
were viable after LPS induced stimulation for 24h. The myricetin 3-galloylrhamnoside
(2) was found to have the highest IL-6 inhibition activity, with 296.71
ρg/mL at high concentration (200 μg/mL)
followed by: myricetin
3-rhamnoside (343.20
ρg/mL)> unknown (350.33 ρg/mL) > myricetin (381.64 ρg/mL), (Table 3).
Anti-inflammatory
activities of solvent extract fractions of R. emodi
NO inhibition
activity of R. emodi
solvent extract fractions on RAW 264.7 cells: The results showed that water and organic solvent
fractions with various concentration (50, 100 and 200 µg/mL)
significantly inhibited LPS-induced NO production in a concentration-dependent manner. At the concentration of 50 µg/mL, the NO inhibition activity from R. emodi
fraction varied from 45.0 to 50.1 µg/mL. The ethyl acetate fraction (50 µg/mL)
was found to have the highest NO inhibition activity (45.04 µg/mL);
while Dichlloromethane fraction at 200 µg/mL exhibited the strongest NO
inhibition activity (4.09 µg/mL) than rest of the fractions (Table 4).
IL-6 inhibition
activity of R. emodi
solvent extract fractions on RAW 264.7 cells: The IL-6 production was measured in the medium of RAW 264.7 cells
cultured with LPS in the presence or absence of five fractions. The results showed that all the fractions significantly
inhibited LPS-induced IL-6 production in a concentration-dependent manner. However, the response of each fraction varied greatly at different
concentration. For instance, at 50 µg/mL ethyl acetate fraction had the highest IL-6
inhibition activity. Likewise, ethyl acetate and n-Hexane fractions of R. emodi exhibited strong IL-6 inhibition activity at 100 µg/mL;
while in dichloromethane fraction at 200 µg/mL possessed the strongest IL-6 inhibition activity (0.88 µg/mL) (Table 4). The water
fraction exhibited the lowest IL-6 inhibition
activity compared to all organic solvent fractions at all concentrations.
IL-1β inhibition activity
of R. emodi solvent
extract fractions on RAW 264.7cells: All
the solvent extract R. emodi fractions
significantly inhibited LPS-induced IL-1β production in a concentration-dependent manner. The IL-1β inhibition activity of R. emodi fraction
varied from 159.5 to 201.3 ρg/mL at low concentration (50 µg/mL).
The ethyl acetate fraction was found to have the
highest IL-1β
inhibition activity at 50 µg/mL; while n-Hexan fraction was most
efficient in inhibiting the IL-1β activity at 100 µg/mL. Nevertheless, at high
concentration (200 µg/mL) R. emodi fractions progressively enhanced the IL-1β inhibition activity and complete
inhibition of IL-1β
activity in RAW 264.7 cells were observed with
dichloromethane fraction (Table 4).
TNF-α
inhibition activity of R. emodi solvent extract fractions on RAW 264.7cells: The LPS-induced TNF-α production was significantly inhibited by all solvent
extract fractions in a concentration-dependent manner. At low concentration (50
µg/mL) there was no significant difference among R. emodi fractions for TNF-α inhibition
activity, while at 100 µg/mL water fraction substantially enhanced the
TNF-α inhibition activity. Moreover, at the concentration of 200 µg/mL,
the n-butanol
fraction was found to have the highest TNF-α inhibition activity
(309.3 ρg/mL) and possess the strongest anti-inflammatory activity among
all other fractions (Table 4).
