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ESEARCH ARTICLE
R ScienceAsia 45 (2019): 127–137
doi: 10.2306/scienceasia1513-1874.2019.45.127
Effects of drying and extraction methods on phenolic
compoundsandinvitro assays of Eclipta prostrata
Linn leaf extracts
a a a b
WorananNakbanpote , Munjit Ruttanakorn , Kannika Sukadeetad , Niramol Sakkayawong ,
b,∗
Somchit Damrianant
a Department of Biology, Faculty of Science, Mahasarakham University, Maha Sarakham 44150 Thailand
b Department of Biotechnology, Faculty of Science and Technology, Thammasat University,
Pathum Thani 12120 Thailand
∗Corresponding author, e-mail: somchit@tu.ac.th
Received 31 Aug 2018
Accepted 30 Apr 2019
ABSTRACT: Eclipta prostrata Linn leaves have a potential to be an alternative source of commercial herbal extracts.
The effects of different drying methods (oven-drying at 60°C, freeze drying, and microwave drying) on polyphenol
and antioxidant activities of E. prostrata were reported. The extractions with absolute ethanol and freeze-drying
preserved higher levels of total phenolic content and total flavonoid content than those of the oven and microwave-
drying methods. Correlation analysis indicated that phenolic and flavonoid compounds were the major antioxidants
in E. prostrata extracts. Drying methods were a critical factor following the order of freeze-dried, microwave dried,
oven-dried leaf extracts, for which the extraction methods seemed to have no correlation with the properties of the
extracts. The E. prostrata ethanolic extracts contained flavonoids (luteolin, luteolin glucoside, luteolin sulphate,
wedelolactone, dimethylwedelolactone, dimethylwedelolactone sulphate, 3′-hydroxybiochanin A), phenolic acids
(gallic acid, protocatechuicacid, caffeoylquinicacid, dicaffeoylquinicacid), phenolicaldehyde(protocatechualdehyde),
andtriterpenoids(eclalbasaponin). Highperformancethinlayerchromatographyofthecrudeextractsfromthefreeze-
driedleavesandsoxhletextractiongavethemainwedelolactone,chlorophyll,andsomenon-phenoliccompounds. The
IC values of the crude extracts for anti-proliferation of HaCaT cells indicated less efficiency for psoriasis treatment.
50
However, an appropriate extraction of E. prostrata leaves to obtain high luteolin and wedelolactone contents has been
suggested for a further development of local and systemic treatments for other inflammatory skin diseases.
KEYWORDS:Ecliptaprostrata, phenolic, flavonoid, drying method, anti-proliferation
INTRODUCTION tional and translational levels9. Wedelolactone
Eclipta prostrata L. belongs to the therophyte herb has been demonstrated a significant retrieval of
of the family Asteraceae, and it is widely distributed anti-oxidative enzymes, its effects on alleviating
throughout Thailand. This plant is widely applied inflammatorymarkersrelatedtoUVBexposure,and
in traditional Thai and Chinese medication, and it early tumour stimulating incidences in murine skin
describing feasible role of the NF-κB pathway10.
is also a functional food1,2. It has been reported
to possess antioxidant3,4, anti-inflammatory5, and These studies provide a medical exploitation of this
antitumor6 activities. Wedelolactone and lute- plant as a substantial treatment for skin diseases,
olin are major polyphenols of methanol/ethanol especially psoriasis and inflammation.
extracts of E. prostrata6–8. Besides known as a The polyphenol contents in plants are affected
natural compound with potent anti-inflammatory bynumerousfactors,suchasgenetics,age,environ-
properties, luteolin has been firstly reported to mental factors (soil type, sun exposure, and rain-
fall), processing, and storage11. The postharvest
reduce keratinocyte proliferation, which is re- dryingmethod,extraction,anddistillationaffectthe
lated to an autoimmune disease psoriasis, with- qualitative and quantitative aspects of the bioactive
out affecting the viability and intracellular aspects. compoundsfromplantmaterials. Differentposthar-
Moreimportantly, luteolin decreases nuclear factor- vest methods have been used for drying different
kappa B (NF-κB) activation, typically associated medicinal plants. Oven drying is the most cost
in inflammatory mediators, at both the transcrip-
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128 ScienceAsia 45 (2019)
and time effective for drying most plant materi- MATERIALSANDMETHODS
als. Microwave drying can shorten the drying time Plant materials and chemicals
and moderately lower the energy consumption, but
sometimesitcausesdegradationofphytochemicals. Like other weeds, E. prostrata was simply collected
Freeze-drying is a sublimation process that a plant from a paddy field in Koeng Subdistrict, Muang
′ ′′
sample is frozen prior to lyophilization, then re- District, Maha Sarakham, Thailand (16°12 30 N,
′ ′′
duced surrounding pressure allows frozen moisture 103°17 42 E), in September 2014. The soil prop-
to sublime directly out of the sample. Despite erties were pH 6.72±0.06, electrical conductivity
high cost, freeze-drying causes less damages of sub- 16.67±5.77 mS/cm, organic matter 3.28%, nitro-
stances than other high temperature drying, result- gen 0.17±0.02%, phosphorus 27.71±2.89 mg/kg,
ing in general applications to preserve food and to potassium 114.52±6.40 mg/kg and cation ex-
increaseshelflifeofagriculturalandpharmaceutical change capacity 17.46±0.55 cmol/kg. This plant
products. Nevertheless, this drying led to loss some was authenticated and a voucher specimen was
volatile compounds capable of sublimation12,13. Al- deposited at Department of Biology, Faculty of Sci-
though modern extraction techniques, e.g., pres- ence, Mahasarakham University, Maha Sarakham,
surized liquid extraction and supercritical fluid ex- Thailand. Collected whole plants were immediately
traction, have been developed, existing practical transferredintopolyethylenebagsandstoredat4°C
techniques (soxhlet extraction, maceration and per- for 20 min of transportation. Healthy leaves (not
colation) have been consistently applied for crude including seriously damaged or diseased leaves)
extraction14,15. The drying and extraction methods werewashedwithanexcessoftapwater. Thewater
are major factors contributing to radical scaveng- waswipedoffwithcleantissue paper before drying
ing activities of herbal plants and their extracts, processes.
