1 Running title: Pharmcological activities of Actinidia callosa var. ephippioides
Chemical compositions, Anti-inflammatory, Antiproliferative and
Radical-scavenging Activities of the methanol extracts of Actinidia
callosa var. ephippioides
Jung-Chun Liao†, Δ, Jeng-Shyan Deng‡, Δ, Chuan-Sung Chiu¶, Shyh-Shyun Huang†,
Wen-Chi Hou＃
, Wang-Ching Lin†, Guan-Jhong Huang§, *
†
School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan
‡
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan
¶
Nursing Department, Hsin Sheng College of Medical Care and Management, Taoyuan 325, Taiwan
＃
Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 250, Taiwan
§
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan
*
Corresponding author Guan-Jhong Huang
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan.
Tel: +886- 4- 2205-3366. Ext: 5508. Fax: +886- 4-2208-3362, E-mail address: gjhuang@mail.cmu.edu.tw
ABSTRACT.
Oxidative stress and inflammation are related to several chronic diseases including cancer. Actinidia callosa var. ephippioides (ACE) is a special folk medicinal plant in Taiwan. The aim of this study is to evaluate the antioxidant, anti-inflammatory, and antiproliferative activities of the methanol extract and fractions from the stem of
ACE. Trolox Equivalent Antioxidant Capacity (TEAC),
1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, total phenolic content, flavonoid content, inhibition on nitric oxide (NO) productions by LPS-induced RAW264.7 cell, and on lung cancer cell proliferation were employed. Among all fractions, ethyl-acetate fraction (EA-ACE) showed higher TEAC, DPPH radical scavenging activities, polyphenol and flavonoid contents, respectively. EA-ACE also decreased the LPS-induced NO production and expressions of inducible nitric-oxide synthase (iNOS) in RAW264.7 cells. EA-ACE had the highest antiproliferative activity with an IC50 (The concentrations required for inhibition of 50% of cell
viability) of 469.17 ± 3.59 μg/mL. Catechin also had good effects in the antioxidant and anti-inflammatory activities. Catechin might be an important bioactive compound in the stem of ACE. The above experimental data indicated that the stem of ACE is a potent antioxidant medicinal plant, and such efficacy may be mainly attributed to its polyphenolic compounds.
Keywords: Actinidia callosa var. ephippioides; Antioxidant activity;
3 Introduction
The reactive oxygen species play an important role in the degenerative or
pathological processes of various serious diseases, such as cancer, coronary heart
disease, Alzheimer’s disease, neurodegenerative disorders, atherosclerosis, cataracts,
inflammation and aging (Huang et al., 2012). The use of traditional medicine is
widespread and plants still represent a large source of natural antioxidants that might
serve as leads for the development of novel drugs. Several anti-inflammatory,
digestive, antinecrotic, neuroprotective, and hepatoprotective drugs have recently been shown to have an antioxidant activity (Huang et al., 2008).
Macrophages play an important role in inflammatory disease through the release
of inflammatory factors such as reactive oxygen species (ROS) and cytokines.
Production of these macrophage mediators has been determined in many
inflammatory tissues, following exposure to immune stimulants including bacterial
endotoxin lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Inappropriate
macrophage activation is responsible for the pathology of acute or chronic
inflammatory disease (Deng et al., 2011). Chronic inflammation leads to the
up-regulation of signaling proteins in affected tissues and cells. Among the
proinflammatory enzymes, the inducible nitric-oxide synthase (iNOS) and cyclooxygenase (COX-2), are known to be involved in various chronic diseases
including multiple sclerosis and Parkinson's and Alzheimer's diseases, as well as lung cancer (Lai et al., 2009). Thus, agents that suppress iNOS and COX-2 overexpression
have potential therapeutic value when associated with inflammation and carcinogenic
processes.
Actinidia callosa var. ephippioides (Actinidiaceae; ACE) is a deciduous
tuberous plant, which is distributed in oriental countries. The stems of the plant have
been extensively employed to treat various ailments like leprosy, abscess, rheumatism,
arthritis inflammation, jaundice, and abnormal leucorrhea and were also useful for the
treatment of cancers, especially those of lung, liver, and digestive system (Gan, 1993).