Classification
pattern for isolated compounds and solvent extract fractions of R. emodi for their anti-inflammatory activities
Table 4: Comparison of NO, IL-6, IL-1β
and TNF-α inhibition activities of solvent extract fractions
concentrations of R. emodi on RAW 264.7 cells
|
(µg/mL) |
Fraction |
NO
inhibition activity (µg/mL) |
(µg/mL) |
IL-1β
inhibition activity (ρg/mL) |
TNF-α
inhibition activity (ρg/mL) |
|||
|
n-Hexan |
47.25 ±
1.49ab |
8.04 ±
2.32b |
189.58 ± 6.54a |
692.54 ± 49.94 |
|
|||
|
n-Butanol |
50.12 ±
1.34b |
9.19 ±
1.71ab |
172.41 ± 36.34ab |
643.52 ± 31.90 |
|
|||
|
50 |
Ethylacetate |
45.04 ±
6.17a |
6.78 ±
0.95a |
159.46 ± 36.65ab |
656.48 ± 105.87 |
|
||
|
Dichloromethane |
47.65 ±
0.63ab |
9.07 ±
0.87ab |
201.25 ± 13.44b |
686.47 ± 116.91 |
|
|||
|
Water |
47.17 ±
1.78ab |
11.66 ±
1.97c |
198.39 ± 7.17b |
699.92 ± 161.45 |
|
|||
|
n-Hexan |
34.60 ±
8.90 |
3.83 ±
0.95a |
27.50 ± 24.44a |
595.08 ± 94.86b |
|
|||
|
n-Butanol |
26.17 ±
2.34 |
5.69 ±
1.01b |
33.13 ± 8.46ab |
579.18 ± 46.09ab |
|
|||
|
100 |
Ethylacetate |
26.66 ±
15.4 |
3.80 ±
0.85a |
39.46 ± 14.05ab |
620.66 ± 48.97b |
|
||
|
Dichloromethane |
29.65 ±
1.00 |
4.99 ±
0.71ab |
53.75 ± 16.41bc |
560.25 ± 41.26ab |
|
|||
|
Water |
30.27 ±
1.18 |
8.35 ±
1.10c |
65.80 ± 11.67c |
514.92 ± 20.87a |
|
|||
|
n-Hexan |
7.56 ±
1.49b |
2.84 ±
0.47c |
4.64 ± 1.03c |
468.61 ± 71.58b |
|
|||
|
n-Butanol |
6.38 ±
1.00b |
2.53 ±
0.53c |
5.27 ± 1.74c |
309.34 ± 52.58a |
|
|||
|
200 |
Ethylacetate |
7.51 ±
1.62b |
1.76 ±
0.4b |
1.21 ± 1.13ab |
469.59 ± 59.31b |
|
||
|
Dichloromethane |
4.09 ±
2.41a |
0.88 ±
0.30a |
0a |
489.84 ± 52.04b |
|
|||
|
|
Water |
6.40 ±
1.14b |
4.17 ±
0.69d |
2.23±1.79b |
470.98 ± 7.75b |
|
||
Each value represents means ± SD
values within a column for each
concentration followed by different letters are significantly different
(p<0.05)
The individual score showed its pattern, change and cluster formation
that contained anti-inflammatory activities of compounds from R. emodi.
Accordingly, as shown in Fig. 9, the correlation of anti-inflammatory activities and compounds (Myricetin
3-o-rhamnoside, Myricetin
3-galloylrhamnoside, Myricetin and unknown) were
expressed through PLS-DA score plotting (Fig. 9). They could be grouped in four
different groups. The compounds belong to left cluster than the ones on right
cluster showed comparably higher anti-inflammatory activities. The cluster for
blue color in compound (Myricetin) was located on the left side.
The others make cluster for red color in Myricetin 3-galloylrhamnoside was
located on the top-right side and green color in Myricetin 3-o-rhamnoside was
located on the bottom (Fig. 9). As
a result, anti-inflammatory activities are related to the structure of compound
and its contents, and it meant that the compound belong to myricetin
cultivar had higher anti-inflammatory activities that the other compounds
belong to Myricetin 3-o-rhamnoside, Myricetin 3-galloylrhamnoside and Unknown cultivars. Also,
the Myricetin 3-galloylrhamnoside showed that
the gallate acylation on the glycoside moiety on a flavonoid had higher anti-inflammatory activities than the Myricetin 3-o-rhamnoside; however, the
potency did not exceed the activity of corresponding aglycon.
%201161-1172_files/image019.jpg)
Fig. 9: Score plotting
chart of principal component 1 and 2 of the PLS-DA results obtained from the
data set by compounds profiling on the anti-inflammatory compounds. This chart
showed classification by origin on all samples
(Green circle: Myricetin 3-rhamnoside, Purple
circle: Unknown, Red circle: Myricetin- 3-galloylrhamnoside, Blue circle:
Myricetin)
The correlation of anti-inflammatory activities and fractions (n-Hexan, n-Butanol, Ethyl acetate, Dichloromethane, and
water) were expressed through PLS-DA score
plotting. The fractions belong to right cluster
(green and blue circle) showed comparably higher anti-inflammatory activity
than the ones on left cluster (purple and red circle) showed comparably higher
anti-inflammatory activities. Especially, it was confirmed that Water and
n-Butanol belong to left cluster (yellow green), and Ethyl acetate and
Dichloromethane belong to right cluster (yellow), but n-Hexane fraction was
mixed up from other fractions (Fig. S4).