i.e., Cinnamomum zeylanicum16, Betula pendula17, The solvents and chemicals used in this study
Alpinia zerumbet, Etlingera elatior, Curcuma longa, were analytical grade. Standard chemicals and
Kaempferia galangal18, and Thunbergia laurifolia19. mobile phases for HPLC and LC-MS/MS analyses
Since there are notable differences in secondary were HPLC grade from Sigma-Aldrich and Fluka.
metabolites in plants, a particular plant needs suit- Chemicals and media for in vitro assays were from
able drying and extracting methods to achieve high GibcoTMThermoFisher Scientific.
levels of beneficially targeted phytochemicals. Drying and extraction
This study investigated the effects of the
postharvest drying processes (freeze drying, oven Thecleanleaves were dried in a hot air oven (RI 53
drying, and microwave drying) and conventional Binder, Germany) at 60°C for 20–24 h. Microwave
extraction processes (soxhlet extraction, macer- drying wasperformedbyadigitalmicrowave(Sam-
ation, and percolation) on the total phenolic sung J7EV, Malaysia) at 600 watts for 4 min, in
content (TPC), total flavonoid content (TFC), which the leaves were flattened on a few pieces of
and free radical scavenging activity (FRSA) of tissue paper laid between the leaves and a ceramic
E. prostrata extracts. The levels of marker com- plate to absorb water vapour. For freeze-drying,
pounds (wedelolactone and luteolin) were anal- the leaf samples were pre-frozen by liquid nitrogen
ysed by high-performance liquid chromatogra- before lyophilizing overnight at −40°C, 0.5 psi in
phy (HPLC). Liquid chromatography-electrospray a freeze dryer (Heto Power Dry PL3000, Thermo
ionization-quadrupole-time of flight-tandem mass Fisher Scientific, Japan). The dried leaves were
spectrometry (LC-ESIQTOF-MS/MS) was used to ground to powder with a grinder, followed by stor-
characterize the phenolic compounds and their gly- age in a dark closed container with silica absorber.
cosides. Cell cytotoxicity and anti-proliferation The extraction methods of soxhlet, maceration
assays of the E. prostrata extracts were assessed and percolation were carried out with the dried
using HaCaT cells. Our experimental data could leaf powder and 99.9% (v/v) ethanol in a ratio of
introduce a proper choice for producing E. prostrata 1:100 (w/v). Dried samples (2.5 g) were packed
crude extracts, which would be further developed in a cellulose thimble (33×80 mm) (Whatman, GE
andapplied for skin health and treatment products, Healthcare, UK) with soxhlet apparatus and Mantle
cooperating by small and medium-sized enterprises (MS-EAM M-TOP, Indonesia), and extracted with
(SMEs). 250mloftheethanolfor10h(onecycleperhour)at
80°C. Maceration was performed by filling 0.2 g of
driedleafpowderinatightlyclosed50mlglasstube
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ScienceAsia 45 (2019) 129
containing 20 ml ethanol, then shaken at 150 rpm FRSA (%) = (A −A )/A ×100, where A is the
0 1 0 0
for 24 h at room temperature (30±5°C). A perco- absorbance of the control and A1 is the absorbance
lation column was formed in a disposable syringe of the test sample.