Actinidia species had showed some pharmacological effects such as
anti-inflammatory activity from the fruit of Actinidia polygama (Kim et al., 2003),
antitumor and immunomodulatory activity from the roots of Actinidia eriantha (Xu et
al., 2009). In this study, we examine the inhibitory effect of the antioxidant, anti-inflammatory, and anti-proliferative activities of the methanol extracts of
Actinidia callosa var. ephippioides (MACE) and fractions. Consequently, the
objective of the present study is to determine the effects of ACE against antioxidant, anti-inflammatory, and anti-proliferative activities and its active compounds.
5 Materials
Lipopolysaccharide (LPS, Escherichia coli O127:B8),
1,1-Diphenyl-2-picrylhydrazyl (DPPH), N-(1-naphthyl) ethylenediamine
dihydrochloride, sulfanilamide, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS), thiobarbituric acid (TBA), 3-[4,5-dimethyl-thiazol- 2-yl]-2,5-diphenyl
tetrazolium bromide (MTT), and other chemical reagents were purchased from
Sigma–Aldrich (St. Louis, MO, USA). Anti-iNOS, anti-COX-2, and anti-β-actin
antibodies (Santa Cruz, CA, USA) were obtained as indicated. Plant materials were
collected from Taichung country in Taiwan. They were identified and authenticated by
Dr. Yuan-Shiun Chang, Professor, School of Chinese Pharmaceutical Sciences and
Chinese Medicine Resources, College of Pharmacy, China Medical University. A
plant specimen was deposited in the Institute.
Extraction and Fractionation
The coarse powder of the stem of Actinidia callosa var. ephippioides (1 kg) was
extracted with methanol three times (10 L). The extract was evaporated under reduced
pressure using a rotavapor, and then stored under light protection. A yield equivalent
to 5.6 % of the original weight was obtained. Next, methanol extract of ACE (46 g)
partitioned in sequence with n-hexane, chloroform, ethyl acetate and n-butanol (800
mL each for three times). Under reduced pressure, fractions were yielded and
collected: n-hexane fraction (19.4 g, 42.2%), chloroform fraction (4.9 g, 10.7%), ethyl
acetate fraction (12.2 g, 26.5%), n-butanol fraction (5.2 g, 11.3%) and aqueous
fraction (4.3 g, 9.3%). All extracts were stored in the refrigerator before the use.
Fingerprint chromatogram of EA-ACE extracts by HPLC
HPLC was performed with a Hitachi Liquid Chromatography (Hitachi Ltd., Tokyo,
Japan), consisting of two model L-5000 pumps, and one model L-7455 photodiode
array detector (254 nm). Samples (10 mg/mL) were filtered through a 0.45 μm
PVDF-filter and injected into the HPLC column. The injection volume was 10 μL and
the separation temperature was 40°C. The column was a Mightysil RP-18 GP (5 μm,
250 mm × 4.6 mm I.D.). The method involved the use of a binary gradient with
mobile phases containing: (A) phosphoric acid in water (0.6‰, v/v) and (B) MeOH
(v/v). The solvent gradient elution program was as follows: from 88% A to 78% A in
30 min, from 78% A to 68% A in 15 min. The flow-rate was kept constant at
1.0 mL/min. A precolumn of μ-Bondapak™ C18 (Millipore, Milford, MA, USA) was
7 wavelengths of phenolic compounds at their respective maximum absorbance
wavelength can monitored at the same time. Identification is based on retention times
and on-line spectral data in comparison with authentic standards. Quantification is
performed by establishing calibration curves for each compound determined, using
the standards.
Determination of antioxidant activity by ABTS·+ scavenging ability
The ABTS·+ scavenging ability was determined according to the method of
Huang et al., (2006). The antioxidative activity assay that uses ABTS is called
the TEAC assay. Aqueous solution of ABTS (7 mM) was oxidized with potassium
peroxodisulfate (2.45 mM) for 16 hrs in the dark at room temperature. The ABTS·+
solution was diluted with 95% ethanol to an absorbance of 0.75 ± 0.05 at 734 nm
(Beckman UV-Vis spectrophotometer, Model DU640B). An aliquot (20 μL) of each
sample (125 μg/mL) was mixed with 180 μL ABTS·+ solution and the absorbance was
read at 734 nm after 1 min. Trolox was used as a reference standard. A standard curve
was constructed for Trolox at 0, 15.625, 31.25, 62.5, 125, 250, 500 μM concentration.