Discussion
The currently used all therapies for cancer treatment, particularly
chemical synthesized drugs exhibit significant cytotoxicity to normal cells
(Gupta et al. 2013). This study
showed the potential of different fractions (organic and aqueous) of R. emodi
rhizome extracts against anticancer and anti-inflammatory activities. In
current study, the aqueous and organic solvent fractions of R. emodi exhibited strong anticancer activities. The
aqueous and organic solvent extract fractions significantly (P < 0.05) enhanced the anticancer
activities against A549 and MCF-7 cancer cell lines which can be attributed to
the presence of compounds such as anthraquinones, polyphenols (flavonoids),
antra glycosides etc. in R. emodi fractions
known for their antifungal, antibacterial and lipid homeostasis activities. Moreover, the activities of R. emodi fractions were strongly influenced by the solvent
used. These differences in activities of different solvents can be ascribed to
existence of distinctive protective metabolites extracted by the different
solvents and solubility or stereoselectivity of the fractions (Yu et al.
2002).
Some naturally occurring flavonoids have exhibited selective toxicity to
human cancer cells accompanied by very low toxicity to normal cells (Sak 2014).
The positive effects of flavonoids in cancer treatment are associated to their
strong antioxidant ability, including their capability to scavenge ROS. Several
natural flavonoids targets caspases and therefore potent candidate as cancer
chemotherapeutic agents (Raffa et al. 2017). One such flavonoid is
myricetin (a dietary flavonoid) present in fruits, vegetables, medicinal plants
including R. emodi (Fig. 1–5; Devi et al.
2015). Myricetin has been found to exert several antitumor roles in various
types of cancers which include, antiproliferation, apoptosis induction and
anti-metastatic activities (Iyer et al. 2015; Xu et al. 2015;
Raffa et al. 2017) In present study, the isolated compounds (Myricetin
and its derivatives) from R. emodi extracts
efficiently inhibited the proliferation and induced the apoptosis in MCF-7 and A549 cancer
cells (Table 1 and Fig. 7–8), which can be ascribed to the involvement of
myricetin in upregulation of pro-apoptosis proteins and downregulation of
ERK1/2 and AKT phosphorylation levels, Bcl-2 protein in cancer cell lines
(Raffa et al. 2017; Park et al. 2020) and activation of caspase-3
in cancer cell lines (Raffa et al. 2017) . The isolated compounds
(myricetin 3-O-rhamnoside, myricetin 3-O-galloyrhamnoside, myricetin and unknown) in present study exhibited the
anticancer activities in a dose dependent manner as higher concentration of
myricetin exhibit the antiproliferative activities against cancer cells (Sun et
al. 2012). Moreover, myricetin reduces the Matrix metalloproteinase 9
(MMP9) (member of MMP family of 24 zinc-dependent endopeptidases involved in
induction of pro-IL-1β cleavage and microglial activation) expression
showing the antimetastatic effect (Raffa et al. 2017). The present study
suggests that the isolated compounds from the R. emodi
extract can be used as potential cancer therapeutics.
Inflammation is the key part of innate immunity and
inflammatory response of a living tissue caused by noxious chemical stimuli,
physical injury or infection (Wahab et al. 2018). The acute and chronic inflammation has been treated with different plant
extracts in traditional medicine.
Here, we observed that the aqueous and organic solvents extract fractions of R.
emodi
inhibited the LPS induced NO production in RAW 264.7 cells. The RAW 264.7 cells
are macrophages used for evaluation of anti-inflammatory responses and drug screening
(Lee et al. 2014). This inhibition of NO production in macrophage RAW
264.7 cells by R. emodi fractions may be accredited to
presence of flavonoids in these fractions (Table 4).
These plant extracts often have active constituent of flavonoids and related
compounds, which possesses strong anticancer, antiinflammation, antiviral and
immunomodulatory activities etc. (Russo et al.
2000; Havsteen 2002). The association
of an aromatic ring to heterocyclic ring and functional group oxidation state
of heterocyclic ring serves as the bases for flavonoids classification (Beecher
2003). The flavanols are the most abundant
flavonoids; usually present in large quantities in vegetables and fruits in the
form of aglycone or glycoside including myricetin, quercetin and kaempferol
(Shukla et al. 2019). Myricetin (myricetin-3-glucoside,
myricetin-3-rhamnoside) is an important bioflavonoid known for their
anti-inflammatory activities (Table; Fig; Skrovankova et al. 2015). In
Raw 264.7 macrophages, LPS induced pro-inflammatory mediators and cytokines
play crucial role in inflammation induction. Nitric oxide (NO) is well known
mediator and reacts with superoxide derived from macrophages in response to
stimuli and produce cytotoxic peroxynitrite, which is involved in the inflammatory
matrix metalloproteinase and cytokines production (Jarvinen et al.