(0.5 cm in diameter and 10 cm height) (NIPRO, LC-MS/MSandHPLC
Japan). A set was 0.1 g of leaf powder packed into
the syringe to obtain a 0.2 ml bed volume. The AnextractwasanalysedbyreversephaseHPLC(LC-
effluent of 10 ml loading was collected as a fraction 20AC Shimadzu, Japan), modified from the previ-
with the flow rate controlled at 0.1–0.2 ml/min by ous method23. Each extract was filtered through a
a vacuum manifold (12-Port Teknokrama, Spain). 0.22 µm nylon filter (VertiClean Vertical, Thailand)
All extracts were filtrated through Whatman no. 4 and injected onto the Inertsil ODS-3C18 column
paper, and the volume was made up to compensate (4.6×250 mm, 5 µm, Hichrom Limited, UK) with
for evaporation. All samples were kept in separated an injection volume of 20 µl. The mobile phases
amber glass bottles with tight stoppers at 4°C until were 3% (v/v) acetic acid (mobile phase A) and
analysis. Then 100 ml of each sample from soxhlet 99.9%(v/v)methanol(mobilephaseB),withaflow
extraction were evaporated and dried at 60°C to rate of 1 ml/min. The components of the extract
obtain a dried crude extract. were separated using gradient elution at 40°C and
TPC, TFC and FRSA detected at 280 and 360 nm with a UV-diode ar-
ray detector (SPD-M20A, Shimadzu, Japan). Peak
The TPC was determined using a modified Folin- identification was performed by comparing the re-
Ciocalteu method20. Briefly, a 100 µl extract was tention times (RT) with the standard compounds.
pipetted into 1.5 ml Eppendorf tube and 500 µl of Wedelolactone and luteolin, which were confirmed
10%(v/v) Folin-Ciocalteu reagent was added. The the peaks by the RT of LC-MS, were quantitatively
mixture was left to stand in the dark for 3 min and determined by external standard methods. The
then 100 µl of 7.5% (w/v) Na CO and 300 µl of same HPLC conditions were operated with various
2 3
deionized water were filled in. After 2 h, the ab- concentrations for calibration curves, which were
sorbance was measured at 731 nm using a UV/vis- generated by linear regression based on the peak
ible spectrometer (Beckman Coulter DU 730 Life area.
Science, USA). A standard curve was constructed The LC-MS/MS was conducted following a
from 5, 20, 40, 80, and 100 mg/l of caffeic acid. modified method24. The main components were
The TPC was expressed in terms of a caffeic acid assayed by LC-MS/MS, with quadrupole-time of
equivalent (µmol CAE/g dry wt). flight mass analysers. The LC-QTOF-MS/MS anal-
The TFC was analysed using a modified colori- ysis was performed on an Agilent HPLC 1260 series
metric method21. Aliquots of 500 µl of deionized coupled with a QTOF 6540 UHD accurate mass
water and 100 µl of leaf extract were added to (Agilent Technologies, Waldbronn, Germany). The
1.5 ml Eppendorf tube. Then 30 µl of 5% (w/v) separation of the sample solution was carried on a
NaNO was added. The mixtures were kept in the Luna C18(2) 150×4.6 mm, 5 µm (Phenomenex,
2
dark for 5 min before adding 60 µl of 10% (w/v) USA). The solvent flow rate was 500 µl/min, and
AlCl3. After standing for 6 min, 200 µl of 1 M NaOH 5 µl of the sample solution was injected into the
and 110 µl of deionized water were added. After LC system. The binary gradient elution system was
5 min in the dark, the absorbance was measured at composed of water as solvent A and acetonitrile
510 nm. A standard curve was prepared from 10, as solvent B, and both contained 0.1% formic acid
20, 40, 80, and 100 mg/l of epicatechin. The TFC (v/v). The linear gradient elution was 5–95% for
wasexpressedintermsofanepicatechinequivalent solvent B at 35 min and a post run for 5 min. The
(µmol EPE/g dry wt). columntemperaturewassetat35°C.Theconditions
The FRSA was evaluated based on the 2,2- for the negative ESI source were as follows: drying
diphenyl-1-picrylhydrazyl free radical (DPPH·) gas(N )flowrate10l/min,dryinggastemperature
22 2
method . An amount of 100 µl of the leaf 350°C, nebulizer 30 psig, fragmentor 100 V, capil-
extract or blank was pipetted into separated lary voltage 3500V,andscanspectrafromm/z100–
1.5 ml Eppendorf tube and 900 µl of 80 µM 1500 amu. The auto MS/MS for the fragmentation
DPPH solution was added. The mixture was was set with collision energies of 10, 20, and 40 V.
kept in the dark for 30 min, the absorbance was All dataanalyseswerecontrolledusingAgilentMass
measured at 515 nm. The ability of the extract HunterQualitativeAnalysisSoftwareB06.0(Agilent
to scavenge DPPH free radicals was calculated by Technologies, CA, USA).