The effects of crude extracts and positive controls (BHT) on DPPH radicals were
estimated according to the method of Huang et al., (2006). Aliquot (20 μL) of crude
extracts at various concentrations were each mixed with 100 mM Tris-HCl buffer (80
μL, pH 7.4) and then with 100 μL of DPPH in ethanol to a final concentration of 250
μM. The mixture was shaken vigorously and left to stand at room temperature for 20
min in the dark. The absorbance of the reaction solution was measured
spectrophotometrically at 517 nm. The percentages of DPPH decolorization of the
samples were calculated according to the equation: % decolorization = [1- (ABS sample
/ABS control)] ×100. EC50 value was the effective concentration at which DPPH
radicals were scavenged by 50% and was obtained by interpolation from linear
regression analysis.
Determination of total polyphenol content
The total polyphenol contents of crude extracts were determined according to the
method of Huang et al (2008). 20 μL of each extract was added to 200 μL distilled
water and 40 μL of Folin-Ciocalteu reagent. The mixture was allowed to stand at
room temperature for 5 min and then 40 μL of 20 % sodium carbonate was added to
the mixture. The resulting blue complex was then measured at 680 nm. Catechin was
9 using the linear equation based on the calibration curve. The total polyphenol content
was expressed as mg catechin equivalence (CE)/g dry weight.
Determination of Total Flavonoid Content
The flavonoid content was determined according to the method of Lamaison and
Carnet (1990). 100 μL aliquots of the extract and fractions were added to equal
volumes of 2% AlCl3·6H2O solutions. The mixtures were shaken vigorously and left
incubating for 10 minutes before the absorbance was read at 430 nm. Rutin was used
as standard for the calibration curve, by which a linear equation was derived to
determine total flavonoid contents of the samples. Total flavonoid data were expressed
in mg of rutin equivalents per gram of dry weight.
Cell culture
A murine macrophage cell line RAW264.7 (BCRC No. 60001) and A549 (BCRC No. 60074) were purchased from the Bioresources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in plastic dishes containing Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA) in a CO2 incubator (5% CO2 in air) at 37°C and subcultured
every 3 days at a dilution of 1:5 using 0.05% trypsin–0.02% EDTA in Ca2+-, Mg2+- free phosphate-buffered saline (DPBS).
Cell viability
Raw 264.7 cells (2 x 105) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with the methanol extract and fractions in the presence of 100 ng/mL LPS for 24 h. After that, the cells were washed twice with DPBS and incubated with 100 L of 0.5 mg/mL MTT for 2 h at 37°C testing for cell viability. A549 were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL of penicillin, 100 mg/mL streptomycin, and 1 mM sodium pyruvate. The cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The cells were then treated with
the methanol extract, fractions, and its active compounds for 24 h. Each concentration was repeated three times. After a period of incubation, the medium was removed, and then the cells were washed with PBS. The medium was then discarded and 100 L dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read using a microplate reader.
Measurement of Nitric oxide/Nitrite
Nitrite levels in the cultured media, which reflect intracellular NO synthase activity, were determined by Griess reaction (Huang et al., 2007). The cells were incubated with samples in the presence of LPS (100 ng/mL) at 37°C for 24 hrs. Then, cells were dispensed into 96-well plates, and 100 L of each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min. By using sodium nitrite to generate a standard curve, the concentration of nitrite was measured form absorbance at 540 nm.
11 Protein Lysate Preparation and Western blot Analysis of iNOS and COX-2
Total protein was extracted with a RIPA solution (radioimmuno-precipitation assay
buffer) at -20°C overnight. We used BSA (bovine serum albumin) as a protein
standard to calculate equal total cellular protein amounts. Protein samples (30g)
were resolved by denaturing sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) using standard methods, and then were transferred to
PVDF membranes by electroblotting and blocking with 1% BSA. The membranes
were probed with the primary antibodies (iNOS, COX-2, and -actin) at 4°C
overnight, washed three times with PBST, and incubated for 1 h at 37 °C with
horseradish peroxidase conjugated secondary antibodies. The membranes were
washed three times and the immunoreactive proteins were detected by enhanced
chemiluminescence (ECL) using hyperfilm and ECL reagent (Amersham
International plc., Buckinghamshire, U.K.). The results of Western blot analysis were
quantified by measuring the relative intensity compared to the control using Kodak
Molecular Imaging Software and represented in the relative intensities.