2008). Further, the isolated Myricetin derivatives at higher doses attenuated
the NO production (Table 2), which can be ascribed to role of these compounds
in iNOS expression downregulation (Cho et al. 2016).
The pro-inflammatory cytokines such as IL-6, IL-1β
and TNF-α are well known, which are involved in inflammation induction.
Bavia et al. (2015) revealed that LPS induced acute lung injury through
increased production of IL-6, IL-1β and TNF-α along with
histopathological changes, alveolar wall thickening etc. which can be counteracted by bioactive compounds isolated from
plants (Niu et al. 2014). In present study, R. emodi solvent
extract fractions inhibited the IL-6, IL-1β and TNF-α activity
similar to NO in RAW 264.7 macrophage cells (Table 4). Here, all R. emodi fractions substantially inhibited the IL-6,
IL-1β and TNF-α activities in dose dependent manner. Ethyl acetate
was more effective at lower concentration (50 and 100 µg/mL) for IL-6,
IL-1β inhibition activity; while dichloromethane was most effective
fractions at higher dose (200 µg/mL) and even complete inhibition of
IL-1β activity was observed in RAW 264.7 macrophages. The differential response of these fractions may be due
presence of different metabolites in these fractions and their solubility.
Furthermore, the compounds (myricetin and its derivatives) isolated from the
ethyl acetate fraction substantially inhibited the activities of
pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) (Table 3); which
can be attributed to Myricetin role in limiting NF-κB activity through
subduing the IκBα degradation, nuclear translocation of NF-κB
(p65 subunit) and NF-κB DNA binding activity in LPS stimulated macrophages
(RAW264.7) (Cho et al. 2016); as myricetin administration has been
testified for inhibition of pro-inflammatory cytokines activities (Niu et al.
2014; Cho et al. 2016). Furthermore, the higher antiinflamatory
activities of isolated compounds are due to myricetin involvement in HO-1
expression induction via Nrf2 translocation. The isolated compounds (myricetin,
myricetin-3-O-galactoside and myricetin-3-O-rhamnoside) from ethyl acetate
fraction of R. emodi indicate that these
compounds can be used for treatment of inflammation disorders.
PLS-DA
is much more advantageous to distinguish the characteristics of pre-defined
groups compared to the existing PCA (Perez-Enciso and Tenenhaus 2003). PLS-DA is discriminating the assigned cluster characteristics. It is
variance that affects the cluster formation or considered as important of
compound information. Metabolite analysis data can be visualized through range;
standardization and
major component score (Cho 2013). The Galloyl flavanol
glycosides protect macrophages from the inflammatory response through the
limiting the expression of COX-2. The Galloyl group showed enhanced COX-2
expression than their parent compounds (Kim et al. 2004). Although this
discrepancy in the results cannot fully be explained at this stage, it may come
from different assay system used or, possibly, to the different attachment
position of the galloyl group. It can be recognized as the promising flavonoid
anti-inflammatory with R. emodi being a good source of the flavonoids.
Conclusion
The result of present study showed the potential of aqueous and organic
solvent R. emodi rhizome fractions and
isolated flavanoids for anti-cancer and anti-inflammatory activities. The
Flavonoids (myricetins) isolated and identified from ethyl acetate fraction of R.
emodi exhibited strong cytotoxicity and
anti-inflammatory activities on cancer cell lines (MCF-7 and A549) and LPS
stimulated macrophages (RAW 264.7), respectively. The higher concentration
(≤ 100 µM) aqueous/organic fractions and isolated compounds strongly inhibited
the LPS stimulated TNF-α, IL-6 and IL-1β and NO production in macrophages and induced the apoptosis and restricted
the proliferation in cancer cell lines. However, myricetin and unknown
compounds were most effective at lower concentration (50 µg/mL) for
anti-inflammatory and anticancer response, suggesting that the isolated
compounds can be used for drug development for cancer and inflammation
treatment.
Author Contributions
Sang KP and SY Im conceived the idea,
Sang KP conducted the study, SY Im analyzed the data and prepared the
manuscript draft for submission
Conflict of Interest
The authors delare no conflict of
interests
Data Availability
The data supporting the findings of
this study are available within the article and its supplementary materials.
Ehics Approval
All procedures performed in this study
involving human or animal cells were in accordance with the ethical standards
of the institution at which the studies were conducted
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