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130 ScienceAsia 45 (2019)
High performance thin layer chromatography Table1 Percentagesofwetweightanddryweightofeach
†
(HPTLC) analysis plant part per whole plant.
The phenolic and flavonoid compounds were in- Plant part Wet weight (%) Dry weight (%)
vestigated by an HPTLC system (CAMAG, Mut- Root 7.1±2.2 11.8±3.9
tenz, Switzerland) with TLC visualizer linked to Leaf 24.2±1.5 35.1±0.4
vision CATS software. A plant crude extract and Flower 6.8±0.1 8.7±1.1
each chemical standard were dissolved in DMSO Stem 61.9±3.6 44.3±3.2
(dimethyl sulphoxide) 30 and 20 mg/ml, respec- † Data are given as mean±SD (n=3).
tively. The filtered solutions were applied to silica
60F 254 on aluminium sheet, 10×20 cm (Merck,
Darmstadt, Germany), and the conditions were sy- 50 ng/ml of TNF-α to stimulate cell inflammation.
ringe delivery speed of 10 s/µl; injection volumes After that, the cells were washed with phosphate
of 2 µl for plant extract and 1 µl for standards; buffer saline (PBS) before treatments with different
bandwidth8mm;anddistancefrombottom8mm. concentrationsoftheplantextracts(62.5,125,250,
The HPTLC plate was developed in a horizontal 500, and 1000 µg/ml) for 24 h. Two marker com-
chamber after saturation in a mobile phase of pounds,wedelolactoneandluteolin,wereemployed
toluene: ethyl acetate: formic acid (11:6:1 v/v/v) as standards at the concentrations of 6.3, 12.5, 25,
for 5 min at room temperature25. The length of 50, and 100 µg/ml. Paclitaxel, a chemotherapy
the chromatograms was 75 mm from the applied medication for cancer treatment, at the concentra-
spots. The developed plate was allowed to dry tions of 0.3, 0.6, 1.3, 2.5, and 5.0 µg/ml was also
for 1 min before derivatizing with natural product used as a positive control.
reagent I (1% (w/v) ethanolamine diphenyl borate After incubation for 24 h, the medium was
in methanol) and natural product reagent II (5% removed from each treatment. Then the cells were
(v/v) polyethylene glycol-100 in ethanol)26. The washed twice with PBS and replaced in each well
plate before and after derivatization was observed with 110 µl of MTT (3-(4,5-dimethylthiazol-2-yl)-
underaUVlampat254and365nm. Thestandards 2,5-diphenyltetrazoliumbromide)solutionatafinal
of chlorogenic acid, caffeic acid, p-coumaric acid, concentration of 0.5 mg/ml, before incubating in
rutin, wedelolactone and luteolin were used for the dark for 2 h. The MTT solution was removed,
identification. washed with PBS, and replaced with 100 µl of
DMSOtodissolvetheintracellularlyformedcrystals
Cell cytotoxicity, anti-proliferation and MTT of dark-blue formazan. The absorbance at 540 nm
assays was measured for the cell viability27. Cell survival
Dried crude extracts were dissolved in DMSO as rate (%) was calculated from the fraction of alive
stock solutions at 50 mg/ml. The concentrations cells relative to that of the control for each point, as
cell survival rate (%) = (A −A )/(A −A )×100,
of DMSO were controlled to be less than 1%. The S B C B
crude extracts were filtered through a 0.2 µm filter where AS, AB, and AC are the absorbances of the
(Corning Inc., Corning, NY, USA). HaCaT cells sample, blank, and control, respectively.
(Cell Line Service, Heidelberg, Germany), the im- Statistical analysis
mortalized human epidermal keratinocyte cell line, The data were reported as the mean±standard
were cultured in Dulbecco’s Modified Eagle Medi- deviations (SD) and were analysed using ANOVA.
um/High glucose (DMEM/HG) (GibcoTM Thermo Significant differences between the means were
Fisher Scientific, USA) with 10% (v/v) fetal bovine determined by Duncan’s new multiple range test
serum, 100U/mlpenicillin, and 100 µg/mlstrepto- (DMRT). Statistical analyses were performed using
mycin. The cells were cultured at 37°C in a humid- SPSS statistical software (SPSS 14, SPSS Inc., IL,
ified atmosphere, and 5% CO2 for 24 h that gave USA).
80% cell confluence. For the cell cytotoxicity test,
the cells were trypsinized with 3 ml of 5% (w/v) RESULTSANDDISCUSSION
trypsin for 15 min and seeded in a 96 well plate Plant parts
at a cell density of 5×104 cells/ml for 24 h that
gave a cell confluence of about 50%. For the anti- WholeplantsofE.prostratawereseparatedtoroots,
proliferation test, the cells were seeded for 12 h, leaves,flowersandstems,andtheyweredriedtode-
then replaced with fresh medium that contained terminethedrymassproductionperplant(Table 1).
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