Statistical analysis
three parallel measurements. Statistical evaluation was carried out by one-way
analysis of variance (ANOVA followed by Scheffe's multiple range tests). Statistical
significance is expressed as *
p < 0.05, **p < 0.01, and ***p < 0.001.
Results and Discussion
Fingerprint Analysis by HPLC
To establish the fingerprint chromatogram for the quality control of EA-ACE,
protocatechuic acid, catechin, and epicatechin were used as markers. An optimized
HPLC-PAD technique was employed. Meanwhile, HPLC chromatograms showed
three marker components present in EA-ACE. As shown in Fig. 1, these phenolic
components have been identified as protocatechuic acid, catechin, and epicatechin
by their retention time and UV absorbance of purified standards. According to the
plot of peak-area ratio (y) vs. concentration (x, g/mL), the regression equations of
three constituents and their correlation coefficients (r) were determined as follows:
protocatechuic acid, y = 0.622x + 4.301 (r2 = 0.999); catechin, y = 0.443x +3.104
(r2 = 0.999); epicatechin, y = 0.836x + 3.893 (r2 = 0.999). The relative amounts of
three phenolic compounds found in EA-ACE were in the order of epicatechin
13 extract), respectively.
The contents of phytochemicals extracted and the antioxidant activities of EA-ACE.
Phenolic compounds occur ubiquitously in plants and are active components in
many herbs. Numerous studies have shown that these phenolic compounds possess
antioxidant and anti-inflammatory activity (Huang et al., 2011). Table 1 shows ABTS
and DPPH scavenging activities of MACE and fractions. EA-ACE exhibited the
stronger antioxidant activities in scavenging DPPH radicals, with EC50 values of
225.29 ± 4.93 g/mL. And TEAC value of EA-ACE was 159.59 ±1.17 g /mg extract.
The results also showed that EA-ACE had the highest phenolic contents of 627.56 ±
1.15 g CE/mg (Table 1). Total flavonoid content was expressed as mg of rutin
equivalent per gram of dry weight. As shown in Table 1, the total flavonoid content of
EA-ACE was 14.31 ± 0.38 g RE/mg.
As shown in Table 1, reference compounds protocatechuic acid, catechin, and
epicatechin in the EA-ACE showed TEAC value of 1252.14 ± 2.87, 1513.36 ± 4.53,
and 857.56 ± 3.59 g/mg extract, respectively. In addition, the reference compounds
protocatechuic acid, catechin, and epicatechin in the EA-ACE in the EAAC showed
DPPH radical scavenging with an EC50 value of 11.55 ± 0.26, 9.38 ± 0.32, and 21.32
Polyphenols act as antioxidants via several mechanisms including the scavenging
of free radicals, chelation of transition metals, as well as the mediation and inhibition
of enzymes (Chang et al., 2009). The radical scavenging activity of EA-ACE seems to
be correlated with its polyphenolic constituents through active components that could play important roles in its antioxidative effect. In this paper, we demonstrated that
EA-ACE inhibited radical scavenging. And the reference compound of protocatechuic
acid, catechin, and epicatechin in the EA-ACE also had the antioxidant activities.
Relationship between Total Antioxidant Power with Respect to Total Phenolic and
Total Flavonoid Contents
Correlation coefficients (R2) of the total antioxidant power with respect to total
phenols and total flavonoid contents of ACE were estimated in this study. As shown in
Figure 2, the correlation coefficient (R2) of TEAC and total phenolic content was 0.96
(Fig. 2A). The R2 value of TEAC and total flavonoid content was 0.47 (Fig. 2B). The
results revealed high correlations between TEAC and total phenolic contents.
15 The effect of MACE and fractions on RAW264.7 cell viability was determined
by a MTT assay. Cells cultured with MACE and fractions at the concentrations (0,
62.5, 125, and 250 g/mL) used in the presence of 100 ng/mL LPS for 24 h did not
change cell viability, significantly (Fig. 3A).
In a cellular model of inflammation, the NO inhibitory activity of MACE and
fraction were determined by using the LPS activated macrophages to produce NO
radicals measured as nitrites in the culture medium by the Griess reaction. As shown
in Fig. 3B, EA-ACE reduced the NO production of activated macrophages with an
IC50 value of 96.29 ± 0.28 g/mL, respectively. This suggests EA-ACE could be a
potential inhibitor of NO related inflammation pathway (Table 2; partial data of table
2 is based on Figure 3A). And the reference compounds of protocatechuic acid,
catechin, and epicatechin in the EA-ACE also showed the NO inhibitory activity
induced by LPS in RAW264.7 macrophages (Table 2). Protocatechuic acid and
epicatechin had weak or no anti-inflammatory activity induced by LPS in RAW264.7 macrophages, respectively. Whatever, we evaluated the reference compounds in the
EA-ACE that catechin exhibited good anti-inflammatory activities by LPS-induced
NO production in RAW264.7 macrophages with an IC50 value of 35.53 ± 0.31 g/mL
Inhibition of LPS-induced iNOS and COX-2 Protein by EA-ACE.
The results showed that incubation with EA-ACE in the presence of LPS for 24
hrs inhibited iNOS protein expression in mouse macrophage RAW264.7 cells in a
dose-dependent manner (Fig. 4A). The intensity of protein bands were analyzed and
showed an average of 86.2% and 10.6% down-regulation of iNOS and COX-2
proteins, respectively, after treatment with EA-ACE at 250 g/mL compared with the
LPS-alone (Fig. 4B).
Excessive production of NO plays a critical role in the aggravation of circulatory
shock and chronic inflammatory diseases, such as septic shock, inflammatory hepatic
dysfunctions, inflammatory lung disease and colitis (Huang and Ho, 2010; Chang et
al., 2011). As many of these conditions exhibit rapid onset and development, often
resulting in the failure of conventional anti-inflammatory therapies and extremely
high mortality rates, a simultaneous suppression of NO production pathways, as
shown by EA-ACE and catechin may satisfy the control of the rapid progression of
the inflammatory process.
Cell Viability
MTT assay was used to investigate whether MACE and fractions affected the
17 400, 600, 800, and 1000 μg/mL of the methanol extract and fractions were assayed by
MTT. After exposing A549 cells to any of the samples with various concentrations,
the cell viabilities decreased significantly as compared to the control (100%),
indicating cytotoxic effect on A549 cells. Among all the fractions, ethyl-acetate
fraction (IC50= 469.17 ± 3.59) fractions showed excellent inhibitory effects on A549
cells, as shown in Table 3. Protocatechuic acid, catechin, and epicatechin showed little
inhibitory effects on A549 cells. The components which are responsible for the
antiproliferative activity of the ACE are still unclear. Therefore, further researches
must be performed to isolate and identify these components.
Phenolic contents were not only indicated in this experiment to be directly
proportional with antioxidant activity, as suggested by Huang et al. (2008), these
phytochemicals may also possess unique or synergic activities on the inhibition of
tumor cell proliferation in vitro. In previous phytochemical investigations,
water-eluted fraction showed the strongest inhibitory effect on the growth of
S180 sarcoma transplanted in mice from Actinidia eriantha (Xu et al., 2009)
and the extracts from roots of Actinidia indochinensis possessed the role of
antitumors in cell culture (Zhong et al., 2005). It is possible that at least part of
the antioxidant or inflammation effects observed in the present work may be due to phenolic compounds.
Conclusion
This study revealed that ethyl-acetate fractions of Actinidia callosa var.
ephippioides exhibited good antioxidant activities, anti-inflammatory activities and
inhibited the growth of A549 cell. These activities may be attributed to the high
polyphenolic contents in these fractions. This study also demonstrated that Actinidia
callosa var. ephippioides had a wide safety dosage range and has a potential for local
therapeutic applications in chronic diseases. Further studies should be carried out to correlate the pharmacological activities with the chemical constituents
Acknowledgement
The authors want to thank the financial supports from the National Science
Council (NSC100-2313-B-039-004- and NSC 100-2320-B-039-033-), China Medical
University (CMU) (CMU99-TC-35, CMU99-COL-10, and CMU100-TC-11) and
Taiwan Department of Heath Clinical Trial and Research Center of Excellence
(DOH101-TD-B-111-004).
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